Toi ! Assu

Renata Alb

Mameli E Marinella

Anna The Mameli's Salva

Kri

Cadm3, Cd200, Cgn, Dio2, Dner, Endod1, Entpd1, Faah, Grem2, Itga5, Klhdc8a, Lhx2, Lsamp Aldh1a1

Genes increased in the Nac D2 neurons

Keywords: Cocaine-induced gene transcription Btaf1, C2cd2, Cep162, Clip2, Cmbl, Crtac1, Cryz, Cstf2t, Cux2 Tmem62 Tmsb10, Tnrc6c, Tor1aip1 Zfhx2, Zfp385b Swi5 Szt2, Timm17b, Tmem59

pouvait parler toujours de Dante » ; on ne peut pas mais si tu savais quel bonheur

-Acknowledgments -

Dear Jean-Antoine, it has been a long but instructive journey untill here. I should insert another long table to list all the reasons that I have to thank you. Thanks for the possibility of joining our lab. Thanks for having "obliged" me to discuss every single experiment that I thought to do. I learned the importance of having a clear idea of what you wish to do, as well as the importance of knowing how to defend it. I also learned how fundamental is to be rigorous and so to be patient. The work that you did on me during these 4 years, and the work that you have done for my thesis in this last month is unvaluable. Even if there is always something new to learn to do this job properly, I'm sure that now I have the good imprinting for the future. Thank you.

Cher Denis, thank you, thank you for having found always the time to discuss and give me real precious advises. Thank you very much for being always direct in the comments on my work as well as on my experiments. Not less important, I really have to thank you for being always that kind, generous, and accessible in the everyday lab life.

I would like to thank, Paul Grennegard. The collaboration with the Greengard's lab was foundamental to realizationon of my thesis work. Thank you as well for having supported me, and make me feel as part of the lab.

I would like to thank all the members of my thesis comeetee, Jocelyne Caboche, Alban de Kerchove, Emmanuel Valjent, Miquel Martinez, and Michael Kreutz for accepting to my jury and to rewiev my work.

Michael Kreutz, I really have several reasons to thank you. First thank you for being part of my thesis commetee, thank you for the scientific and personal adivices that you gave me during the last years, and most of all thank you and Christina Spilker for the amazing work that you did with the NPLAST. You really took care of us, and it has been a unique, formative, challenging, and excellent experience. Thank you.

Dear Miquel Martinez, thank you for having accept to be part of my thesis, thank you for your patience, and most of all, for all the help that you an Emmanuel gave me in setting up the operant protocol. You can see now how important was for my work. #5thfloor. Lieng je commence par toi, parfois trouver une langue commun n'a pas été facile, mais on y est arrivés. Merci pour avoir essayé et réussi a te mettre sur ma longueur d'onde et pour le soutien et surtout pour tout ce que on a produit ensemble. Sophie, t'es mon daily rappel au calme. Comment te remercier pour ça ? Merci aussi pour ton amitié sincère et pour ta confiance. Moshi moshi Yuki, when you arrived « madreusement » you found me a bit stressed but we made a good team! Thank for having brought a little japanise organization in my lab-life, the help and the good time. Yukari, welcome on board, you'll be amazing, I know it already! Merci Nico, merci pour tes conseils, ta -Summary -Dopamine (DA) controls movement execution, action selection, and incentive learning by regulating the activity and plasticity of corticostriatal transmission.

Long-term modifications require changes in gene transcription. The aim of this work is to study the changes in transcriptions following an operant learning protocol or mimicking stimulation of the reward system with cocaine.

The largest neuronal population of the striatum is comprised of medium-size spiny striatal projection neurons (SPNs), which can be divided into two different populations based on the expression of the D1 or D2 DA receptor. Although these two populations share many morphological characteristics and functional properties, they participate in distinct pathways, the direct pathway for D1-SPNs and indirect pathway for D2-SPNs, which have opposite functional effects on their target regions.

Therefore it is crucial to distinguish responses in the two populations.

To do so, we used transgenic mice that express a tagged ribosomal protein (L10a-EGFP) under control of the D1 or D2 receptor promoter to isolate currently translated mRNA and nuclei from each population of SPNs, as well as from D1 pyramidal neurons of the prefrontal cortex. following passive stimulation of the reward system (chronic treatment with cocaine) and active recruitment of the reward system (operant learning for food). For the latter, we developed an operant conditioning protocol in which mildly food-deprived mice learned to nose poke to obtain either regular food or highly palatable food. The results in trained mice were compared to yoked controls which receive the same food but non-contingently.

The first part of this work was dedicated to the comparison of the basal gene expression in the different neuronal populations characterized by the expression of D1 or D2 receptors and their regional localization in the ventral striatum (nucleus accumbens), dorsal striatum, or prefrontal cortex. We thus identified several hundreds of differentially expressed mRNA which provide a precise characterization of the cellular and regional differences in dopaminoceptive neurons.

In the second part, we characterized the changes induced in each neuronal population by a 1-week exposure to cocaine. In the third part, the pattern of gene alterations in each condition and neuronal population are currently being analyzed and compared to other parameters including morphological alteration of spines. - ------------------------------------------------------------------------------------------------- -----------------------------------------------------------------FIGURE 6: ANATOMICAL PART OF THE STRIATUM: -------------------------- --------------------------------------------- --------------------------------------------- ---------------------------------------------- --------------------------------------------------------------------- ------------------------------------------------------------ -------------------------------------------------------------------------------- ------------------------------------------ ---------------------------------- ------------------------------------------------------------ ------------------------------------------------------------ ------------------------------------------------- ----------------------------------------------------------------------------- ----------------------------------------------------------------------------- - ----------------------------------------------------------------------------------------------- -------------------------------FIGURE 36: GRAPHICAL CONCLUSION AIM-1 ----------------------------------FIGURE 37 GRAPHICAL CONCLUSION AIM-2. ----------------------------------FIGURE 38 GRAPHICAL CONCLUSION AIM-3 ---------------------------------- --------------------------------------------- important roles in the brain as well as in rest of the body. In the nervous system, DA acts as neurotransmitter by regulating the activation of the dopamine receptors, and affecting the neural plasticity of several types of neurons. Dopamine signalling within its different circuit is associated with key mechanisms such as locomotor activity or goal-directed behaviours. The main dopaminergic cell groups are located in ventral mesencephalic tegmentum and project to the more anterior part of the brain via the medial forebrain bundle. Based on the localisation of the dopaminergic cells it is possible to distinguish two principal populations of DA neurons that form a gradient of projections to different areas of the brain: the DA cells of the substantia nigra pars compacta (SNc, A9) that principally project to the striatum via the nigrostriatal system, and the DA cells in the ventral tegmental area (VTA, A10) that project to the nucleus accumbens (NAc) and olfactory tubercle by the mesolimbic system, and to the amygdala, hippocampus and prefrontal cortex (PFC) via mesocortical system. The different dopaminergic projections are implicated in the regulation of different mechanisms that are completely dependent of the receiving area. The nigrostriatal circuit (A9>A10) has been traditionally implicated in the regulation of motor function [START_REF] Graybiel | The Basal Ganglia and Adaptive Motor Control[END_REF] [START_REF] Graybiel | The Basal Ganglia and Adaptive Motor Control[END_REF]) and its deregulation results in motor-related disease such as Parkinson's disease (PD). The mesolimbic circuit (A10>A9) is associated with goal directed behaviours [START_REF] Wise | Brain Dopamine and Reward[END_REF]) and motivation, and high jacking this system results in different forms of addiction. Finally the mesocortical circuit is mostly associated with higher cognitive functions such as learning and memory. A third of group of DA cells is found at the level of the hypothalamus. The cells in this area project to the pituitary gland where DA inhibits the prolactin secretion, and this circuit is associated with important process such as pregnancy or nurturing behaviour. This pathway hasn't been investigated in the present work; therefore it will be not further discussed. stimulatory (Gα s ) or inhibitory (Gα i ) subunit. In striatal neurons, the stimulatory subunit is the Gα-olf isoform [START_REF] Hervé | G(olf) and Gs in Rat Basal Ganglia: Possible Involvement of G(olf) in the Coupling of Dopamine D1 Receptor with Adenylyl Cyclase[END_REF][START_REF] Zhuang | G(olf)alpha Mediates Dopamine D1 Receptor Signaling[END_REF][START_REF] Corvol | Galpha(olf) Is Necessary for Coupling D1 and A2a Receptors to Adenylyl Cyclase in the Striatum[END_REF]. Gα-olf mediates the coupling of D1 receptors to the AC and is coded by an independent gene [START_REF] Corvol | Galpha(olf) Is Necessary for Coupling D1 and A2a Receptors to Adenylyl Cyclase in the Striatum[END_REF]. The activation of the receptor via DA stimulation results in the detachment of the Gα subunit from the β and γ subunits.

Gαs/olf exerts a positive effect on AC and leads to production of cAMP, whereas Gαi/o inhibits AC and decreases cAMP production [START_REF] Albert | Coupling of a Cloned Rat Dopamine-D2 Receptor to Inhibition of Adenylyl Cyclase and Prolactin Secretion[END_REF]). In the striatum, the activation of D1Rs leads to the Golf-mediated stimulation of adenylyl cyclase. Adenylyl cyclase catalyzes the conversion of ATP to cAMP, which binds to the regulatory subunits of the PKA holoenzyme to disinhibit the catalytic subunits.

The activation of cAMP signalling promotes transmission trough the α-amino-3hydroxyl-5-methyl-4-isoxazole-propionate (AMPA) and N-methyl-D-aspartate (NMDA) glutamate receptors [START_REF] Surmeier | Modulation of Calcium Currents by a D1 Dopaminergic Protein Kinase/phosphatase Cascade in Rat Neostriatal Neurons[END_REF][START_REF] Blank | The Phosphoprotein DARPP-32 Mediates cAMP-Dependent Potentiation of Striatal N-Methyl-D-Aspartate Responses[END_REF][START_REF] Snyder | A dopamine/D1 Receptor/protein Kinase A/dopamine-and cAMP-Regulated Phosphoprotein (Mr 32 kDa)/protein Phosphatase-1 Pathway Regulates Dephosphorylation of the NMDA Receptor[END_REF]. The final output of D1R activation is to elevate the ability of sustained release of glutamate to promote the excitability of the neurons carrying the D1 receptor [START_REF] Surmeier | Calcium, Ageing, and Neuronal Vulnerability in Parkinson's Disease[END_REF]. In striatal neurons, cAMP is degraded by several phosphodiesterases, including PDE1B and PDE10A, which are highly enriched in medium -sized spiny neurons [START_REF] Fujishige | Striatum-and Testis-Specific Phosphodiesterase PDE10A Isolation and Characterization of a Rat PDE10A[END_REF][START_REF] Polli | Expression of a Calmodulin-Dependent Phosphodiesterase Isoform (PDE1B1) Correlates with Brain Regions Having Extensive Dopaminergic Innervation[END_REF] where they play key role in regulating cAMP signalling [START_REF] Nishi | Distinct Roles of PDE4 and PDE10A in the Regulation of cAMP/PKA Signaling in the Striatum[END_REF]). As mentioned above, the action of the activation of the D2R is coupled with the Gi/o-mediated inhibition of adenylyl cyclase, which decreases cAMP synthesis [START_REF] Stoof | Opposing Roles for D-1 and D-2 Dopamine Receptors in Efflux of Cyclic AMP from Rat Neostriatum[END_REF]. The decrease of cAMP synthesis will in turn affect the PKA substrates phosphorylation. In addition D2R activation is able to regulate the L-type Ca 2+ currents trough Ca 2+ channels [START_REF] Hernandez-Lopez | D2 Dopamine Receptors in Striatal Medium Spiny Neurons Reduce L-Type Ca2+ Currents and Excitability via a Novel PLC[END_REF] and activate K + channels [START_REF] Wickman | Recombinant G-Protein Beta Gamma-Subunits Activate the Muscarinic-Gated Atrial Potassium Channel[END_REF][START_REF] Kuzhikandathil | Human Dopamine D3 and D2L Receptors Couple to Inward Rectifier Potassium Channels in Mammalian Cell Lines[END_REF][START_REF] Hopf | Cooperative Activation of Dopamine D1 and D2 Receptors Increases Spike Firing of Nucleus Accumbens Neurons via G-Protein Betagamma Subunits[END_REF]. The coordinated modulation of ion channels exerted by D2R via activation of Gi/o protein leads to a reduced responsiveness of MSNs to glutamate, and therefore reduced excitability [START_REF] Surmeier | Calcium, Ageing, and Neuronal Vulnerability in Parkinson's Disease[END_REF]. on dopaminergic nerve terminals, where they are involved in the regulation of the DA synthesis and release (Jaber et al., 1996).

-Chapter 2 -

The Basal Ganglia

The striatum

The striatum is the main recipient of afferents of the basal ganglia. The term striatum refers to its striped appearance produced by the abundant fiber bundles that pass through it. Although the striatum is a complicated and heterogeneous structure, in mice, it is possible to define a gross division of this structure in two sub-regions that correspond to the DS and the ventral striatum that includes NAc and olfactory tuberculum. In primates the presence of the internal capsule allows an additional separation of the DS into two additional components the caudate nucleus, being the main target of the prefrontal cortex inputs, and the putamen that is mostly targeted by sensorimotor and motor cortices. This separation is not found in rodents, even though it is possible to find a regional distribution of the inputs coming from the afferent regions. In rodents the dorsal striatum is also referred to as the caudate-putamen (CP) and can be further sub-divided in dorsomedial and dorsolateral striatum. The NAc can be subdivided in two main sub-regions: the core and the shell [START_REF] Alheid | New Perspectives in Basal Forebrain Organization of Special Relevance for Neuropsychiatric Disorders: The Striatopallidal, Amygdaloid, and Corticopetal Components of Substantia Innominata[END_REF]Voorn et al. 2004). While the core was proposed to be closely related to the DS in terms of connections and functions, the shell is considered to be related to the extended amygdala. The shell itself is highly heterogeneous [START_REF] Gangarossa | Distribution and Compartmental Organization of GABAergic Medium-Sized Spiny Neurons in the Mouse Nucleus Accumbens[END_REF].

The dorso-ventral division was initially based on the differences in the afferent connections received by these two striatal regions, since DS and NAc are histologically indistinguishable. Important functional differences arise from these afferent connections from the other brain areas. The DS receives a massive excitatory glutamatergic input from most cortical regions and the thalamus, as well as a DA input from the SNc (Kitai et al., 1976, Donoghue andHerkenham, 1986;Nakano, 2000;Herrero et al., 2002). On the other hand the ventral striatum mainly receive DA innervation from the ventral tegmental area as well as glutamatergic input from limbic cortices, amygdala and thalamic nuclei.

-Introduction -The basal ganglia 2 dorsomedial to dorsoventral gradient. While the dorsolateral striatum collects projections from the sensorimotor cortical areas, the ventromedial part of the striatum receives a massive innervations from the prelimbic and infralimbic prefontal cortex [START_REF] Berendse | Topographical Organization and Relationship with Ventral Striatal Compartments of Prefrontal Corticostriatal Projections in the Rat[END_REF]. Amygdala and hippocampus, as the cortical area connect to the striatum. In general, the dorsal striatum collects projections from the neurons located in the dorsal part of the hippocampus while the more ventral neurons in the hippocampus form a major input in the ventral part of the striatum [START_REF] Groenewegen | Convergence and Segregation of Ventral Striatal Inputs and Outputs[END_REF]. The same pattern of projections is maintained for the amygdaloid nuclei: the rostral basal nuclei of the amygdala project to the more lateral striatum and are linked to associative functions, whereas the caudal basal nuclei that associate with viscerolimbic functions target the ventral part of the striatum. The same type of gradient is maintained at the level of the thalamic connections: while the motor associated posterior-lateral intralaminar thalamic nuclei mostly connect with the dorsolateral part of the striatum, the paraventricular nucleus, located more ventrally in the thalamic complex innervates predominantly the NAc.

Organization of the dopaminergic projections to the striatum

DA is the principal modulatory neurotransmitter that is released in the striatum.

The main dopaminergic cell groups are located in the midbrain and project in a topographic fashion [START_REF] Beckstead | Efferent Connections of the Substantia Nigra and Ventral Tegmental Area in the Rat[END_REF]Mattiace et al. n.d. 1979). The dorsal lateral striatum mostly collects the projections coming from two distinct populations of DA cells: the DA neurons that originate in the retrorubral area (A8), and the DA cells of SNc (A9) that project to a more intermediary part of the striatum via the nigro-striatal system. Ventromedial striatum and NAc are mostly reached by the DA cells located in the VTA (A10). [START_REF] Guyenet | Antidromic Identification of Dopaminergic and Other Output Neurons of the Rat Substantia Nigra[END_REF][START_REF] Beckstead | Efferent Connections of the Substantia Nigra and Ventral Tegmental Area in the Rat[END_REF][START_REF] Veening | The Topical Organization of the Afferents to the Caudatoputamen of the Rat. A Horseradish Peroxidase Study[END_REF][START_REF] Albanese | Organization of the Ascending Projections from the Ventral Tegmental Area: A Multiple Fluorescent Retrograde Tracer Study in the Rat[END_REF][START_REF] Gerfen | The Neostriatal Mosaic: II. Patch-and Matrix-Directed Mesostriatal Dopaminergic and Non-Dopaminergic Systems[END_REF]).

-Introduction -The basal ganglia 2

The patch/matrix organization in the striatum

The striatum in organized into two anatomically distinct compartments called patches (striosomes) and matrix (extratriosome) [START_REF] Pert | Opiate Receptor: Autoradiographic Localization in Rat Brain[END_REF][START_REF] Graybiel | Histochemically Distinct Compartments in the Striatum of Human, Monkeys, and Cat Demonstrated by Acetylthiocholinesterase Staining[END_REF] .

The very first study suggesting an histological compartmentalization in the striatum dates back to 1976, when Pert and collaborators showed a patchy distribution of the µ-opiate receptors in a matrix characterized by a lower receptor density [START_REF] Pert | Opiate Receptor: Autoradiographic Localization in Rat Brain[END_REF]Herkenham & Pert 1981) [START_REF] Pert | Opiate Receptor: Autoradiographic Localization in Rat Brain[END_REF][START_REF] Kent | Ontogeny of Opiate Receptors in Rat Forebrain: Visualization by in Vitro Autoradiography[END_REF]). In general, differential staining pattern are used for visualizing the patch matrix organization, for example the acetylcholinesterase staining as well as calbindin immunoreactivity, is poorer in patches than in the matrix while dopamine transporter (DAT), and Nr4a1 (nuclear receptor subfamily 4, group A, member 1) staining are higher in the patches [START_REF] Graybiel | Histochemically Distinct Compartments in the Striatum of Human, Monkeys, and Cat Demonstrated by Acetylthiocholinesterase Staining[END_REF][START_REF] Holt | Neurochemical Architecture of the Human Striatum[END_REF][START_REF] Prensa | Chemical Heterogeneity of the Striosomal Compartment in the Human Striatum[END_REF]Gerfen n.d.;[START_REF] Davis | Nr4a1-eGFP Is a Marker of Striosome-Matrix Architecture, Development and Activity in the Extended Striatum[END_REF]Jill R Crittenden & Graybiel 2011). The matrix compartment represents approximately 85% of the striatum (Johnston et al., 1990 andMikula et al., 2009) and the striosomal compartment comprises the 15% of the striatum and is associated with limbic circuits [START_REF] Eblen | Highly Restricted Origin of Prefrontal Cortical Inputs to Striosomes in the Macaque Monkey[END_REF]Gerfen n.d.;[START_REF] Kincaid | Corticostriatal Innervation of the Patch and Matrix in the Rat Neostriatum[END_REF] However, as shown in the work of Davis and Puhl in 2011 these ratios can vary across the striatum [START_REF] Davis | Nr4a1-eGFP Is a Marker of Striosome-Matrix Architecture, Development and Activity in the Extended Striatum[END_REF].

Patches and matrix features

Patches and matrix differ from each other in several ways, including neurotransmitter enrichment [START_REF] Graybiel | Histochemically Distinct Compartments in the Striatum of Human, Monkeys, and Cat Demonstrated by Acetylthiocholinesterase Staining[END_REF][START_REF] Holt | Neurochemical Architecture of the Human Striatum[END_REF] connectivity [START_REF] Gerfen | The Neostriatal Mosaic: II. Patch-and Matrix-Directed Mesostriatal Dopaminergic and Non-Dopaminergic Systems[END_REF]Gerfen 1989), neuronal organization, development [START_REF] Van Der Kooy | Neuronal Birthdate Underlies the Development of Striatal Compartments[END_REF][START_REF] Graybiel | Chemospecificity of Ontogenetic Units in the Striatum: Demonstration by Combining [3H]thymidine Neuronography and Histochemical Staining[END_REF][START_REF] Liu | Heterogeneous Development of Calbindin-D28K Expression in the Striatal Matrix[END_REF], and gene expression [START_REF] Moratalla | Differential Vulnerability of Primate Caudate-Putamen and Striosome-Matrix Dopamine Systems to the Neurotoxic Effects of 1-Methyl-4-Phenyl-1,2,3,6-Tetrahydropyridine[END_REF][START_REF] Grande | Chronic Treatment with Atypical Neuroleptics Induces Striosomal FosB/DeltaFosB Expression in Rats[END_REF] The patch matrix organization during the development: the DA innervation in patches within the striatum can be observed already at the early stages of the post-2 -Introduction -The basal ganglia 36 natal development [START_REF] Tennyson | The Developing Neostriatum of the Rabbit: Correlation of Fluorescence Histochemistry, Electron Microscopy, Endogenous Dopamine Levels, and ( 3 H)dopamine Uptake[END_REF]. The neurons that are first innervated in the striatum are known as "dopamine island" and mostly receive innervations from the substantia nigra [START_REF] Olson | Heterogeneity of Striatal and Limbic Dopamine Innervation: Highly Fluorescent Islands in Developing and Adult Rats[END_REF][START_REF] Edley | Comparative Development of Striatal Opiate Receptors and Dopamine Revealed by Autoradiography and Histofluorescence[END_REF][START_REF] Van Der Kooy | Neuronal Birthdate Underlies the Development of Striatal Compartments[END_REF]. The patches neurons of striatum will develop earlier than most matrix neurons that are born later in the embryogenesis.

Organization of the patch-matrix connectivity: the glutamatergic projections to the striatum differentially innervates patch and matrix in the striatum: while the patches receive mostly the innervation from the limbic circuit (orbitofrontal, anterior cingulate, and insular cortices) [START_REF] Graybiel | Histochemically Distinct Compartments in the Striatum of Human, Monkeys, and Cat Demonstrated by Acetylthiocholinesterase Staining[END_REF], the projections from the other neocortical areas such as somatosensory, motor, and association cortices terminate mainly in the matrix [START_REF] Ragsdale | A Simple Ordering of Neocortical Areas Established by the Compartmental Organization of Their Striatal Projections[END_REF]Flaherty & Graybiel 1994;[START_REF] Eblen | Highly Restricted Origin of Prefrontal Cortical Inputs to Striosomes in the Macaque Monkey[END_REF][START_REF] Kincaid | Corticostriatal Innervation of the Patch and Matrix in the Rat Neostriatum[END_REF]Lévesque & Parent n.d.).

The glutamatergic inputs can terminate on dendritic shafts or spines [START_REF] Lacey | GABA(B) Receptors at Glutamatergic Synapses in the Rat Striatum[END_REF][START_REF] Raju | Differential Synaptology of vGluT2-Containing Thalamostriatal Afferents between the Patch and Matrix Compartments in Rats[END_REF], however, the inputs on the shaft are mainly enriched in the matrix (Fujiyama et al. 2006;[START_REF] Raju | Differential Synaptology of vGluT2-Containing Thalamostriatal Afferents between the Patch and Matrix Compartments in Rats[END_REF]. According to the classical view of the patch-matrix afferents, the inputs from the thalamic nuclei are denser in the matrix (Fujiyama et al. 2006). However, lately it has been shown that the intralaminar thalamic nuclei mainly target the matrix, whereas the midline thalamic nuclei target preferentially the patches [START_REF] Unzai | Quantitative Analyses of the Projection of Individual Neurons from the Midline Thalamic Nuclei to the Striosome and Matrix Compartments of the Rat Striatum[END_REF]. DA projections, like the glutamatergic inputs, differentially target the patches and the matrix. While neurons originating from the SNc preferentially target the patch compartment, the cells originating from VTA, lateral SNc and retrobrural area principally connect to the matrix [START_REF] Gerfen | The Neostriatal Mosaic: II. Patch-and Matrix-Directed Mesostriatal Dopaminergic and Non-Dopaminergic Systems[END_REF]. Given this difference in innervation, a recent work compared the release of DA in the striosome and matrix compartments using fast-scan cyclic voltammetry in Nr4a1-eGFP transgenic mice. In particular in the DS, dopamine release in striosomes was less than in the matrix, while the opposite way was observed in the ventral striatum. Salinas and collaborators also found that cocaine administration enhanced the DA levels more in the patches than in matrix regions [START_REF] Salinas | Dopamine Dynamics and Cocaine Sensitivity Differ between Striosome and Matrix Compartments of the Striatum[END_REF] Lastly, concerning the projections arising from the striatum, the matrix compartment preferentially targets the GP and the SNr, while patches synapse preferentially with the SNc [START_REF] Kawaguchi | Projection Subtypes of Rat Neostriatal Matrix Cells Revealed by Intracellular Injection of Biocytin[END_REF][START_REF] Giménez-Amaya | Modular Organization of Projection Neurons in the Matrix Compartment of the Primate Striatum[END_REF]Lévesque & Parent n.d.;[START_REF] Chuhma | Functional Connectome of the Striatal Medium Spiny Neuron[END_REF]). According to this scheme of projections, it can be argue that the striatal projection neurons in the striosomes may -Introduction -The basal ganglia 2 have a role in regulating the release of dopamine in the striatum [START_REF] Fujiyama | Exclusive and Common Targets of Neostriatofugal Projections of Rat Striosome Neurons: A Single Neuron-Tracing Study Using a Viral Vector[END_REF][START_REF] Gerfen | The Neostriatal Mosaic: II. Patch-and Matrix-Directed Mesostriatal Dopaminergic and Non-Dopaminergic Systems[END_REF][START_REF] Watabe-Uchida | Whole-Brain Mapping of Direct Inputs to Midbrain Dopamine Neurons[END_REF] Figure 7: The different release of DA in the patch and matrix compartments of the striatum.

A. Coronal section from Nr4a1-eGFPmice stained with an antibody against the µ-opiate receptors. B Scheme of the dorso ventral release of DA in the patch and the matrix Differential gene expression in patch and matrix compartments: the different profiles of gene expression in the patch and the matrix represent another useful criterion to distinguish those two compartments. More than 60 genes are reported to have a specific enrichment in striosome or matrix (reviewed in (Jill R. Crittenden & Graybiel 2011)). Already in 1992 it has been shown that the administration of different drugs can regulate the gene expression in matrix and patch in different fashion. For example, amphetamine and a neuroleptic are able to specifically induce different profiles of expression of immediate early genes (IEG) in the patches or the matrix [START_REF] Grande | Chronic Treatment with Atypical Neuroleptics Induces Striosomal FosB/DeltaFosB Expression in Rats[END_REF][START_REF] Moratalla | Differential Vulnerability of Primate Caudate-Putamen and Striosome-Matrix Dopamine Systems to the Neurotoxic Effects of 1-Methyl-4-Phenyl-1,2,3,6-Tetrahydropyridine[END_REF][START_REF] Saka | Compartmental Changes in Expression of c-Fos and FosB Proteins in Intact and Dopamine-Depleted Striatum after Chronic Apomorphine Treatment[END_REF][START_REF] Adams | Distinct Effects of Methamphetamine and Cocaine on Preprodynorphin Messenger RNA in Rat Striatal Patch and Matrix[END_REF][START_REF] Miura | Compartment-Specific Modulation of GABAergic Synaptic Transmission by Mu-Opioid Receptor in the Mouse Striatum with Green Fluorescent Protein-Expressing Dopamine Islands[END_REF].

-Introduction -

The basal ganglia 38 2.2.2. Neuronal composition of the striatum In the striatum, the DA afferents originating from the midbrain mostly innervate two populations of GABAergic neurons named medium sized spiny neurons (MSNs) or striatal projection neurons (SPNs). In the rat SPNs represent up to 97.7 % of the striatal neuronal population [START_REF] Rymar | Neurogenesis and Stereological Morphometry of Calretinin-Immunoreactive GABAergic Interneurons of the Neostriatum[END_REF]), the remaining 2.3 % being mostly GABAergic and cholinergic interneurons (Kreitzer 2009;[START_REF] Tepper | Heterogeneity and Diversity of Striatal GABAergic Interneurons[END_REF]. SPNs are GABAergic neurons characterized by a large and extensive dendritic tree, and a medium sized body of 10-20 µm in diameter [START_REF] Kawaguchi | Projection Subtypes of Rat Neostriatal Matrix Cells Revealed by Intracellular Injection of Biocytin[END_REF]).

Striatal GABAergic interneurons

The striatal GABAergic interneurons represent roughly 2-3% of the interneurons in the striatum, and were firstly identified in 1979 as non-spiny neurons stained for glutamic acid decarboxylase 67 (GAD67) [START_REF] Ribak | The GABA Neurons and Their Axon Terminals in Rat Corpus Striatum as Demonstrated by GAD Immunocytochemistry[END_REF][START_REF] Bolam | Glutamate Decarboxylase-Immunoreactive Structures in the Rat Neostriatum: A Correlated Light and Electron Microscopic Study Including a Combination of Golgi Impregnation with Immunocytochemistry[END_REF].

Five different classes of interneurons are identifiable on the basis of the combination of markers that they express [START_REF] Kawaguchi | Physiological, Morphological, and Histochemical Characterization of Three Classes of Interneurons in Rat Neostriatum[END_REF][START_REF] Kawaguchi | Cholinergic and GABAergic Interneurons in the Striatum[END_REF] Somatostatin-positive interneurons. The somatostatin-containing interneurons are medium sized aspiny cells of 12-25 µm in diameter. This class of interneurons is positive for somatostatin, neuropeptide Y, and nitric oxide synthase.

Compared to the other classes of interneurons, their axonal arborizations are less dense within the region of the dendritic field, although they extend over longer distances within the striatum [START_REF] Kawaguchi | Physiological, Morphological, and Histochemical Characterization of Three Classes of Interneurons in Rat Neostriatum[END_REF]. The somatostatin-containing interneurons collect projections from GP, SN and cortex [START_REF] Kubota | Neostriatal GABAergic Interneurones Contain NOS, Calretinin or Parvalbumin[END_REF][START_REF] Bevan | Selective Innervation of Neostriatal Interneurons by a Subclass of Neuron in the Globus Pallidus of the Rat[END_REF], are located in both patch and matrix but innervate mainly the striatal matrix. From an electrophysiological point of view, these neurons are characterized by low threshold spikes (LTS) and a prolonged calcium dependent plateau potential [START_REF] Kawaguchi | Physiological, Morphological, and Histochemical Characterization of Three Classes of Interneurons in Rat Neostriatum[END_REF]. The activation of these cells results in the production of inhibitory postsynaptic currents in the SPNs, and their capacity to release nitric oxide has been proposed as possible regulators of the corticostriatal synaptic plasticity [START_REF] Centonze | Dopamine, Acetylcholine and Nitric Oxide Systems Interact to Induce Corticostriatal Synaptic Plasticity[END_REF] Parvalbumin positive neurons. Parvalbumin-positive interneurons have been identified in the striatal tissue by their specific content in the calcium binding protein parvalbumin [START_REF] Kita | Parvalbumin-Immunoreactive Neurons in the Rat Neostriatum: A Light and Electron Microscopic Study[END_REF][START_REF] Gerfen | The Neostriatal Mosaic: Compartmental Distribution of Calcium-Binding Protein and Parvalbumin in the Basal Ganglia of the Rat and Monkey[END_REF] and are cells of 16-18 µm in diameter that give rise to aspiny dendrites that branch modestly in the striatum.

Paravalbumin neurons receive projections from cortex, thalamus and globus pallidus [START_REF] Gerfen | The Neostriatal Mosaic: Compartmental Distribution of Calcium-Binding Protein and Parvalbumin in the Basal Ganglia of the Rat and Monkey[END_REF][START_REF] Bevan | Selective Innervation of Neostriatal Interneurons by a Subclass of Neuron in the Globus Pallidus of the Rat[END_REF][START_REF] Luk | GABA Promotes Survival but Not Proliferation of Parvalbumin-Immunoreactive Interneurons in Rodent Neostriatum: An in Vivo Study with Stereology[END_REF] and mostly project to the SPNs [START_REF] Kita | GABAergic Circuits of the Striatum[END_REF][START_REF] Bennett | Synaptic Input and Output of Parvalbumin-Immunoreactive Neurons in the Neostriatum of the Rat[END_REF][START_REF] Kubota | Dependence of GABAergic Synaptic Areas on the Interneuron Type and Target Size[END_REF] on which they exert a strong inhibition through multiple perisomatic synapses. Paravalbumin interneurons are themselves electrotonically coupled through gap junctions [START_REF] Koós | Inhibitory Control of Neostriatal Projection Neurons by GABAergic Interneurons[END_REF][START_REF] Koos | Comparison of IPSCs Evoked by Spiny and Fast-Spiking Neurons in the Neostriatum[END_REF][START_REF] Kita | Parvalbumin-Immunoreactive Neurons in the Rat Neostriatum: A Light and Electron Microscopic Study[END_REF]). Due to their electrophysiological 2 -Introduction -The basal ganglia 40 properties, the paravalbumin interneurons are commonly referred to as fast-spiking interneurons (FSIs). Compared to SPNs, parvalbumin interneurons present a lower threshold of activation [START_REF] Koós | Inhibitory Control of Neostriatal Projection Neurons by GABAergic Interneurons[END_REF] Calretinin-positive interneurons. The calretinin-positive interneurons are GABAergic interneuron, relatively sparse in the caudal striatum [START_REF] Bennett | Characterization of Calretinin-Immunoreactive Structures in the Striatum of the Rat[END_REF]) that express the calcium binding protein calretinin. The calretinin interneurons are characterized by medium-sized cell bodies and few non-spiny dendrites, and exert a powerful monosynaptic inhibition on the SPNs. Although the electrophysiological profile of those neurons is not fully described yet, they share some of the characteristics of the PLTS neurons [START_REF] Kawaguchi | Physiological, Morphological, and Histochemical Characterization of Three Classes of Interneurons in Rat Neostriatum[END_REF], such as their fired prominent LTSs [START_REF] Petryszyn | Distribution and Morphological Characteristics of Striatal Interneurons Expressing Calretinin in Mice: A Comparison with Human and Nonhuman Primates[END_REF].

NPY-neurogliaform (NGF) interneurons. The NPY-neurogliaform interneurons

are GABAergic interneurons of 13 µm in diameter that have been firstly identified in 2011. NPY-NGF interneurons differ in several features from the somatostatin interneurons. Contrary to the NPY-PLTS interneurons, NPY-NGF neurons are characterized by a dense, compact, highly branched dendritic and local axonal arborizations. Furthermore, NPY-neurogliaform interneurons exhibit a lower input resistance and hyperpolarized membrane potential, as well as the lack of depolarizations plateau or low-threshold spikes. Also, contrary to the NPY-PLST, NPY-NGF interneurons do not react to immunostaining for somatostatin or NOS.

The major target of NPY-NGF are the SPNs on which are able to exert a slow GABA(A) receptor-mediated IPSC [START_REF] Ibáñez-Sandoval | A Novel Functionally Distinct Subtype of Striatal Neuropeptide Y Interneuron[END_REF].

Late-spiking (LS) neuropeptide-Y (NPY)-negative neurogliaform (NGF) and

LTS-lke interneurons. Those two last populations of neurons are both reacting to the 5HT3a marker and were both described by [START_REF] Muñoz-Manchado | Novel Striatal GABAergic Interneuron Populations Labeled in the 5HT3a(EGFP) Mouse[END_REF][START_REF] Muñoz-Manchado | Novel Striatal GABAergic Interneuron Populations Labeled in the 5HT3a(EGFP) Mouse[END_REF]. Although those cells do not show striking electrophysiological difference they mainly differ one from the other in their pharmacological responses: LTS-like cells are characterized by a robust response to nicotine administration, while NPY-NGF-5HT3 cells type shows little or no response.

-Introduction -The basal ganglia 2

Striatal cholinergic interneurons

The cholinergic interneurons in the striatum were firstly identified in the late 1800s by Kolliker by using the Golgi staining. In the beginning those giant cells, were wrongly identified as projection neurons. Cholinergic interneurons are easily identifiable by the presence of the choline acetyltransferase enzyme (ChAT), and from their cell body of 40 µm. ChAT interneurons are often referred as tonically active neurons (TANs) based on their slow and regular firing characterized by a long action potential and slow spike after the phase of hyperpolarization. ChAT interneurons receive excitatory input from the cortex and the thalamus as well as DA inputs form SN and GABAergic inhibition from SPNs. Although they represent a minor fraction of the striatal neurons (1-2%), the synchronic activation of this population provides a tight control of the striatal input via the generation of large inhibitory currents within the striatum. Even if few in number, the ChAT interneurons have an enormous and dense dendritic arborisation that account for the fact that they represent the neurons with the higher level of expression of Ach acetylcholine and ChAT [START_REF] Macintosh | The Distribution of Acetylcholine in the Peripheral and the Central Nervous System[END_REF][START_REF] Hebb | Biochemical Evidence for the Neural Function of Acetylcholine[END_REF]. The major targets of the cholinergic interneurons are the SPNs. The ChAT neurons are able to drive an inhibitory response on the SPNs in different fashions. A direct regulation consists in a direct synaptic contact onto distal dendrites and dendritic spine necks of the SPNs [START_REF] Bolam | Characterization of Cholinergic Neurons in the Rat Neostriatum. A Combination of Choline Acetyltransferase Immunocytochemistry, Golgi-Impregnation and Electron Microscopy[END_REF]. A second, indirect, regulation on SPNs is provided by a previous excitatory synaptic input onto the nicotinic synapses on GABAergic interneurons [START_REF] English | GABAergic Circuits Mediate the Reinforcement-Related Signals of Striatal Cholinergic Interneurons[END_REF]. Importantly, due to the high density of their arborisation, and their exact position within the striatum, cholinergic axon terminals can also exert influence via volume transmission [START_REF] Koós | Dual Cholinergic Control of Fast-Spiking Interneurons in the Neostriatum[END_REF][START_REF] Descarries | Diffuse Transmission by Acetylcholine in the CNS[END_REF], [START_REF] Zhou | Endogenous Nicotinic Cholinergic Activity Regulates Dopamine Release in the Striatum[END_REF]). Lastly, several recent studies have pointed out that the nigrostriatal DA terminals are good candidates of being activated by the cholinergic neurons (Cachope, Mateo, Brian N. Mathur, et al. 2012;[START_REF] Exley | Presynaptic Nicotinic Receptors: A Dynamic and Diverse Cholinergic Filter of Striatal Dopamine Neurotransmission[END_REF][START_REF] Threlfell | Striatal Dopamine Release Is Triggered by Synchronized Activity in Cholinergic Interneurons[END_REF][START_REF] Zhou | Endogenous Nicotinic Cholinergic Activity Regulates Dopamine Release in the Striatum[END_REF]). In particular it has been shown that the optogenetic stimulation of the cholinergic neurons is able to induce the DA release in the striatum (Cachope, Mateo, Brian N Mathur, et al. 2012). Based on these evidences, recently, Nelson and colleagues suggestsed that the cholinergic interneurons are able to exert a neuromodulatory control of the striatal output by controlling the DA signaling [START_REF] Nelson | Striatal Cholinergic Interneurons Drive GABA Release from Dopamine Terminals[END_REF]

-Introduction -The basal ganglia

42

Medium sized spiny striatal projection neurons (SPNs)

Spiny neurons receive DA inputs from the SNc at the base of the dendrites spines via symmetric synapses. The DA input to the SPNs represent the 13% of the total number of synapses. The thalamic and cortical inputs represent roughly 80% of the synapses in the striatum and are mainly taking place at the level of the tips of the spines [START_REF] Freund | Synaptic Connections, Axonal and Dendritic Patterns of Neurons Immunoreactive for Cholecystokinin in the Visual Cortex of the Cat[END_REF][START_REF] Smith | Synaptic Relationships between Dopaminergic Afferents and Cortical or Thalamic Input in the Sensorimotor Territory of the Striatum in Monkey[END_REF][START_REF] Hanley | Synaptology of the Nigrostriatal Projection in Relation to the Compartmental Organization of the Neostriatum in the Rat[END_REF]. It has been estimated that each SPN receives from 5 to 10 thousand excitatory inputs. The convergent action of several inputs is necessary to trigger the activation of a single neuron. Importantly, SPNs are able to make synaptic symmetric contacts with cholinergic interneurons in the striatum itself, and with serotoninergic projections from the raphe [START_REF] Izzo | Cholinergic Synaptic Input to Different Parts of Spiny Striatonigral Neurons in the Rat[END_REF][START_REF] Soghomonian | Serotonin Innervation in Adult Rat Neostriatum. II. Ultrastructural Features: A Radioautographic and Immunocytochemical Study[END_REF]. Lastly, SPNs receive a major inhibitory input from the striatal cholinergic and GABAergic interneurons and from the collateral SPNs [START_REF] Wilson | GABAergic Inhibition in the Neostriatum[END_REF]. Importantly, SPNs are really sensitive to the inhibition acted by interneurons. The combination of the weak signal transferred from the thalamo-cortical synapses, the strong inhibitory signal due to interneurons, and of the conductance properties of the SPNs results in low firing signal in vivo [START_REF] Kreitzer | Endocannabinoid-Mediated Rescue of Striatal LTD and Motor Deficits in Parkinson's Disease Models[END_REF]. artificial chromosome (BAC) transgenic mice expressing enhanced green fluorescent protein (eGFP) under the control of promoters for the D1 and D2 receptors allowed a clear distinction of the two populations [START_REF] Day | Selective Elimination of Glutamatergic Synapses on Striatopallidal Neurons in Parkinson Disease Models[END_REF][START_REF] Kreitzer | Endocannabinoid-Mediated Rescue of Striatal LTD and Motor Deficits in Parkinson's Disease Models[END_REF][START_REF] Ade | Differential Tonic GABA Conductances in Striatal Medium Spiny Neurons[END_REF][START_REF] Cepeda | Differential Electrophysiological Properties of Dopamine D1 and D2 Receptor-Containing Striatal Medium-Sized Spiny Neurons[END_REF].

Taking advantage of this technology, at least 2 different works showed that the dendritic tree of the D 2 SPNs is significantly smaller than D 1 SPNs [START_REF] Gertler | Dichotomous Anatomical Properties of Adult Striatal Medium Spiny Neurons[END_REF][START_REF] Fujiyama | Exclusive and Common Targets of Neostriatofugal Projections of Rat Striosome Neurons: A Single Neuron-Tracing Study Using a Viral Vector[END_REF]. By making use of whole-cell patch-clamp recordings in D 1 and D 2 SPNs in brain slices from BAC transgenic mice, Tracy S. and collaborators were able to correlate this anatomical dichotomy with the differences in excitability observed in the 2 populations of SPNs. As already shown by Kreitzer and Malenka in 2007 -due to the smaller dendritic tree -D2 SPNs are more excitable than the D1 SPNs [START_REF] Kreitzer | Endocannabinoid-Mediated Rescue of Striatal LTD and Motor Deficits in Parkinson's Disease Models[END_REF].

As an additional level of complication, several studies pointed out the existence of some differences between the SPNs in the DS and in the NAc. The different projections of the SPNs that belong to the DS or the NAc, account for the different behaviours mediated by those two regions. Although SPNs in the DS and NAc look identical in phenotypes, they differ in their output connections (reviewed in [START_REF] Yager | The Ins and Outs of the Striatum: Role in Drug Addiction[END_REF]) and gene expression profile (Montalban E. et al., this thesis). It is widely accepted that in the DS, D1 SPNs mainly project to the SNr and to the medial part of the GP, whether the D2 project to the lateral part of the GP [START_REF] Valjent | Looking BAC at striatal signaling: cell-specific analysis in new transgenic mice[END_REF][START_REF] Bertran-Gonzalez | What Is the Degree of Segregation between Striatonigral and Striatopallidal Projections?[END_REF], however, it is important to mention that, recently, Gangarossa and collaborators -taking advantage of the BAC technology -described a specific region in the caudal striatum, adjacent to the GPe, that is enriched D1R SPNs, cholinergic and GABAergic interneurons, while lacking markers for indirect pathway neurons [START_REF] Gangarossa | Distribution and Compartmental Organization of GABAergic Medium-Sized Spiny Neurons in the Mouse Nucleus Accumbens[END_REF]. This last study corroborated some of the results that have previously described the existence of D1 outputs to the lateral GP [START_REF] Kawaguchi | Projection Subtypes of Rat Neostriatal Matrix Cells Revealed by Intracellular Injection of Biocytin[END_REF][START_REF] Fujiyama | Exclusive and Common Targets of Neostriatofugal Projections of Rat Striosome Neurons: A Single Neuron-Tracing Study Using a Viral Vector[END_REF]. It is thought that the D1 outputs to the eGP may serve as a bridge between the direct and indirect pathways [START_REF] Cazorla | Dopamine D2 Receptors Regulate the Anatomical and Functional Balance of Basal Ganglia Circuitry[END_REF]. Nevertheless it has been proposed that D2 neurons in DS control the D1 output to the GPe: an increase in the bridging collaterals is associated with enhanced inhibition of pallidal neurons in vivo, while the chronic inhibition of the D2 receptor with haloperidol decreases the amount of bridging collaterals [START_REF] Cazorla | Dopamine D2 Receptors Regulate the Anatomical and Functional Balance of Basal Ganglia Circuitry[END_REF].

Importantly, it has been recently suggested that, in contrast with the classical scheme of the basal ganglia circuit, both D1 and D2 are able to disinhibit the cortex by -Introduction -The basal ganglia 2 inhibiting the acteyltransferase positive neurons in the eGP that send direct inhibitory projection to the frontal cortex [START_REF] Saunders | A Direct GABAergic Output from the Basal Ganglia to Frontal Cortex[END_REF].

This "re-vision" of the connectivity of the basal ganglia circuit has been recently proposed by the group of Sabatini. Here, Saunders and collaborators described a direct projection from the eGP to the frontal cortex that comprises cholinergic and GABA-inhibitory cells. Importantly, D1 and D2 SPNs are both able to inhibit the neurons making up this projection. Thus, the direct connection globus pallidumfrontal cortex is under the direct control of striatum [START_REF] Saunders | A Direct GABAergic Output from the Basal Ganglia to Frontal Cortex[END_REF]2015).

The NAc is the only region of the striatum hosting the D3-SPNs. The D3 dopamine SPNs are enriched in the shell of the NAc in the region enriched in D1-SPN [START_REF] Gangarossa | Distribution and Compartmental Organization of GABAergic Medium-Sized Spiny Neurons in the Mouse Nucleus Accumbens[END_REF]. The organization of the SPNs in the NAc, as for DS, challenges the classical view of the basal ganglia circuit. As mentioned above, two regions, the core and the shell, are distinguished in the NAc, based on histochemical differences, as well as differences in their afferents and efferents [START_REF] Groenewegen | The Specificity of the 'Nonspecific' Midline and Intralaminar Thalamic Nuclei[END_REF]Voorn et al. 2004;[START_REF] Zahm | An Integrative Neuroanatomical Perspective on Some Subcortical Substrates of Adaptive Responding with Emphasis on the Nucleus Accumbens[END_REF][START_REF] Zahm | On the Significance of Subterritories in The 'accumbens' part of the Rat Ventral Striatum[END_REF]. Both core and shell project to the SNc and VTA, although the shell sends prominent reciprocal projections to the ventral pallidum [START_REF] Zahm | Ventral Striatopallidothalamic Projection: IV. Relative Involvements of Neurochemically Distinct Subterritories in the Ventral Pallidum and Adjacent Parts of the Rostroventral Forebrain[END_REF], but also to the lateral hypothalamus, the pedunculopontine tegmental nucleus and other several subcortical areas that are not part of the basal ganglia. [START_REF] Winn | On the Relationships between the Striatum and the Pedunculopontine Tegmental Nucleus[END_REF][START_REF] Zahm | An Integrative Neuroanatomical Perspective on Some Subcortical Substrates of Adaptive Responding with Emphasis on the Nucleus Accumbens[END_REF][START_REF] Zahm | On the Significance of Subterritories in The 'accumbens' part of the Rat Ventral Striatum[END_REF]. Because of the GABAergic nature of both NAc shell and ventral pallidum projection neurons it is possible to hypothesize that the shell of the NAc could act on their targets by mechanisms of disinhibition and inhibition similar to the one that are taking place in the direct and indirect pathway in the DS.

The NAc core as the shell projects to ventral pallidum but in different districts. As for the indirect pathway of the DS, the ventral pallidum reciprocally connects to the STN and projects to both the mediodorsal nucleus of the thalamus and to brainstem similarly to motor areas. On the other hand, the VP also projects to the STN, a characteristic shared by the indirect path dorsal striatal target [START_REF] Groenewegen | The Specificity of the 'Nonspecific' Midline and Intralaminar Thalamic Nuclei[END_REF][START_REF] Zahm | On the Significance of Subterritories in The 'accumbens' part of the Rat Ventral Striatum[END_REF]. Based on those connections it is possible to hypothesise that the ventral pallidum could control the mediodorsal nucleus of the thalamus by two different pathways: a "direct" path made up of D2 SPNs [START_REF] Kupchik | Coding the direct/indirect pathways by D1 and D2 receptors is not valid for accumbens projections[END_REF] consisting in a first connection of the NAc to -Introduction -The basal ganglia 2 striatal afferents originating from different population of striatal SPNs and project to different outputs. The GP is mainly composed by GABAergic neurons characterized by a large cell body of 20-50 µm, and a dense dendritic arborisation [START_REF] Difiglia | A Golgi and Ultrastructural Study of the Monkey Globus Pallidus[END_REF]. SPNs in the striatum are the major input on the GP, however, the external globus pallidus also receives glutamatergic projections from the subthalamic nucleus (STN) and, although to lesser extent, from the cerebral cortex, GPi, raphe nucleus, pedunculopontine tegmentum and SN [START_REF] Hazrati | Evidence for Interconnections between the Two Segments of the Globus Pallidus in Primates: A PHA-L Anterograde Tracing Study[END_REF][START_REF] Fink-Jensen | A Direct Neuronal Projection from the Entopeduncular Nucleus to the Globus Pallidus. A PHA-L Anterograde Tracing Study in the Rat[END_REF][START_REF] Kita | The Morphology of Globus Pallidus Projection Neurons in the Rat: An Intracellular Staining Study[END_REF][START_REF] Deschênes | A Single-Cell Study of the Axonal Projections Arising from the Posterior Intralaminar Thalamic Nuclei in the Rat[END_REF][START_REF] Yasukawa | Rat Intralaminar Thalamic Nuclei Projections to the Globus Pallidus: A Biotinylated Dextran Amine Anterograde Tracing Study[END_REF][START_REF] Kita | Globus Pallidus External Segment[END_REF].

The GPe mainly projects to the STN but also to the GPi/SNr and the striatum.

The GPi targets the motor thalamus and the ventral medial and parafascicular thalamic nuclei [START_REF] Deniau | Synaptic Organization of the Basal Ganglia: An Electroanatomical Approach in the Rat[END_REF][START_REF] Deniau | The Pars Reticulata of the Substantia Nigra: A Window to Basal Ganglia Output[END_REF]).

The subthalamic nucleus

Although relatively small and with relatively few neurons, the STN is the second major port of entry of basal ganglia. Positioned on the medial side of the internal capsule and cerebral peduncle, the STN is the only excitatory structure of the BG that provide glutamatergic projections to the GPe [START_REF] Bevan | Neurons Projecting from the Entopeduncular Nucleus to the Thalamus Receive Convergent Synaptic Inputs from the Subthalamic Nucleus and the Neostriatum in the Rat[END_REF]) GPi, and SNr [START_REF] Nakanishi | Intracellular Study of Rat Substantia Nigra Pars Reticulata Neurons in an in Vitro Slice Preparation: Electrical Membrane Properties and Response Characteristics to Subthalamic Stimulation[END_REF][START_REF] Parent | Organization of Efferent Projections of the Subthalamic Nucleus in the Squirrel Monkey as Revealed by Retrograde Labeling Methods[END_REF]. The STN receives three main inputs: a major GABAergic input (1) from GPe, and from the mesopontine tegmentum [START_REF] Smith | Neurons of the Substantia Nigra Reticulata Receive a Dense GABA-Containing Input from the Globus Pallidus in the Rat[END_REF][START_REF] Bevan | Cholinergic, GABAergic, and Glutamate-Enriched Inputs from the Mesopontine Tegmentum to the Subthalamic Nucleus in the Rat[END_REF], a glutamtergic input (2) from prefrontal cortex and the intralaminar thalamic nuclei [START_REF] Kitai | Cortical Inputs to the Subthalamus: Intracellular Analysis[END_REF][START_REF] Nambu | Dual Somatotopical Representations in the Primate Subthalamic Nucleus: Evidence for Ordered but Reversed Body-Map Transformations from the Primary Motor Cortex and the Supplementary Motor Area[END_REF][START_REF] Bevan | GABAergic Control of the Subthalamic Nucleus[END_REF], and a third (3) input from the DA fibres arising by the SNc [START_REF] Brown | A Direct Role of Dopamine in the Rat Subthalamic Nucleus and an Adjacent Intrapeduncular Area[END_REF][START_REF] Lavoie | Dopaminergic Innervation of the Basal Ganglia in the Squirrel Monkey as Revealed by Tyrosine Hydroxylase Immunohistochemistry[END_REF][START_REF] Cragg | Synaptic Release of Dopamine in the Subthalamic Nucleus[END_REF]). The cortico-subthalamic pathway bypasses the striatum and is often referred as the hyperdirect pathway, the fastest route by which cortical and thalamic information can influence activity in the output nuclei. Importantly, it has been proposed that this pathway could be involved in the inhibition of initiated movements, providing a STOP signal [START_REF] Aron | Cortical and Subcortical Contributions to Stop Signal Response Inhibition: Role of the Subthalamic Nucleus[END_REF].

The substantia nigra

The substantia nigra (SN) is a structure lying dorsally to the cerebral peduncle in the ventral midbrain. This structure can be sub-divided in two different compartments: the pars compacta (SNc) composed by a compact and a diffuse clusters of neurons, and the pars reticulata (SNr), which is characterized by lower density of cells which are interspaced within a dense neuropil of radiating dendrites originating from both SNr and SNc neurons. The SN, together with ventral tegmental area (VTA) and the retrorubral area, constitutes the major input of DA to the striatum and other forebrain structures. The SN receives connections both D1 and D2 SPNs, from GABAergic neurons in the GPe, and glutamatergic inputs from the striatum.

The brain reward system: neuroanatomical and general principles

In the study of behaviour, the term reward describes an event that produces a pleasant or positive affective experience. The rewarding effect of a stimuzlus can be measured by the willingness of the subject to work, in order to gain access to this goal. Along this idea the responses to stimuli that produce positive effects are likely to be repeated [START_REF] Thorndike | Animal Intelligence: An Experimental Study of the Associative Processes in Animals[END_REF]. The idea that behavioural responses can be a direct measure of the rewarding properties of a certain object is the basis of the theory of reinforcement initially proposed by Skinner in 1938. In his pioneer work Skinner proposed that in an operant conditioning model -a behaviour paradigm that involves choice and reward measurement -the response strength can be determined by measuring the frequency and the intensity of behavioural responses. By measuring the motivation to work for a certain goal operant conditioning can offer an index of the rewarding properties of the object.

The operant training is the behavioural paradigm used when the reward circuit was first discovered by [START_REF] Olds | Positive Reinforcement Produced by Electrical Stimulation of Septal Area and Other Regions of Rat Brain[END_REF] In this important study the authors showed that rats are willing to work in order to self-administer electrical stimulation in specific brain regions [START_REF] Olds | Positive Reinforcement Produced by Electrical Stimulation of Septal Area and Other Regions of Rat Brain[END_REF]. Later studies have shown that rats will perform the task particularly well if the electrical stimulation is elicited in the medial forebrain bundle (MFB) [START_REF] Briese | REINFORCING BRAIN STIMULATION AND MEMORY IN MONKEYS[END_REF] and in the midbrain extension of this structure. More regions that seem to also be part of these reward circuits were later highlighted, and include the orbitofrontal cortex, the lateral hypothalamus, the NAc, the VTA and some brain stem structures [START_REF] Corbett | Intracranial Self-Stimulation in Relation to the Ascending Noradrenergic Fiber Systems of the Pontine Tegmentum and Caudal Midbrain: A Moveable Electrode Mapping Study[END_REF]). An amount of studies of self-stimulation helped to clarify which brain structures are mainly involved in the reward system and how they are connected with each other. The neural system that mediates the experience of reward consists of a complex network of several brain regions. The meso-corticolimbic pathway is a central component of this system. The meso-corticolimbic pathway arises from dopaminergic neurons located in the VTA, in the midbrain, that send projections to target areas in the limbic forebrain, particularly to the NAc, the DS, and the PFC. The

-Introduction -Long-lasting changes induced by cocaine

52

PFC provides descending projections to the NAc and the VTA. Experiments in mice have been confirmed by imaging studies in human in which the striatum is reported to be activated in response to food (DS) [START_REF] Small | Feeding-Induced Dopamine Release in Dorsal Striatum Correlates with Meal Pleasantness Ratings in Healthy Human Volunteers[END_REF], drugs [START_REF] Breiter | Acute Effects of Cocaine on Human Brain Activity and Emotion[END_REF]) money [START_REF] Wilson | Effect of Smoking Opportunity on Responses to Monetary Gain and Loss in the Caudate Nucleus[END_REF]) and romantic love (Acevedo et al. 2012), stimuli that all present reward-like properties in humans.

The nature of the major neurotransmitter produced by the VTA to modulate striatal function -DA -and the central role that the VTA plays in the reward network suggest that DA is involved in reward mechanisms. Elevated dopamine levels in the nucleus accumbens of rats were found following exposure to food [START_REF] Hernandez | Food Reward and Cocaine Increase Extracellular Dopamine in the Nucleus Accumbens as Measured by Microdialysis[END_REF], sweets [START_REF] Hajnal | Accumbens Dopamine Mechanisms in Sucrose Intake[END_REF]) sex [START_REF] Pfaus | Sexual Activity Increases Dopamine Transmission in the Nucleus Accumbens and Striatum of Female Rats[END_REF] , and self-administered drugs (e.g., cocaine, morphine, and ethanol) [START_REF] Di Chiara | Reinforcing Drug Seeking[END_REF]. Of note, for sugar and drugs, the levels of DA are directly proportional to the increasing concentration of the stimulus. Dopamine's action in the striatum is neuromodulatory. It could reinforce synaptic strength in the area where it is secreted by VTA terminals. It is thus well positioned to modulate excitatory projections of cortical neurons onto striatal neurons.

Introduction to addiction

Drug addiction, also called substance use disorder, is an illness that can be described as loss of control over taking a legal or illegal drug or medication.

Addiction is a complex phenomenon, which depends on both pharmacological and socio-cultural mechanisms. Genetic factors account for only half of the vulnerability to drugs; the other 50% of the risk for addiction is due to the interaction between environmental factors with the individual's genetic set. Although different types of environmental influences are considered to be fundamental to the development of the addiction, the necessary factor is exposure to a drug of abuse. Estimates from twins and adoption studies give ranges of 40% to 60% to heritability. Inherited contributions to addiction result from complex genetic differences; however, several studies in human addicts identified some of the genes that are clearly associated with cocaine addiction, those coding for: D2 receptor [START_REF] Noble | Allelic Association of the D2 Dopamine Receptor Gene with Cocaine Dependence[END_REF]), dopamine -Introduction -Long-lasting changes induced by cocaine 3 transporter DAT [START_REF] Gelernter | Genetic Association between Dopamine Transporter Protein Alleles and Cocaine-Induced Paranoia[END_REF][START_REF] Guindalini | A Dopamine Transporter Gene Functional Variant Associated with Cocaine Abuse in a Brazilian Sample[END_REF]), CB1 cannabinoid receptor [START_REF] Comings | Cannabinoid Receptor Gene (CNR1): Association with I.v. Drug Use[END_REF], prodynorphin [START_REF] Chen | Potentially Functional Polymorphism in the Promoter Region of Prodynorphin Gene May Be Associated with Protection against Cocaine Dependence or Abuse[END_REF], µ-opioid receptor [START_REF] Zubieta | Increased Mu Opioid Receptor Binding Detected by PET in Cocaine-Dependent Men Is Associated with Cocaine Craving[END_REF], the serotonin transporter [START_REF] Mash | Serotonin Transporters Upregulate with Chronic Cocaine Use[END_REF] and myelin-related genes [START_REF] Albertson | Distinctive Profiles of Gene Expression in the Human Nucleus Accumbens Associated with Cocaine and Heroin Abuse[END_REF] In vulnerable individuals, the repeated exposure to addictive drugs can lead to stable maladaptive neural changes in specific regions of the brains over time that account for potentially life long abnormalities. Of note, in 2014, 5 % of the US population was composed of addicted people in the age of 12-17(Samsha 2014). Several different drugs seem to share the same mechanisms of action in the brain for both humans and mice. Lehrmann and collaborators showed that in human post-mortem brain samples, cocaine, cannabis, and phencyclidine all decrease transcription of calmodulin-related genes and increase transcription of genes connected to lipid/cholesterol and Golgi/endoplasmic reticulum function in the anterior PFC [START_REF] Lehrmann | Transcriptional Changes Common to Human Cocaine, Cannabis and Phencyclidine Abuse[END_REF]). In mice, cocaine, nicotine, morphine, and THC (Valjent et al. 2004), cannabinoids [START_REF] Tonini | ERK-Dependent Modulation of Cerebellar Synaptic Plasticity after Chronic Delta9-Tetrahydrocannabinol Exposure[END_REF], alcohol, MDMA [START_REF] Salzmann | Importance of ERK Activation in Behavioral and Biochemical Effects Induced by MDMA in Mice[END_REF], phencyclidine [START_REF] Kyosseva | Differential and Region-Specific Activation of Mitogen-Activated Protein Kinases Following Chronic Administration of Phencyclidine in Rat Brain[END_REF]) and nicotine [START_REF] Schroeder | Cue-Induced Reinstatement of Alcohol-Seeking Behavior Is Associated with Increased ERK1/2 Phosphorylation in Specific Limbic Brain Regions: Blockade by the mGluR5 Antagonist MPEP[END_REF]) can activate the ERK pathway. Thanks to studies that used both passive and active drug administration, we know today that drug addiction involves gene expression, neurochemical, neurophysiological, and structural changes in many different brain cell populations. The following section will focus on the neural changes induced by cocaine.

Cocaine

Cocaine is a strong and addictive stimulant, made from the leaves of coca plants, which acts as an indirect agonist of the monoaminergic system. Cocaine, can be inhaled, smoked or injected into the bloodstream. Its effects include euphoria (which might eventually turn into anxiety), hyperactivity, suppression of appetite, and -due to its capacity to block the voltage-dependent sodium channel -local anaesthesia [START_REF] Reith | Locomotor Effects of Cocaine, Cocaine Congeners, and Local Anesthetics in Mice[END_REF]. Intensity and duration of the effects depend on the route of administration. Once administered, cocaine can easily cross the blood-brain barrier, get into the central nervous system, and inhibit the dopamine-(DAT), norepinephrine-(NET), and serotonin-(SERT) re-uptake transporters, thereby -Introduction -Long-lasting changes induced by cocaine 3 while opioids and alcohol have been shown to be directly self-administered into the VTA, cocaine is preferentially self-administered into the frontal cortex [START_REF] Mcbride | Localization of Brain Reinforcement Mechanisms: Intracranial Self-Administration and Intracranial Place-Conditioning Studies[END_REF]. Importantly, even if the mechanisms of action is specific to each drug, most drugs of abuse increase dopaminergic release from VTA to other regions of the reward circuitry. It is commonly recognized that the DA innervation of the nucleus accumbens and the associated ventral parts of the striatum accounts for the primary reinforcing effect of the cocaine administration (Di [START_REF] Chiara | Dopamine and Drug Addiction: The Nucleus Accumbens Shell Connection[END_REF][START_REF] Wise | Dopamine, Learning and Motivation[END_REF][START_REF] Ikemoto | The Functional Divide for Primary Reinforcement of D-Amphetamine Lies between the Medial and Lateral Ventral Striatum: Is the Division of the Accumbens Core, Shell, and Olfactory Tubercle Valid?[END_REF][START_REF] Wise | Dopamine and Reward: The Anhedonia Hypothesis 30 Years on[END_REF]). Indeed, the acquisition of cocaine selfadministration is impaired by DA depletion in the NAc or DA receptor blockade [START_REF] Roberts | Disruption of Cocaine Self-Administration Following 6-Hydroxydopamine Lesions of the Ventral Tegmental Area in Rats[END_REF][START_REF] Ito | Differential Control over Cocaine-Seeking Behavior by Nucleus Accumbens Core and Shell[END_REF]). It has been proposed that cocaine can hijack the normal reward circuit by increasing the dopaminergic transmission within corticostriatal systems that are normally involved in learning and memory processes in the context of natural rewards [START_REF] Everitt | Neural Systems of Reinforcement for Drug Addiction: From Actions to Habits to Compulsion[END_REF][START_REF] Belin | Addiction: Failure of Control over Maladaptive Incentive Habits[END_REF]). In vulnerable individuals, this leads to the habitual and compulsive drug use from the initial voluntary drug use. The transition from goal-directed behaviour to habits would reflect shifts from ventral to dorsal striatal control over behaviour, while the loss of control would be linked to the loss of the inhibitory control mediated by the afferents of the PFC to the DS (reviewed in [START_REF] Everitt | Neural Systems of Reinforcement for Drug Addiction: From Actions to Habits to Compulsion[END_REF]. Drug taking is often associated with environmental stimuli. Once associated to the drug, the presentation of an environmental stimulus alone can predict drug availability, evoke memories of the effect of the drug and ultimately elicit the drug taking [START_REF] Garavan | Cue-Induced Cocaine Craving: Neuroanatomical Specificity for Drug Users and Drug Stimuli[END_REF][START_REF] Everitt | Review. Neural Mechanisms Underlying the Vulnerability to Develop Compulsive Drug-Seeking Habits and Addiction[END_REF]. Those mechanisms are highly reproducible in animal models undergoing a classical pavlovian conditioning, in which the drug-associated stimulus is called conditioned stimulus (CS). It has been shown that in the presence of a CS, the instrumental learning for cocaine self-administration is selectively interrupted by disruption of the connection between NAc and basolateral amygdala [START_REF] Whitelaw | Excitotoxic Lesions of the Basolateral Amygdala Impair the Acquisition of Cocaine-Seeking Behaviour under a Second-Order Schedule of Reinforcement[END_REF], or by selective lesions of both NAc core [START_REF] Ito | Differential Control over Cocaine-Seeking Behavior by Nucleus Accumbens Core and Shell[END_REF], and the connection between NAc core specifically and basolateral amygdala (Di [START_REF] Ciano | Direct Interactions between the Basolateral Amygdala and Nucleus Accumbens Core Underlie Cocaine-Seeking Behavior by Rats[END_REF]). While the NAc seems to be more related to the instauration of goaldirected actions, two interesting studies of Yin and collaborators showed that lesions of the dorsolateral striatum, the striatal region mostly implicated in habits formation, preserved the outcome expectancy but disrupted habits formation in relation with cocaine conditioning [START_REF] Yin | Lesions of Dorsolateral Striatum Preserve Outcome Expectancy but Disrupt Habit Formation in Instrumental Learning[END_REF]Yin et al. 2006). These hypotheses are strengthened by several other results that involve approaches different from neuronal lesions. Evidences from in vivo microdialysis measurements of extracellular DA, showed a shift in the location of the DA release in rats trained over two months in a cocaine self-administration paradigm: self-administered cocaine increased extracellular DA levels in the NAc and DS However, while the presentation of the stimulus alone at the beginning of training increased DA only in the core of the NAc, the release of DA shifted to the DS when the stimulus was presented after a long training period [START_REF] Ito | Dopamine Release in the Dorsal Striatum during Cocaine-Seeking Behavior under the Control of a Drug-Associated Cue[END_REF][START_REF] Ito | Dissociation in Conditioned Dopamine Release in the Nucleus Accumbens Core and Shell in Response to Cocaine Cues and during Cocaine-Seeking Behavior in Rats[END_REF]. Indeed DA receptor antagonist αflupenthixol, decreases cocaine self-administration under a second-order schedule of reinforcement when infused into the DS [START_REF] Vanderschuren | Involvement of the Dorsal Striatum in Cue-Controlled Cocaine Seeking[END_REF]). In the same period Porrino and collaborators used autoradiography of selected markers of the DA system to evaluate the neuronal adaptations of the striatum during chronic cocaine self-administration in monkeys. This instructive study showed that the response to cocaine self-administration within the striatum shifts dramatically over time (5 days, 3.3 months, and 15-22 months). The increase in the duration to the cocaine exposure correlates with both changes in functional activity and alterations in the dopamine system by involving larger and larger portions in the more dorsal part of striatum. In particular, a decrease of the D2 and D3 dopamine receptor density has been observed in the dorsal striatum following chronic, but not acute, cocaine self-administration [START_REF] Porrino | The Expanding Effects of Cocaine: Studies in a Nonhuman Primate Model of Cocaine Self-Administration[END_REF][START_REF] Porrino | The Expanding Effects of Cocaine: Studies in a Nonhuman Primate Model of Cocaine Self-Administration[END_REF]. The same results have been reported in monkeys, after months but not weeks of cocaine self-administration. [START_REF] Letchworth | Progression of Changes in Dopamine Transporter Binding Site Density as a Result of Cocaine Self-Administration in Rhesus Monkeys[END_REF]. More recent studies have proved that -contrary to the medial part of the dorsal striatum -the dorsolateral striatum in not involved in the instatement of the cocaine seeking behaviour: the administration of a DA antagonist in the dorsolateral striatum does not affect at all cocaine self-administration [START_REF] Murray | N-Acetylcysteine Reduces Early-and Late-Stage Cocaine Seeking without Affecting Cocaine Taking in Rats[END_REF]. The DS, with the PFC and the basolateral amygdala, is also involved in the mechanism of relapse: the specific inactivation of the dorsolateral striatum attenuates cocaine seeking after abstinence [START_REF] Fuchs | Different Neural Substrates Mediate Cocaine Seeking after Abstinence versus Extinction Training: A Critical Role for the Dorsolateral Caudate-Putamen[END_REF]. Also, in 2007 See and collaborators used a gamma-aminobutyric acid agonist in order to inhibit SN, VTA, DS, and NAc in rat that learned to self-administer cocaine: while the inactivation of the dorsal striatum and midbrain regions attenuated cocaine seeking, inactivation of the ventral striatum had no such effects. Interestingly, subsequent training sessions under extinction conditions revealed a rebound in cocaine seeking in animals that had undergone inactivation in all regions except the dorsolateral striatum [START_REF] See | The Role of Dorsal vs Ventral Striatal Pathways in Cocaine-Seeking Behavior after Prolonged Abstinence in Rats[END_REF]).

-Introduction -Long-lasting changes induced by cocaine 3

The PFC is another region of the reward system highly involved in the drug addiction. Structural MRI pointed out that cocaine-addicted patients are -among other regions -affected by a significant decrease of the gray matter in the medial prefrontal cortex [START_REF] Ersche | Abnormal Brain Structure Implicated in Stimulant Drug Addiction[END_REF]) Importantly, the down-regulation of the D2 receptors in the striatum correlates with the hypometabolism of the orbitofrontal cortex, which is represented by the medial prefrontal cortex in its medial part [START_REF] Ersche | Abnormal Brain Structure Implicated in Stimulant Drug Addiction[END_REF]. Different works have shown a direct reciprocal connection between VTA and PFC [START_REF] Carr | Projections from the Rat Prefrontal Cortex to the Ventral Tegmental Area: Target Specificity in the Synaptic Associations with Mesoaccumbens and Mesocortical Neurons[END_REF]. The activity of the neurons of the VTA is regulated by the direct input from the PFC. Glutamate increases the activity of the dopaminergic neurons in the VTA [START_REF] Taber | Electrical Stimulation of the Prefrontal Cortex Increases Dopamine Release in the Nucleus Accumbens of the Rat: Modulation by Metabotropic Glutamate Receptors[END_REF] and facilitates DA release from the presynaptic terminals in the NAc [START_REF] Floresco | Basolateral Amygdala Stimulation Evokes Glutamate Receptor-Dependent Dopamine Efflux in the Nucleus Accumbens of the Anaesthetized Rat[END_REF]. Thus, electrical or chemical stimulation of the PFC accounts for both dopamine release in the NAc and burst the firing of dopamine neurons [START_REF] Murase | Prefrontal Cortex Regulates Burst Firing and Transmitter Release in Rat Mesolimbic Dopamine Neurons Studied in Vivo[END_REF][START_REF] Taber | Electrical Stimulation of the Prefrontal Cortex Increases Dopamine Release in the Nucleus Accumbens of the Rat: Modulation by Metabotropic Glutamate Receptors[END_REF][START_REF] Tong | Stimulation of the Prefrontal Cortex in the Rat Induces Patterns of Activity in Midbrain Dopaminergic Neurons Which Resemble Natural Burst Events[END_REF][START_REF] You | Electrical Stimulation of the Prefrontal Cortex Increases Cholecystokinin, Glutamate, and Dopamine Release in the Nucleus Accumbens: An in Vivo Microdialysis Study in Freely Moving Rats[END_REF][START_REF] Jackson | Stimulation of Prefrontal Cortex at Physiologically Relevant Frequencies Inhibits Dopamine Release in the Nucleus Accumbens[END_REF]. Cocaine stimulates glutamate release in the PFC and NAc [START_REF] Reid | Evidence for Sensitization of Cocaine-Induced Nucleus Accumbens Glutamate Release[END_REF]. The stimulation is potentiated by repeated cocaine exposure [START_REF] Reid | Evidence for Sensitization of Cocaine-Induced Nucleus Accumbens Glutamate Release[END_REF]. In the PFC cocaine binds also norepinephrine leading to increased levels of extracellular norepinephrine. [START_REF] Han | Comparison of the Monoamine Transporters from Human and Mouse in Their Sensitivities to Psychostimulant Drugs[END_REF] showed that cocaine can bind with an overall equal affinity to both the DA and the norepinephrine receptor [START_REF] Han | Comparison of the Monoamine Transporters from Human and Mouse in Their Sensitivities to Psychostimulant Drugs[END_REF]. This would lead to activation of alpha1-noradrenergic receptors, and possibly influence the signal transmitted by the PFC to the VTA altering the action-potential dependent DA release. The PFC seems to be fundamental also in the inhibitory control of relapse.

Lastly, it is now widely accepted that the connection between medial PFC (mPFC) and the posterior medial striatum accounts for the goal-directed system in both rats and humans [START_REF] Shiflett | Acquisition and Performance of Goal-Directed Instrumental Actions Depends on ERK Signaling in Distinct Regions of Dorsal Striatum in Rats[END_REF]. On the other hand, the habit system implicates the anterior dorsolateral striatum, and perhaps motor cortical areas [START_REF] Balleine | Human and Rodent Homologies in Action Control: Corticostriatal Determinants of Goal-Directed and Habitual Action[END_REF]

Effect of cocaine on neuroplasticity

General introduction.

Neuroplasticity is often referred as the capacity of the brain to functionally 3 -Introduction -Long-lasting changes induced by cocaine 58 remodel its neuronal circuits in response to experience and the environment.

Although neuroplasticity can occur at a variety of levels, ranging from molecular changes in synapses to large scale changes involved in neurocircuitry remapping, it is possible to define two main different classes of neuroplasticity: (1) the activitydependent alteration of connections among neurons, such as the creation of new synapses and the pruning of the existing ones, and (2) the changes in the intrinsic excitability of neurons [START_REF] Malenka | LTP and LTD: An Embarrassment of Riches[END_REF][START_REF] Lammel | Projection-Specific Modulation of Dopamine Neuron Synapses by Aversive and Rewarding Stimuli[END_REF]. It has been proposed that we could look at addiction as a disease of the goal-directed learning [START_REF] Hyman | Addiction: A Disease of Learning and Memory[END_REF][START_REF] Redish | A Unified Framework for Addiction: Vulnerabilities in the Decision Process[END_REF]. According to this model, repeated exposure to drugs could promote learning of drug-related behaviours with such efficacy that they become compulsive. In other words, drugs would override the mechanisms that are taking place during normal reward learning [START_REF] Redish | Addiction as a Computational Process Gone Awry[END_REF]. All these considerations make the synaptic plasticity a good candidate for the persistence of addiction-related behaviour. Synaptic plasticity can be classified in two different forms: short-term and long-term synaptic plasticity. The short-term synaptic plasticity acts within milliseconds or minutes, and accounts only for transient and fast modification such as synaptic facilitations. Long-term synaptic plasticity acts within hours or day.

Repeated stimulations, such as drug exposure, could turn the short-term synaptic plasticity into long-term synaptic plasticity. The best-characterized forms of synaptic plasticity are the activity dependent or N-methyl-D-aspartate receptor (NMDAR)mediated long-term potentiation (LTP), and long-term depression (LTD) [START_REF] Watt | A Proportional but Slower NMDA Potentiation Follows AMPA Potentiation in LTP[END_REF]) LTP occurs when the presynaptic stimulation coincides with the postsynaptic depolarization (Hebb, D.O. (1949). The Organization of Behavior. New York: Wiley & Sons; [START_REF] Nakamura | Monoclonal Antibodies to the Extracellular Domain of HIV-1IIIB gp160 That Neutralize Infectivity, Block Binding to CD4, and React with Diverse Isolates[END_REF])) while LTD occurs when presynaptic activation comes along with postsynaptic inactivity [START_REF] Kullmann | Long-Term Synaptic Plasticity in Hippocampal Interneurons[END_REF].

Evidences of cocaine-dependent regulation of the synaptic plasticity in the VTA

A single non-contingent dose of cocaine is able to induce an augmentation of the AMPAR/NMDAR ratio, and so a considerable potentiation of the excitatory synaptic transmission, in the DA neurons of the VTA [START_REF] Ungless | Single Cocaine Exposure in Vivo Induces Long-Term Potentiation in Dopamine Neurons[END_REF]. The potentiation can be measured 24 h after the cocaine injection, is lost one week later, and is -Introduction -Long-lasting changes induced by cocaine 3 restricted to the DA neurons projecting to the Nac [START_REF] Lammel | Projection-Specific Modulation of Dopamine Neuron Synapses by Aversive and Rewarding Stimuli[END_REF]. [START_REF] Mameli | Cocaine-Evoked Synaptic Plasticity: Persistence in the VTA Triggers Adaptations in the NAc[END_REF]collaborators in 2011, and[START_REF] Yuan | Expression of Cocaine-Evoked Synaptic Plasticity by GluN3A-Containing NMDA Receptors[END_REF]collaborators in 2013 showed that the increase in the ratio is due to an increase of the AMPAR-dependent currents, related to the insertion of Ca 2+ -permeable Glu2A subunit containing AMPARs and to the reduction of the NMDAR-dependent currents, related to the insertion of the semi-Ca 2+impermeable NMDARs containing GluN3A and GluN2B subunits [START_REF] Yuan | Expression of Cocaine-Evoked Synaptic Plasticity by GluN3A-Containing NMDA Receptors[END_REF]. mGluR5 and mGluR1 are two other receptors implicated in the mechanisms of synaptic plasticity in the context of cocaine action. Several studies have shown that the induction of mGluR1-dependent LTD is able to reverse the synaptic plasticity induced by cocaine [START_REF] Bellone | Cocaine Triggered AMPA Receptor Redistribution Is Reversed in Vivo by mGluR-Dependent Long-Term Depression[END_REF][START_REF] Mameli | Rapid Synthesis and Synaptic Insertion of GluR2 for mGluR-LTD in the Ventral Tegmental Area[END_REF]). In the VTA, mGluR1 binds to isoforms of the scaffolding protein Homer to induce LTD.

When this interaction in the VTA is disrupted, the plasticity response to a single injection of cocaine in the NAc becomes comparable to the synaptic adaptations that are normally obtained by chronic cocaine injections. Interestingly, the opposite is true in the NAc, where mGluR1 is a positive modulator of synaptic plasticity [START_REF] Knoflach | Positive Allosteric Modulators of Metabotropic Glutamate 1 Receptor: Characterization, Mechanism of Action, and Binding Site[END_REF][START_REF] Mameli | Cocaine-Evoked Synaptic Plasticity: Persistence in the VTA Triggers Adaptations in the NAc[END_REF].

Evidences of the cocaine regulation of the synaptic plasticity in the NAc

Unlike in the VTA, multiple non-contingent doses of cocaine administration are required to elicit synaptic plasticity in excitatory synapses in the NAc [START_REF] Thomas | Long-Term Depression in the Nucleus Accumbens: A Neural Correlate of Behavioral Sensitization to Cocaine[END_REF] Two different studies showed that in the NAc, 10 to 14 days of repeating cocaine administration cause a reduction in the AMPAR/NMDAR ratio [START_REF] Thomas | Long-Term Depression in the Nucleus Accumbens: A Neural Correlate of Behavioral Sensitization to Cocaine[END_REF][START_REF] Beurrier | Enhanced Inhibition of Synaptic Transmission by Dopamine in the Nucleus Accumbens during Behavioral Sensitization to Cocaine[END_REF]. Later, in 2008 Thomas and collaborators showed a repression of the AMPAR currents in brain slices from rats injected with cocaine for 8 days. Furthermore, the activation of D1Rs by DA leads to an enhanced reduction in the AMPAR-mediated synaptic transmission, but not NMDARmediated synaptic transmission in the NAc shell [START_REF] Beurrier | Enhanced Inhibition of Synaptic Transmission by Dopamine in the Nucleus Accumbens during Behavioral Sensitization to Cocaine[END_REF].

Interestingly, it has been recently shown that in the NAc, non-contingent exposure to cocaine leads to the generation of so-called silent synapses after one or two days of withdrawal [START_REF] Huang | In Vivo Cocaine Experience Generates Silent Synapses[END_REF]). The number of synapses is increasing with the duration of cocaine injections and goes back to normal after a long period of withdrawal. Two different studies in 2013 and 2014 respectively showed an increasing number of silent synapses in the 2 major glutamatergic inputs to the NAc: amygdala and PFC [START_REF] Lee | Maturation of Silent Synapses in Amygdala-Accumbens Projection Contributes to Incubation of Cocaine Craving[END_REF][START_REF] Ma | Bidirectional Modulation of Incubation of Cocaine Craving by Silent Synapse-Based Remodeling of Prefrontal Cortex to Accumbens Projections[END_REF]). In both conditions, extended withdrawal led to the recruitment to the AMPA receptors.

Cocaine-induced structural plasticity

Importantly, the regulation of synaptic plasticity described above correlates with a regulation of the structural plasticity. In 2007 Sarti and collaborators showed an increase in synaptic density in the VTA following a single injection of cocaine and in the same cells that exhibited the increase of the AMPAR/NMDAR ratio. Numerous studies reported that cocaine and other drugs of abuse produce persistent changes in the structure of dendrites and dendritic spines in D1 and D2 SPNs in the NAc [START_REF] Lee | Cocaine-Induced Dendritic Spine Formation in D1 and D2 Dopamine Receptor-Containing Medium Spiny Neurons in Nucleus Accumbens[END_REF][START_REF] Li | Cocaine-Induced Dendritic Remodeling Occurs in Both D1 and D2 Dopamine Receptor-Expressing Neurons in the Nucleus Accumbens[END_REF]). Importantly, it appears that the new spines induced by cocaine in the NAc are more stable in D1 SPNs. Interestingly, cocaine induces ΔFosB preferentially in D1 neurons [START_REF] Hope | Induction of a Long-Lasting AP-1 Complex Composed of Altered Fos-like Proteins in Brain by Chronic Cocaine and Other Chronic Treatments[END_REF][START_REF] Nestler | Review. Transcriptional Mechanisms of Addiction: Role of DeltaFosB[END_REF]). Correlating with the persistent increase of spines in D1 neurons, ΔFosB accounts for the transcriptional regulation of several genes involved in the shaping of synapses: Cdk5 [START_REF] Bibb | Effects of Chronic Exposure to Cocaine Are Regulated by the Neuronal Protein Cdk5[END_REF], synaptogamin 6, and microtubule-associated protein 2 [START_REF] Mcclung | Regulation of Gene Expression and Cocaine Reward by CREB and DeltaFosB[END_REF], activity-regulated cytoskeleton-associated protein (ARC) [START_REF] Renthal | Genome-Wide Analysis of Chromatin Regulation by Cocaine Reveals a Role for Sirtuins[END_REF]. Moreover, chronic cocaine administration reduces the activity of Rac1, leading to the intensification of the polymerization rate of filamentous actin in the NAc ΔFosB. Acute cocaine treatment increases the expression of the Fos family transcription factors in the NAc [START_REF] Graybiel | Amphetamine and Cocaine Induce Drug-Specific Activation of the c-Fos Gene in Striosome-Matrix Compartments and Limbic Subdivisions of the Striatum[END_REF][START_REF] Hope | Regulation of Immediate Early Gene Expression and AP-1 Binding in the Rat Nucleus Accumbens by Chronic Cocaine[END_REF]. All the Fos family members are able to form heterodimers with Jun family proteins (c-Jun, JunB, JunD). Once formed, the heterodimer can regulate the transcription of selected genes by binding to the activator protein-1 (AP-1) sites present within the promoters. The maximal induction of Fos proteins occurs within 1-2 h after drug administration and returns to normal levels within 8-12 h. All members of the Fos family proteins are induced by acute cocaine exposure; however, their expression is attenuated upon repeated drug treatment (tolerance). The truncated form of FosB, ΔFosB, accumulates for several weeks after repeated cocaine injections [START_REF] Hope | Induction of a Long-Lasting AP-1 Complex Composed of Altered Fos-like Proteins in Brain by Chronic Cocaine and Other Chronic Treatments[END_REF]. This is due to its long half-life that is even further enhanced when it is phosphorylated on Ser27 (Ulery- [START_REF] Ulery-Reynolds | Phosphorylation of DeltaFosB Mediates Its Stability in Vivo[END_REF]). In particular ΔFosB accumulation seems to occur preferentially in the D1 SPNs subtype. [START_REF] Hope | Induction of a Long-Lasting AP-1 Complex Composed of Altered Fos-like Proteins in Brain by Chronic Cocaine and Other Chronic Treatments[END_REF][START_REF] Moratalla | D1-Class Dopamine Receptors Influence Cocaine-Induced Persistent Expression of Fos-Related Proteins in Striatum[END_REF][START_REF] Kelz | Expression of the Transcription Factor deltaFosB in the Brain Controls Sensitivity to Cocaine[END_REF][START_REF] Nestler | DeltaFosB: A Sustained Molecular Switch for Addiction[END_REF]. ΔFosB stability provides a molecular mechanism by which drug-induced changes in gene expression can persist despite long periods of drug withdrawal [START_REF] Nestler | DeltaFosB: A Sustained Molecular Switch for Addiction[END_REF]. As mentioned above, due to its capacity to induce the transcription of several genes related to the remodelling of the cytoskeleton, ΔFosB is necessary to the instauration of the structural synaptic plasticity induced by cocaine. However, this mechanism requires CaMKIIα, which expression is increased following chronic cocaine exposure as well; importantly those 2 proteins have been found to be up-regulated in the postmortem NAc of patients addicted to cocaine [START_REF] Robison | Behavioral and Structural Responses to Chronic Cocaine Require a Feedforward Loop Involving ΔFosB and Calcium/calmodulin-Dependent Protein Kinase II in the Nucleus Accumbens Shell[END_REF]). Although ΔFosB is only slightly induced after cocaine treatment, its overexpression is able to enhance the rewarding properties of cocaine, as assessed in the conditioned place preference (CPP) for cocaine, the cocaine self-administration, and the cocaine-induced locomotor activity. Of note, the overexpression of ΔFosB in D1 SPNs results in the decrease of the AMPAR/NMDAR ratio, the increase of silent synapses on these neurons in the NAc, and in a decrease of the immature spines [START_REF] Grueter | ∆FosB Differentially Modulates Nucleus Accumbens Direct and Indirect Pathway Function[END_REF].

Conversely, overexpression in D2 R-MSNs results in increased excitatory synaptic strength and in the decrease of silent synapses in the specifically in NAc shell only [START_REF] Grueter | ∆FosB Differentially Modulates Nucleus Accumbens Direct and Indirect Pathway Function[END_REF].

-Introduction -Long-lasting changes induced by cocaine 3 CREB. CREB is a member of the leucine-zipper transcription factors family, that constitutively binds to a specific sequence of DNA called CRE (cAMP-responseelement) in the promoter region of specific genes including c-fos, dynorphin, and encephalin [START_REF] Shaywitz | CREB: A Stimulus-Induced Transcription Factor Activated by a Diverse Array of Extracellular Signals[END_REF]. The phosphorylation of Ser133 by either PKA, CaMK II/IV, p90 ribosomal S6 kinases 1/2 (RSK1/2), mitogen-and stressactivated kinases 1/2 (MSK1/2), or PKC [START_REF] Johannessen | Multisite Phosphorylation of the cAMP Response Element-Binding Protein (CREB) by a Diversity of Protein Kinases[END_REF]) is necessary to activate CREB and to induce its interaction with its co-activators CREB binding protein (CBP) and p300 [START_REF] Lundblad | Adenoviral E1A-Associated Protein p300 as a Functional Homologue of the Transcriptional Co-Activator CBP[END_REF]. Both co-activators have a histone acetyl-transferase activity. The acetylation of histones induced by the 2 co-activators, allows the relaxation of the chromatin and promotes the transcriptional activity of CREB together with the recruitment of RNA polymerase II that triggers mRNA synthesis (Bannister & Kouzarides n.d.;[START_REF] Kwok | Nuclear Protein CBP Is a Coactivator for the Transcription Factor CREB[END_REF]; Acute and chronic cocaine treatment is able to induce the CREB in different brain regions [START_REF] Walters | Differential Distribution of CREB in the Mesolimbic Dopamine Reward Pathway[END_REF][START_REF] Carlezon | The Many Faces of CREB[END_REF]. The induction of CREB activity appears to become greater and more persistent with repeated drug exposures. CREB, unlike ΔFosB, reduces the sensitivity to the rewarding effects of cocaine (tolerance) and increases the self-administration and relapse via negative reinforcement. Virally-mediated overexpression of CREB in the NAc decreases tolerance to cocaine, whereas reduction in CREB activity -via overexpression of a negative-mutant form of CREB in the NAc -has the opposite effects [START_REF] Carlezon | Regulation of Cocaine Reward by CREB[END_REF][START_REF] Barrot | CREB Activity in the Nucleus Accumbens Shell Controls Gating of Behavioral Responses to Emotional Stimuli[END_REF]. The ability of CREB to decrease reward is mediated by the induction of the expression of dynorphin peptide [START_REF] Cole | Neuronal Adaptation to Amphetamine and Dopamine: Molecular Mechanisms of Prodynorphin Gene Regulation in Rat Striatum[END_REF]. Dynorphin acts on κ opiod receptors in VTA neurons to decrease dopamine release in the NAc [START_REF] Spanagel | Opposing Tonically Active Endogenous Opioid Systems Modulate the Mesolimbic Dopaminergic Pathway[END_REF], thus impairing rewarding behaviours [START_REF] Carlezon | Regulation of Cocaine Reward by CREB[END_REF][START_REF] Muschamp | Activation of CREB in the Nucleus Accumbens Shell Produces Anhedonia and Resistance to Extinction of Fear in Rats[END_REF][START_REF] Ehrich | Kappa Opioid Receptor Activation Potentiates the Cocaine-Induced Increase in Evoked Dopamine Release Recorded in Vivo in the Mouse Nucleus Accumbens[END_REF]. Lastly, CREB is necessary for the induction of ΔFosB. The genetic deletion of CREB in the SPNs, increases the cocaine conditioned place preference (CPP) and the cocaine-induced locomotor sensitization [START_REF] Carlezon | Regulation of Cocaine Reward by CREB[END_REF][START_REF] Walters | Differential Distribution of CREB in the Mesolimbic Dopamine Reward Pathway[END_REF][START_REF] Mcclung | Regulation of Gene Expression and Cocaine Reward by CREB and DeltaFosB[END_REF].

Notably, the major limitation of those types of studies is the impossibility to distinguish the transcriptional changes occurring specifically in D1 and D2 SPNs.

Only one pioneer work in 2008 (Heiman et al. 2008), made use of the TRAP technology to immune-precipitate cell specific mRNA from D1 or D2 neurons in the striatum. The mRNA was then used for microarrays analysis in order to elucidate the cocaine-induced transcriptional changes in the two populations of cells. For this study, adult mice were non-contingently treated with cocaine or saline in an acute or chronic paradigm and used for translational profiling of striatonigral (Drd1a) and striatopallidal (Drd2) SPNs Heiman et al. 2008. This work showed hundreds of genes being specifically regulated in the two types of neurons and confirmed some of the cocaine-induced genes already known, by adding additional information on the neurons in which those mRNA are regulated. Some examples of the genes already known as responsive to cocaine: Cartpt [START_REF] Douglass | PCR Differential Display Identifies a Rat Brain mRNA That Is Transcriptionally Regulated by Cocaine and Amphetamine[END_REF] enriched after acute treatment in D1 neurons, Fosb [START_REF] Hope | Regulation of Immediate Early Gene Expression and AP-1 Binding in the Rat Nucleus Accumbens by Chronic Cocaine[END_REF]) up-regulated in acute treatment in both striatonigral and striatopallidal, and only in D1 after chronic cocaine administration; Homer1 [START_REF] Brakeman | Homer: A Protein That Selectively Binds Metabotropic Glutamate Receptors[END_REF], up-regulated by both acute and chronic treatment, in both D1 and D2 neurons; Per2 [START_REF] Yuferov | Differential Gene Expression in the Rat Caudate Putamen After 'binge' cocaine Administration: Advantage of Triplicate Microarray Analysis[END_REF] -Chapter 4-Homeostatic and hedonic mechanisms driving the feeding behaviour

The feeding behaviour

The regulation of an adequate energy intake in the body is the sine qua non condition for surviving. Indeed, the vertebrate's brain has evolved complex neural circuits in order to insure the high priority of the feeding process. The feeding behaviour represents a very complex mechanism involving homeostatic and motivation pathways. The homeostatic control of the food pathway involves the regulation of energy balance by increasing the motivation to eat following exhaustion of the energy store. The hedonic properties of food raise a valuable contribution to the feeding control. The hedonic based control of feeding exhibits a reward-based regulation. The exposure to highly palatable or highly fat food can disrupt the normal appetite regulation (Erlanson-Albertsson 2005) and induce the development of compulsive-like approach to the food leading to obesity [START_REF] Johnson | Dopamine D2 Receptors in Addiction-like Reward Dysfunction and Compulsive Eating in Obese Rats[END_REF] Thus, to understand the rewarding nature of food, it is necessary to comprehend the link between the homeostatic and hedonic brain circuit that is the basis of a correct intake of calories and feeding behaviour

Homeostatic aspects of food intake

The homeostatic control of food intake is mainly related to the regulation of the energy balance. To ensure the availability of a correct amount of food, the brain needs to tightly communicate with the periphery, control the levels of the different nutrients circulating in the blood system, and be informed about the availability of food in the external environment [START_REF] Berthoud | Interactions between The 'cognitive' and 'metabolic' brain in the Control of Food Intake[END_REF].

The hypothalamus is one of the regions of the brain more implicated in collecting and integrating information from the peripheral organs. The hypothalamus can be divided in different nuclei based on anatomical boundaries [START_REF] Bernardis | The Lateral Hypothalamic Area Revisited: Neuroanatomy, Body Weight Regulation, Neuroendocrinology and Metabolism[END_REF], gene expression [START_REF] Broberger | Hypocretin/orexin-and Melanin-Concentrating Hormone-Expressing Cells Form Distinct Populations in the Rodent Lateral Hypothalamus: Relationship to the Neuropeptide Y and Agouti Gene-Related Protein Systems[END_REF][START_REF] Lein | Genome-Wide Atlas of Gene Expression in the Adult Mouse Brain[END_REF]) and function (reviewed in Saper et al. 2002). The main nuclei of the hypothalamus include 4 -Introduction -Homeostatic & hedonic mechanisms of feeding behaviour 68 the arcuate nucleus (ARC), the paraventricular nucleus (PVN), the ventromedial nucleus (VMN), the dorsomedial region (DMV), and the lateral hypothalamic area (LHA). The importance of these nuclei in energy homeostasis was first suggested by classic experiments of lesion performed in rodents (reviewed in [START_REF] Suzuki | Obesity and Appetite Control[END_REF].

According to those studies, ARC is the area that collects information on the body energy state from the blood brain barrier, DMV the area more related to the sense of satiety, and the LH the area involved in the activation of the feeding response (reviewed in [START_REF] Quarta | Rewarding, Reinforcing and Incentive Salient Events Involve Orexigenic Hypothalamic Neuropeptides Regulating Mesolimbic Dopaminergic Neurotransmission[END_REF]. After the evaluation of the amount of nutrients in the body, two different mechanisms can be activated in the hypothalamus: the anabolic pathway or the catabolic pathway.

The catabolic pathway is activated by the peripheral production of anorectic signals and has as final effect the stimulation of the sense of satiety. Leptin is a wellknown example of an anorectic hormone able to shift the system to the interruption of the feeding behaviour. Leptin is synthesized and released by adipose tissue and acts principally in the ARC nucleus of the hypothalamus where it stimulates the secretion of two potent anorexigenic neuropeptides: the melanocyte-stimulating hormone (MSH), and the cocaine and amphetamine-regulated transcript (CART) peptide. Simultaneously, leptin is also able to inhibit neurons expressing the Agoutirelated protein (AgRP) and Neuropeptide Y (NPY)-producing neurons, which coexpress the orexigenic neuropeptides AgRP and NPY, and antagonizes MSH.

Contrary to the catabolic pathway, the anabolic pathway is activated during low energetic states. It involves the production of orexigenic signals such as ghrelin, and has as final output the induction of feeding behaviour [START_REF] Atalayer | Anorexia of Aging and Gut Hormones[END_REF].

Interestingly, the receptors for ghrelin can also be found in the VTA while the leptin receptors are enriched in both VTA and SN, suggesting that these peptides can control even the reward regulation of the feeding [START_REF] Morton | Leptin and the Central Nervous System Control of Glucose Metabolism[END_REF]. 4.2.1 The lateral hypothalamus, a centre of relay of homeostatic and hedonic control of food intake Until 1990, the general mechanism accepted for regulation of food intake was based on a model that attributed to the lateral hypothalamus area (LHA) the drives to eat, and to the ventral hypothalamus the sense of satiety. While lesions of the LHA result in the suppression of both feeding [START_REF] Anand | Hypothalamic Control of Food Intake in Rats and Cats[END_REF] and drinking [START_REF] Montemurro | Adipsia Produced by Hypothalamic Lesions in the Rat[END_REF], the lesion of the ventral hypothalamus promotes feeding and body weight gain [START_REF] Hetherington | Hypothalamic Lesions and Adiposity in the Rat[END_REF].

Although this model is still useful in some aspects, different studies in the last 15 years proved the existence of a much more complex system, in terms of both molecules and circuits involved. The LHA is a heterogeneous structure located anterior to the VTA and through which pass the fibres of the medial forebrain [START_REF] Nieuwenhuys | The Medial Forebrain Bundle of the Rat. I. General Introduction[END_REF]. The LHA contains a plethora of different types of cells, including a mixture of excitatory and inhibitory neurons as confirmed by the enrichment in both vesicular glutamate transporter type 2 (Vglut2) mRNA [START_REF] Collin | Plasma Membrane and Vesicular Glutamate Transporter mRNAs/proteins in Hypothalamic Neurons That Regulate Body Weight[END_REF][START_REF] Rosin | Hypothalamic Orexin (Hypocretin) Neurons Express Vesicular Glutamate Transporters VGLUT1 or VGLUT2[END_REF][START_REF] Ziegler | Distribution of Vesicular Glutamate Transporter mRNA in Rat Hypothalamus[END_REF], and GABAergic markers [START_REF] Karnani | Lateral Hypothalamic GAD65 Neurons Are Spontaneously Firing and Distinct from Orexin-and Melanin-Concentrating Hormone Neurons[END_REF].

Orexin-producing neurons

Orexin is a neuropeptide that regulates arousal, wakefulness and appetite (Davis et al. 2011). The genetic ablation of the Orexin neurons results in narcolepsy, hypophagia and obesity [START_REF] Harris | A Role for Lateral Hypothalamic Orexin Neurons in Reward Seeking[END_REF]). More recently it has been shown a major implication of the Orexin neurons in the regulation of arousal, as optogenetic stimulation of Orx neuron increases wakefulness [START_REF] Adamantidis | Neural Substrates of Awakening Probed with Optogenetic Control of Hypocretin Neurons[END_REF]).

The name orexin comes from orexis, appetite in Greek. Consistently with its name, orexin is thought to be involved in feeding and in reward related behaviours.

Injections of the peptide into the lateral ventricle increases food intake [START_REF] Sakurai | Orexins and Orexin Receptors: Implication in Feeding Behavior[END_REF], while the treatment with Orx receptor antagonists or genetic removal of Orx results in a decrease of consumption [START_REF] Haynes | Anorectic, Thermogenic and Anti-Obesity Activity of a Selective Orexin-1 Receptor Antagonist in Ob/ob Mice[END_REF]. Food restriction cause an increase of the orexin mRNA [START_REF] Clegg | Eating Elicited by Orexin-A, but Not Melanin-Concentrating Hormone, Is Opioid Mediated[END_REF][START_REF] Sakurai | Orexins and Orexin Receptors: Implication in Feeding Behavior[END_REF]) and hypoglycaemia induces an increase of the Orx mRNA expression as well as an increase in Fos expression [START_REF] Cai | Hypothalamic Orexin Expression: Modulation by Blood Glucose and Feeding[END_REF][START_REF] Griffond | Insulin-Induced Hypoglycemia Increases Preprohypocretin (Orexin) mRNA in the Rat Lateral Hypothalamic Area[END_REF][START_REF] Moriguchi | Neurons Containing Orexin in the Lateral Hypothalamic Area of the Adult Rat Brain Are Activated by Insulin-Induced Acute Hypoglycemia[END_REF].

Melanin-concentrating hormone (MCH)-producing neurons

The melanin-concentrating hormone (MCH)-producing neurons constitute another important group of hypothalamic cells distinct from the Orx-producing neurons [START_REF] Broberger | Hypocretin/orexin-and Melanin-Concentrating Hormone-Expressing Cells Form Distinct Populations in the Rodent Lateral Hypothalamus: Relationship to the Neuropeptide Y and Agouti Gene-Related Protein Systems[END_REF]. MCH-producing neurons are predominantly located in the LHA [START_REF] Bittencourt | The Melanin-Concentrating Hormone System of the Rat Brain: An Immuno-and Hybridization Histochemical Characterization[END_REF]) and project widely throughout the brain [START_REF] Elias | Chemically Defined Projections Linking the Mediobasal Hypothalamus and the Lateral Hypothalamic Area[END_REF]. MCH neurons are composed of subsets of inhibitory and excitatory cells as shown by co-expression of MCH with GAD67 or Vglut2 [START_REF] Harthoorn | Multi-Transcriptional Profiling of Melanin-Concentrating Hormone and Orexin-Containing Neurons[END_REF]. As the Orx producing neurons, MCH neurons have also been implicated in the regulation of feeding, and in the sleep-wakefulness balance. However, it has been hypothesized that MCH and orexin neurons have opposite roles in controlling arousal states (reviewed [START_REF] Brown | To Ingest or Rest? Specialized Roles of Lateral Hypothalamic Area Neurons in Coordinating Energy Balance[END_REF]. Indeed, contrary to orexin neurons, the activation of MCH neurons promotes REM sleep [START_REF] Herrera | Hypothalamic Feedforward Inhibition of Thalamocortical Network Controls Arousal and Consciousness[END_REF].

MCH-producing neurons are thought to play a fundamental role in the regulation of feeding behaviour and body weight. Intracerebro-ventricular injections of the peptide increase feeding and body weight in rodents,. MCH mRNA levels are increased by food deprivation, and leptin-deficient OB/OB mice express elevated levels of MCH [START_REF] Qu | A Role for Melanin-Concentrating Hormone in the Central Regulation of Feeding Behaviour[END_REF]. Furthermore, genetic studies have revealed that mice lacking MCH neurons [START_REF] Alon | Late-Onset Leanness in Mice with Targeted Ablation of Melanin Concentrating Hormone Neurons[END_REF] or the MCH gene [START_REF] Shimada | Mice Lacking Melanin-Concentrating Hormone Are Hypophagic and Lean[END_REF]) are hypophagic and lean. On the other hand, the over-expression of MCH results in hyperphagia, resistance to insulin, and obesity [START_REF] Ludwig | Melanin-Concentrating Hormone Overexpression in Transgenic Mice Leads to Obesity and Insulin Resistance[END_REF].

Interestingly, the NAc is a major input of the MCH producing neurons. This direct connection makes MCH neurons and their receptors suitable candidates to link the homeostatic aspects of feeding with the reward aspects of feeding.

-Introduction -Homeostatic & hedonic mechanisms of feeding behaviour 72

Neurotensin-producing neurons

Neurotensin-producing neurons are a group of cells located in the pre-optic and anterior hypothalamic regions. Neurotensin neurons do not co-localize with MCH and Orx, however they present ~95% overlap with galanin-expressing neurons.

Neurotensin producing neurons mediate the anorexigenic leptin action [START_REF] Laque | Leptin Receptor Neurons in the Mouse Hypothalamus Are Colocalized with the Neuropeptide Galanin and Mediate Anorexigenic Leptin Action[END_REF]. The administration of Nts at both peripheral and central level suppresses feeding [START_REF] Cooke | Peripheral and Central Administration of Xenin and Neurotensin Suppress Food Intake in Rodents[END_REF]). In agreement with these results, both the genetic ablation of a subset of Neurotensin neurons [START_REF] Kim | Impaired Anorectic Effect of Leptin in Neurotensin Receptor 1-Deficient Mice[END_REF]) and the knock out of the Neurotensin receptor [START_REF] Leinninger | Leptin Action via Neurotensin Neurons Controls Orexin, the Mesolimbic Dopamine System and Energy Balance[END_REF]) result in hyperphagia and obesity.

Input circuits of the LHA

The LHA collects excitatory and inhibitory inputs from both cortical and subcortical structures: Glutamatergic inputs. The LHA receives two major glutamatergic inputs: the monosynaptic glutamatergic inputs from the hippocampus [START_REF] Nauta | Hippocampal Projections and Related Neural Pathways to the Midbrain in the Cat[END_REF], and the monosynaptic and polysynaptic inputs from the medial PFC [START_REF] Kita | Reciprocal Connections between the Lateral Hypothalamus and the Frontal Complex in the Rat: Electrophysiological and Anatomical Observations[END_REF] GABAergic inputs. The GABAergic afferents represent the major input to the LHA. GABAergic inputs come from several structures of the basal forebrain and from other subcortical fibres via the septum [START_REF] Anthony | Control of Stress-Induced Persistent Anxiety by an Extra-Amygdala Septohypothalamic Circuit[END_REF] Input from the bed nucleus of the stria terminalis (BNST)/pre-optic area. The fibres of the ventral BNST and connected structures send monosynaptic inputs that predominately inhibit postsynaptic LHA glutamate neurons. This pathway seems to be tightly connected with the feeding control, such that the stimulation of this connection via optogenetics results in both the fast initiation and the increase of the feeding behaviour towards highly palatable or highly fat food.

The amplitude of the response is proportional to the dose of stimulation. Mice learn quite fast to self-stimulate this circuit and interestingly, the selfstimulation is majorly regulated by satiety or food deprivation (Stamatakis et al. -Introduction -Homeostatic & hedonic mechanisms of feeding behaviour 4

2013).

Input from the nucleus accumbens shell. The majority of projections from the NAc are arising from the D1 SPNs, although some D2 projection can also be found. The very first work suggesting the communication between the NAc and hypothalamus dates back to 1995. In this work, Maldonado-Irizarry and collaborators investigated the role of excitatory amino acid inputs to the NAc (core and shell) in feeding behaviour of rats. In the first series of experiments, it was shown that blocking AMPA and glutamate receptors with DNQX in the medial shell, but not core, results in an increase of the feeding behaviour. This feeding response was blocked by the local injection of AMPA, while no effects were elicited by NMDA antagonist infusion. Interestingly, the prior administration of D1 or D2 antagonist receptors decrease by half the feeding induced by DNQX, suggesting a major implication of the DA in the feeding.

Lastly, antagonizing directly in the lateral hypothalamus results in a complete inhibition of the ingestive behaviour, suggesting the existence of an important functional link between two major brain regions involved in the homeostatic and hedonic regulation of the food intake: the LHA and the NAc [START_REF] Maldonado-Irizarry | Glutamate Receptors in the Nucleus Accumbens Shell Control Feeding Behavior via the Lateral Hypothalamus[END_REF]. Complementary works showed that local injections of GABA receptor agonists or glutamate receptor antagonists in the nucleus accumbens shell elicit an intense feeding response resembling to the one seen after stimulation of the lateral hypothalamus. Furthermore, injections of a GABA-A receptors agonist in the shell of the NAc increased the number of cells positive to Fos staining in the LH, as well as in the lateral septum, paraventricular hypothalamic nucleus, ventral tegmental area, substantia nigra pars compacta and nucleus of the solitary tract [START_REF] Stratford | GABA in the Nucleus Accumbens Shell Participates in the Central Regulation of Feeding Behavior[END_REF].

Several works during the last decade tried to better describe the function of the connection between NAc and LHA. A work recently published by the group of Lüscher showed that the D1-expressing SPNs in the NAc inhibit GABAergic neurons, but not orexin or MCH producing neurons, in the more ventro-laterlal part of the LHA. The optogenetic inhibition of D1R-MSNs prolongs feeding in satiated mice even in presence of distracting external stimuli, whereas the activation of D1R-MSN terminals in LH is sufficient to override immediate metabolic need and rapidly stop food consumption despite hunger. Furthermore, optogenetic inhibition of postsynaptic LHA-GABA neurons, which are inhibited by D1R-MSNs, suppresses the licking for a palatable reward consumption of food [START_REF] O'connor | Accumbal D1R Neurons Projecting to Lateral Hypothalamus Authorize Feeding[END_REF].

Other inputs to the LHA. Other subcortical fibres innervate the LHA from the lateral septum [START_REF] Anthony | Control of Stress-Induced Persistent Anxiety by an Extra-Amygdala Septohypothalamic Circuit[END_REF], ventral pallidum [START_REF] Root | The Ventral Pallidum: Subregion-Specific Functional Anatomy and Roles in Motivated Behaviors[END_REF] and substantia innominate [START_REF] Grove | Efferent Connections of the Substantia Innominata in the Rat[END_REF]. Projections are also arising from the midbrain and brainstem, including the inputs from the serotoninergic neurons in the raphe [START_REF] Moore | Serotonin Neurons of the Midbrain Raphe: Ascending Projections[END_REF], and from the norepinephrine neurons in the locus coeruleus. Of note, several works described different intra-hypothalamic connections. For example, LHA connects with the arcuate nucleus [START_REF] Betley | Parallel, Redundant Circuit Organization for Homeostatic Control of Feeding Behavior[END_REF]) and, importantly, the stimulation of this pathway can evoke the feeding behaviour. An increase of the feeding behaviour is also obtained by optogenetic stimulation of the GABAergic fibres connecting the LHA with the periventricular hypothalamus [START_REF] Wu | GABAergic Projections from Lateral Hypothalamus to Paraventricular Hypothalamic Nucleus Promote Feeding[END_REF]. A connection with the ventral medial hypothalamus has also been described [START_REF] Thompson | Organization of Projections from the Dorsomedial Nucleus of the Hypothalamus: A PHA-L Study in the Rat[END_REF].

Output circuitry of the LHA

Classical anatomy studies have demonstrated the existence of multiple projections of the LHA to the VTA, periventricular thalamus, lateral habenula, and many other regions [START_REF] Berk | Efferent Connections of the Lateral Hypothalamic Area of the Rat: An Autoradiographic Investigation[END_REF]. Two recent studies confirmed the existence and the importance of a direct output from the LHA to the VTA. In the first work, Nieh and collaborators clarified that the output is arising from both glutamatergic and GABAergic LHA fibres, and functionally innervates both GABAergic and DA neurons of the VTA. The specific stimulation of the LH-GABA-VTA pathway leads to feeding behaviour [START_REF] Nieh | Decoding Neural Circuits That Control Compulsive Sucrose Seeking[END_REF]. The second work showed that hypothalamic neurotensin projections promote reward by enhancing glutamate transmission in the VTA [START_REF] Kempadoo | Hypothalamic Neurotensin Projections Promote Reward by Enhancing Glutamate Transmission in the VTA[END_REF]. Of note, a major target of the orexin-and MCHproducing neurons includes the brainstem motor systems that support behaviours like chewing, licking, and swallowing. Those innervations include trigeminal and reticular neurons engaged during ingestive behaviour [START_REF] Yamamoto | Response Properties of Lateral Hypothalamic Neurons during Ingestive Behavior with Special Reference to Licking of Various Taste Solutions[END_REF]. MCH and orx neurons also innervate critical sites that regulate oesophageal and gastric 4 -Introduction -Homeostatic & hedonic mechanisms of feeding behaviour 76 properties of the food regulate the food intake [START_REF] Berthoud | Interactions between The 'cognitive' and 'metabolic' brain in the Control of Food Intake[END_REF]. While liking responses are mediated primarily by opioid and GABA mechanisms within the brainstem, wanting processes are predominantly mediated by enhanced dopamine transmission within the cortico-mesolimbic system [START_REF] Alcaro | Behavioral Functions of the Mesolimbic Dopaminergic System: An Affective Neuroethological Perspective[END_REF][START_REF] Berridge | Modulation of Forebrain Electroencephalographic Activity in Halothane-Anesthetized Rat via Actions of Noradrenergic Beta-Receptors within the Medial Septal Region[END_REF][START_REF] Björklund | Dopamine Neuron Systems in the Brain: An Update[END_REF]. Lesions impairing dopamine release within the cortico-mesolimbic structures substantially decrease rodents' food intake and body weight [START_REF] Salamone | Characterization of the Impaired Feeding Behavior in Rats given Haloperidol or Dopamine-Depleting Brain Lesions[END_REF][START_REF] Salamone | The Role of Brain Dopamine in Response Initiation: Effects of Haloperidol and Regionally Specific Dopamine Depletions on the Local Rate of Instrumental Responding[END_REF]. Moreover, as discussed above, the mesolimbic DA system is sensitive to many important feeding peptides and chemicals, including ghrelin, leptin and other cannabinoids [START_REF] Liu | Regulation of the Mesolimbic Dopamine Circuit by Feeding Peptides[END_REF].

The enhanced DA transmission within the reward system, induced by heightened VTA dopamine neuron activity, encourages motivational behaviours to obtain and consume food, especially palatable or rewarding foods [START_REF] Lutter | Homeostatic and Hedonic Signals Interact in the Regulation of Food Intake[END_REF].

Signalling pathway underlying the hedonic control of the food

Signalling events downstream the dopamine receptors

Certain foods, such as highly palatable and highly caloric ones, can raise a reward effect even bigger than (that of) cocaine [START_REF] Lenoir | Intense Sweetness Surpasses Cocaine Reward[END_REF]. From an evolutionary point of view, the rewarding properties of food are really advantageous: palatable food induces a major consumption and a major store of energy for the future needs.

However, in modern societies, where food is widely available, this adaptation has become a liability: the major consumption can become overeating and lead to food disorders. Palatable food and environmental cues that predict their delivery, increase DA transmission within the striatum, thereby influencing striato-hypothalamic and striato-pallidal circuits that control the hedonic and incentive properties of food [START_REF] Kenny | Reward Mechanisms in Obesity: New Insights and Future Directions[END_REF]. Specifically, DA neurons in the VTA increase the firing rate and hence the DA release in the NAc in response to an unexpected food [START_REF] Hajnal | Accumbens Dopamine Mechanisms in Sucrose Intake[END_REF][START_REF] Norgren | Gustatory Reward and the Nucleus Accumbens[END_REF]. This DA response habituates with repeated exposure to the food reward, and is gradually transferred onto the stimuli associated with the reward [START_REF] Epstein | Habituation as a Determinant of Human Food Intake[END_REF]. The level of DA released in response to the -Introduction -Homeostatic & hedonic mechanisms of feeding behaviour 4 food reward is regulated by glutamatergic afferents from several other brain region related: a) to the sensory perception of the food (nucleus tractus solitarius and insula for taste, olfactory bulb and pyriform cortex for smell and visual cortices for the appearance of the food), b) to the homeostatic properties of the food (hypothalamus), c) to the reward (NAc), d) to the emotional (amygdala and hippocampus) and e) to the multimodal (OFC for salience attribution) properties of the food. In humans, ingestion of palatable food has been shown to release DA in the dorsal striatum in proportion to the self-reported level of pleasure derived from eating food [START_REF] Small | Feeding-Induced Dopamine Release in Dorsal Striatum Correlates with Meal Pleasantness Ratings in Healthy Human Volunteers[END_REF].

Signalling events downstream the opiod receptors

The endogenous opioid systems can regulate the hedonic value of food intake independently from the ongoing metabolic needs of the individual. β-endorphin and enkephalin positively contribute to the incentive-motivation to acquire food reinforcers. The lack of either enkephalin or β-endorphin peptides leads to a deficit in the ability of food reward to increase lever pressing behaviour, regardless of the palatability and nutrient content of the foods examined [START_REF] Hayward | Selective Reward Deficit in Mice Lacking Beta-Endorphin and Enkephalin[END_REF]. In both humans and mice, the exposure to sugar drives an analgesic response, suggesting an increase of the opiate signalling directly mediated by sugar [START_REF] Lewkowski | Sweet Taste and Blood Pressure-Related Analgesia[END_REF]). Furthermore, it has been shown that it is possible to provoke an opiate withdrawal syndrome similar to that observed in animals chronically exposed to opioid drugs, in rodents previously exposed to a sugar-rich diet reviewed in [START_REF] Avena | Evidence for Sugar Addiction: Behavioral and Neurochemical Effects of Intermittent, Excessive Sugar Intake[END_REF]. The opiate signalling within the NAc and the VP mainly relates to food liking. The mu-opioid stimulates both the NAc shell, NAc core or VP and amplifies both the liking reactions to sweetness and appetitive wanting for food reward. NAc hotspots modulate the expression in the VP and viceversa. The opioid signalling in the basolateral amygdala is implicated in conveying the affective properties of food, which contribute in food wanting as well as in modulating the incentive value of food and reward-seeking behaviour [START_REF] Wassum | Distinct Opioid Circuits Determine the Palatability and the Desirability of Rewarding Events[END_REF].

Signalling events downstream the cannabinoid receptor

Several studies have highlighted the importance of endocannabinoids signalling in the regulation of feeding behaviour. In 2003, it has been shown that delta-9 tetrahydrocannabinol (THC), the active ingredient of cannabis, increases food intake.

In this work, Higgs and collaborators examined the mode of action of cannabinoids on ingestion by studying the effect of CB1 receptor agonists and antagonists, on licking microstructure in rats ingesting a palatable sucrose solution. Contrary to the antagonist, the CB1 agonist decreased instrumental responding for food [START_REF] Rasmussen | Effects of Rimonabant on Behavior Maintained by Progressive Ratio Schedules of Sucrose Reinforcement in Obese Zucker (Fa/fa) Rats[END_REF][START_REF] Zoë | The Cannabinoid CB1 Receptor Antagonist SR141716A Reduces Appetitive and Consummatory Responses for Food[END_REF]. CB 1 receptor knockout mice display a reduced responding for sucrose or fat-enriched but not other types of food reward (Guegan 2012) as well as the absence of the structural plasticity induced by the training for highly sugar food (Guegan 2012).

The CB1 receptor is located on presynaptic terminals releasing GABA or glutamate in the cortex, hippocampus, amygdala, striatum, and hypothalamus. The retrograde activation of CB1 receptors regulates the release and the signalling of different neurotransmitters involved in feeding behaviour, such as DA, or orexin. Of note, when injected in the shell of NAc, the endogenous CB 1 receptor ligand anandamide enhances the liking of a sweet reward [START_REF] Mahler | Endocannabinoid Hedonic Hotspot for Sensory Pleasure: Anandamide in Nucleus Accumbens Shell Enhances 'Liking' of a Sweet Reward[END_REF]). The endocannabinoid signalling elicited by the stimulation of the CB1 receptor takes part in several mechanisms of feeding: the homeostatic regulation (Lattemannand D., 2008) and the hedonic regulation of food intake, and the regulation of the energy expenditure (D. Richard, et al.2009. I. Matias, V. Di Marzo 2007).

-Introduction -Homeostatic & hedonic mechanisms of feeding behaviour 4

Structural plasticity induced by highly palatable food

Surprisingly, although numerous studies have already reported that cocaine and other drugs of abuse produce persistent changes in the structure and number of dendritic spines on D1 and D2 SPNs, few groups investigated if the same changes are taking place following the stimulation of the reward system with natural rewards.

In 2012, the group of Martinez gave a major contribution to the field. Guegan and co-workers showed that operant training for highly palatable food leads to an augmentation of the dendritic spines in the NAc-shell and in the medial PFC but not in the NAc-core. These structural changes seem to be specifically related to learning, as the yoked control groups (non-contingently receiving the same amount of food) did not display any structural rearrangement. In the same paper it was also shown that similar modifications were not induced in CB1R-KO mice, confirming a major implication of the CB1R in the regulation of the reward-related consumption of food (Guegan et al., 2012). In a similar work, Mancino S. and collaborators showed that changes in the density of specific dendritic spines could be found not only in the NAc shell and PFC, but also in the hippocampus (HP) of mice trained in an operant training to obtain chocolate-flavoured pellets. Moreover, the analysis of the spine morphology showed that the operant training increased stubby spines density in the HP and filopodia density in the PFC and NAc shell. Interestingly, the same paper has been also shown that knocking out the gene for the delta opioid receptor 1 (DOR1) results in the abolishment of the operant-induced spine formation. DOR1-KO mice showed also reduced levels of stubby spines in PFC and HP and a decreased filopodia spine density in the PFC and NAc shell. Consumption of high-energy diets may compromise health and may also impair cognition; these impairments have been linked to tasks that require hippocampal function. Conversely, food restriction has been shown to improve certain aspects of hippocampal function, including spatial memory and memory persistence. It has been recently proved that those observations correlate with changes of neuronal plasticity in hippocampus (Babits R., 2016).

All the studies discussed above focused on NAc, cortex and hippocampus. In a recent work, J. Ibias and co-workers examined whether schedule-induced polydipsia (SIP), an adjunctive behaviour in which rats exhibit excessive drinking as 4 -Introduction -Homeostatic & hedonic mechanisms of feeding behaviour 80 a consequence of intermittent feeding, would induce modifications in SPNs in dorsolateral striatum (DLS) and in the basal dendrites of layer V pyramidal cells anterior prefrontal cortex (aPFC) neurons. The reported results showed that SIP caused an increase in dendritic spine density in DLS but not aPFC neurons. The authors hypothesized that SIP-induced structural plasticity in DLS neurons could be related to an inflexible response in compulsive behaviour and demonstrate the involvement of the dorsolateral striatum and anterior prefrontal cortex regions in compulsive disorders and in the control of the feeding behaviour (J. Ibias et al., 2016). Importantly, different studies suggest that this hypothesized inflexibility is due to a structural plasticity specific to the D2-SPNs (Bock R., 2013;Johnson PM., 2010).

In conclusion, in the last years considerable progress has been made in unveiling homeostatic and hedonic pathways that regulate signals for feeding. Today, the existence of a tight communication between the systems of neurotransmitters and peptides in midbrain and corticolimbic areas is unquestionable. The studies of the last 15 years consider the hypothalamus as the centre of the homeostatic control of the food, and the lateral area of the hypothalamus as the direct rely between the homeostatic and the rewarding control of the food intake. It is also clear that the meso-corticolimbic system with DA, 5HT, and opioid and cannabinoid systems, is likely to play prominent roles in the hedonic control of the feeding.

As discussed, the rewarding properties of highly palatable food can override the homeostatic control of food intake and lead to food disorders such as obesity.

Indeed, it has been shown that -as for drugs of abuse -highly palatable food triggers the formation of new spines that could be the substrate of maladaptive neuronal In the following paragraphs we review different techniques currently utilized to elucidate the biological properties of discrete neurons populations

Trangenesis strategies

One of the most promising approaches to study a pure population of cells is still to use mouse genetics methods to identify specific cell populations.

The development of transgenic strategies has permitted the expression of specific genes in both spatially and temporally defined patterns. Early in the 1980's several research groups [START_REF] Gordon | Integration and Stable Germ Line Transmission of Genes Injected into Mouse Pronuclei[END_REF][START_REF] Costantini | Introduction of a Rabbit Beta-Globin Gene into the Mouse Germ Line[END_REF]Harbers et al., 5 -Introduction -Profiling discrite neuronal population 84 1981;EF Wagner et al., 1981, TC Wagner et al., 1981) developed methods to introduce a specific gene into the mouse genome, which could be inherited in a Mendelian fashion. Coding DNA sequences are microinjected into one or both pronuclei of zygote-stage embryos. Microinjected embryos are subsequently transferred into recipient females allowing the embryonic development.

In 1996 Mayford and colleagues, by using the bacterial tetracycline operator system initially developed by Bujard, achieved for the first time an inducible expression of a mutant form of the activated calcium-independent form of calciumcalmodulin-dependent kinase II (CaMKII) that was forebrain-specific [START_REF] Mayford | Control of Memory Formation through Regulated Expression of a CaMKII Transgene[END_REF]. The TetOff tetracycline system involves the tetracycline transactivator (tTA) protein, which is created by fusing one protein, TetR (tetracycline repressor), found in Escherichia coli bacteria, with the activation domain of another protein, VP16, found in the Herpes simplex virus, and the TetO operator placed upstream of the gene of interest [START_REF] Gossen | Tight Control of Gene Expression in Mammalian Cells by Tetracycline-Responsive Promoters[END_REF][START_REF] Gossen | Transcriptional Activation by Tetracyclines in Mammalian Cells[END_REF]. Usually several repeats of TetO sequences are placed upstream of a minimal promoter such as the CMV promoter, forming together a so-called tetracycline response element (TRE). In the presence of a tetracycline (usually doxycycline), binding of tTA to TetO is prevented, and the gene of interest is not transcribed until doxycycline regimen is interrupted. Later on, TetOn systems were developed in which rtTA (a modified form of tTA) binds to TetO only in the presence of doxycycline. In this case the transgene is induced by doxycycline administration. Mayford and collaborators used tTA (TetOff system) to control a portion of the αCaMKII promoter and thus obtained a mouse with region-specific inducible gene expression. In 1998 the same system was used to target a striatal neuronal population. Nestler's group generated two lines of bitransgenic mice that inducibly overexpressed ΔFosB selectively in striatal regions under the control of the tetracycline gene regulation system (Chen et al., 1998;[START_REF] Kelz | Expression of the Transcription Factor deltaFosB in the Brain Controls Sensitivity to Cocaine[END_REF][START_REF] Werme | Delta FosB Regulates Wheel Running[END_REF]. Those studies showed that the overexpression of ΔFosB selectively in D1 SPNs increases response to the rewarding and locomotor effects of cocaine [START_REF] Kelz | Expression of the Transcription Factor deltaFosB in the Brain Controls Sensitivity to Cocaine[END_REF][START_REF] Colby | Striatal Cell Type-Specific Overexpression of DeltaFosB Enhances Incentive for Cocaine[END_REF] as well as to the rewarding effects of morphine (Zachariou et al., 2006Voluntary). Consistently, mice overexpressing ΔFosB predominantly in striatopallidal neurons ran considerably less.

In 2008 first, and then in 2011 the Palmiter's group used the knock-in (KI) technology to gain an insight on the differential output obtained by the artificial modulation of gene expression in D1 or D2 SPNs. In a first paper published in 2008 the group obtained a mouse line expressing the Cre recombinase specifically in the D1 SPNs. These KI mice were then crossed with mice carrying a conditional allele of the Gad1 gene -which encodes GAD67, one of the two enzymes responsible for GABA biosynthesis -in order to achieve the selective reduction of the GABA synthesis in striatonigral neurons [START_REF] Heusner | Deletion of GAD67 in Dopamine Receptor-1 Expressing Cells Causes Specific Motor Deficits[END_REF]. The mice showed mild motor deficits in tasks such as rotarod. The same KI mice have been used to obtain a mouse line in which the NR1 subunit of glutamate receptor NMDA was inactivated in D1 neurons. These KI mice failed to display locomotor sensitization to repeated cocaine administration and have a decreased ability to form a conditioned place preference to cocaine [START_REF] Beutler | Severely Impaired Learning and Altered Neuronal Morphology in Mice Lacking NMDA Receptors in Medium Spiny Neurons[END_REF]. These results suggested that NMDA receptor signalling in the striatonigral pathway is required for the manifestation of behaviours associated with repeated drug exposure [START_REF] Heusner | Deletion of GAD67 in Dopamine Receptor-1 Expressing Cells Causes Specific Motor Deficits[END_REF][START_REF] Beutler | Severely Impaired Learning and Altered Neuronal Morphology in Mice Lacking NMDA Receptors in Medium Spiny Neurons[END_REF].

More recently Lambot and collaborators generated a conditional knock out mouse for the NMDA receptor in the indirect pathway to show a reduction of the corticostriatopallidal synapses both at the level of number and strength. The mice also displayed a reduced habituation, a delay in goal-directed learning, a lack of associative behaviour, and impairment in action selection or skill learning. [START_REF] Lambot | Striatopallidal Neuron NMDA Receptors Control Synaptic Connectivity, Locomotor, and Goal-Directed Behaviors[END_REF].

These results supported the importance of studying the two pathways independently and the opposite role of striatonigral and striatipallidal neurons in natural reward, drug reward and locomotion.

5 -Introduction -Profiling discrite neuronal population 86

BAC strategies

The advent of bacterial artificial chromosome (BAC) transgenic mice, pioneered by Nat Heintz from the Rockefeller University [START_REF] Yang | Homologous Recombination Based Modification in Escherichia Coli and Germline Transmission in Transgenic Mice of a Bacterial Artificial Chromosome[END_REF]Heintz, 2001;[START_REF] Gong | A gene expression atlas of the central nervous system based on bacterial artificial chromosomes[END_REF], allowed the development of high throughput genetic labelling of distinct neuronal populations using the GPCRs and neuropeptides promoters to drive the expression of reporters such as EGFP. BACs are fragments of 100-250 kb of genomic mouse DNA that contain almost all the regulatory sequences necessary for an accurate expression in vivo. The BAC-driven expression of tagged proteins allows an easy and reproducible identification of specific neuronal populations.

The most common transgenic lines used to target the two populations of striatal neurons are eGFP and the Cre recombinase (review in [START_REF] Valjent | Looking BAC at striatal signaling: cell-specific analysis in new transgenic mice[END_REF]. drd1a-EGFP and chrm4-EGFP BAC mice express EGFP in striatonigral cells and their axonal projections to the internal globus pallidus and the sustantia nigra, whereas drd2a-EGFP BAC mostly label the striatopallidal neurons and their projections to the external globus pallidus [START_REF] Gong | A gene expression atlas of the central nervous system based on bacterial artificial chromosomes[END_REF]Lobo et al., 2006;Bertran-Gonzalez et al., 2008;[START_REF] Matamales | Striatal medium-sized spiny neurons: identification by nuclear staining and study of neuronal subpopulations in BAC transgenic mice[END_REF]. The use of the red fluorescent protein tdTomato (tandem dimer Tomato) in drd1a-tdTomato BAC, crossed with the drd2a-GFP lines [START_REF] Gong | A gene expression atlas of the central nervous system based on bacterial artificial chromosomes[END_REF]Shuen et al., 2008), allowed the visualization of the two populations in the same animals. These various reporter mice provide extremely important tools for deciphering the anatomical, electrophysiological and molecular differences of D1 and D2 SPNs.

Laser-capture microdissection (LCM)

The mRNAs of cells of distinct identity can be isolated by microdissection of tissue. The laser-capture microdissection (LCM) is a method to obtain subpopulations of cells by dissecting a tissue under direct microscopic visualization.

The LCM couples a laser to a microscope and defines a trajectory on the tissue. This trajectory can be separated from the adjacent tissue. Harvesting the population of cells of interest or cutting away the unwanted cells will allow obtaining a pure cell -Introduction -Profiling discrite neuronal populations 5 population, RNA seq, cDNA libraries generation, as well as genotyping are some examples of the possible downstream applications that could be performed after LCM (Emmert-Buck MR et al., 1996;Espina V. et al., 2006;Curran, S. et al., 2000).

A few examples of the application of the LCM technologies in different cellular subpopulation of the striatum are summarized below. shown that LCM is accurate enough to perform proteomic studies on specifically defined cell groups. Immunostaining can be used to label specific cells and LCM can be used dissect single cells within a tissue (Moulédous L et al., 2003). As the droplets pass through the laser, a decision is made whether to sort that event.

Flow cytometry and fluorescence-activated sorting

The sorting is typically based on fluorescent labelling. The positive droplet will be charged either positively or negatively by a charging electrode and travel toward positively or negatively charged platinum plates into the appropriate collection tube.

Separated fractions can then be analysed independently and used for downstream applications.

One major issue of the flow cytometry is the involvement of the dissociation of the tissue. In the adult brain, the tissue dissociations lead to the loss of neurites and all the cytoplasmic proteins that could be characteristic of a particular cell population. Furthermore, the preparation of the tissue involves long incubation with proteases and chelators that could potentially lead to the alteration of important posttranslational modification.

Nevertheless, FACS-based separation of different cell populations allowed several important studies of discrete population of neurons properties. In 2006, Lobo and collaborators use the GFP BAC mice to perform high throughput microarray gene expression analysis in the two SPNs subtypes developing the so called FACSarray approach. The FACS-array consists in isolating live EGFP-positive neurons using fluorescence-activated cell sorting (FACS) and purifying RNA from the sorted neurons for microarray profiling. For example, Ebf1, a D1-enriched lineage-specific transcription factor, was found essential for the maturation of D1 but not for D2 SPNs. The confirmation of the selective expression of Slc35d3, a gene encoding an uncharacterized nucleotide sugar transporter, in D1 SPNs, indicated that cell typespecific protein glycosylation may have a role in the function of the striatonigral neurons. The D2 SPNs enrichment of the G-protein coupled-receptor 6 (Gpr6) pointed out the importance of the cAMP signalling in these neurons for mediating in the context of instrumental learning [START_REF] Lobo | Genetic Control of Instrumental Conditioning by Striatopallidal Neuron-Specific S1P Receptor Gpr6[END_REF].

-Introduction -Profiling discrite neuronal populations 5 Guez-Barber and co-workers have also used FACS-array in 2011 to purify adult striatal neurons based on their activation state, as defined by their c-fos promoter induction [START_REF] Guez-Barber | FACS Identifies Unique Cocaine-Induced Gene Regulation in Selectively Activated Adult Striatal Neurons[END_REF][START_REF] Guez-Barber | FACS purification of immunolabeled cell types from adult rat brain[END_REF]. Neurons activated by acute and repeated cocaine injection were isolated and used to compare their unique patterns of gene expression with those in the non-activated majority of neurons. This work confirmed the differential responses of striatal neurons following cocaine treatment. The authors found that the activated neurons showed higher expression of the D1 neuronal marker gene prodynorphin, as compared to the expression of the D2 neuronal marker genes (D2R and A2AR). A differential regulation of the IEGs expression was observed in the activated or inactivated neurons, with the IEGs being induced only in activated neurons, and unchanged or even decreased in the nonactivated majority of neurons. Since many of these IEGs and neural activity markers are also transcription factors, it is likely that very different patterns of gene expression are subsequently induced within these activated neurons that may contribute to the physiological and behavioural effects of cocaine.

In 2009 Kriaucionis and collaborators (Kriaucionis et al.,200) pioneered a protocol to study discrete populations of nuclei. An advantage of this method is that the nuclei dissociation is faster than the cell dissociation and can be done at 4°C.

Recently our group used FACS based technique to evaluate the cocaineinduced epigenetic modifications specifically in striatonigral and striatopallidal neurons that account for the dramatic differences in gene expression between the two cell-types. In this work, E. Jordi and co-workers showed that a single injection of cocaine triggers the acetylation of K 5 H4 the tri-methylation of K 9 H3 and that this increase persists 24 h after a single injection of cocaine in D1 [START_REF] Jordi | Differential effects of cocaine on histone posttranslational modifications in identified populations of striatal neurons[END_REF]. Of note, those results were opposite in the D2 SPNs.

Although flow cytometry is really useful for studying DNA modifications, or the enrichment of nucleolar proteins, one major limitation of the flow cytometry in nuclei is the main loss of the nuclear protein due to the preparation of the samples.

To overcome this problem, we have recently developed a method for isolating and analyzing cell type-specific nuclei from fixed adult brain (fluorescence-activated sorting of fixed nuclei; FAST-FIN) (see paper as Annex 1). The method is based on 5 -Introduction -Profiling discrite neuronal population 90 the same protocol as developed in [START_REF] Jordi | Differential effects of cocaine on histone posttranslational modifications in identified populations of striatal neurons[END_REF], except for a prior fixation of the brain with paraformaldehyde. The fixation of tissue covalently binds to the nuclei the proteins that normally would be lost during homogenization and maintains the more labile posttranslational modifications. Although it decreases the yield of nuclei preparation as compared to unfixed tissue, it has the advantage to be usable in transgenic mice expressing GFP or tdTomato, or after immunolabeling of nuclei.

-Introduction -Profiling discrite neuronal populations 5

Genetic profiling of discrete populations of neurons

One important consequence of the possibility to distinguish discrete populations of neurons is the possibility to combine those techniques with neuronal populationspecific genetic profiling.

Single cell RNA-sequencing

The single-cell RNA sequencing is a method that aims to provide new perspectives to our understanding of genetics by bringing the study of genome expression to the cellular level. The single-cell approach provides high-resolution of a sample's genomic content by reducing the complexity of the genomic signal through the physical separation of cells. Clustering analyses allow the identification of rare cell types within a cell population. The possibility of obtaining an indication of the transcriptional profile of cells content allows the analysis of RNA with low copy number, which may exert important functions in the cells and that is mainly undetected when the sequencing is performed on an averaged cell population. The single cell RNA-seq is a relatively new method that is not yet standardized. Single cells can be obtained following FACS, serial samples-dilution or LCM, combined with microfluidics. Although single cell sequencing is a really promising technique, it introduces several bias compared to other RNA-sequencing methods. For example, FACS preparation, as other methods, requires the separation of cells from their natural milieu. This could possibly cause perturbation in the transcriptional profiles of RNA expression analysis; the serial dilution is susceptible to misidentification of the cells under the microscope and is really difficult to limit the contamination from neighbouring cells in the LCM.

BAC-TRAP

The BAC-TRAP (translating ribosome affinity purification) technology consists of a rapid affinity purification strategy for the isolation of translated mRNA from genetically targeted cell types. In BAC transgenic mice, a fluorescent EGFP is fused to the N terminus of the large subunit ribosomal protein L10a and inserted under the control of the promoter of either Drd1a or Drd2 (or other promoters) (Doyle et al., 2008;Heiman et al., 2008). EGFP-tagged polysomes can be immunoprecipitated with anti-GFP-coated beads and purified mRNA analysed by microarray or RNA sequencing.

The major advantage of the BAC-TRAP is that it allows to study changes within an identified cell population in response to a challenge. . In 2008, Heiman and collaborators combined the BAC-TRAP technology to microarray analysis and identified more than 300 genes differentially expressed between the D1 and D2 SPNs. Some of them were common to the work of Lobo and colleagues but this approach revealed many more differences between the two neuronal populations than those observed with the FACS-array. Furthermore, they evaluated the expression changes in the two populations after acute and chronic cocaine treatments, confirming some of the genes already known as regulated by cocaine and showing a major activation of the D1 SPNs to the cocaine.

-Introduction -Profiling discrite neuronal populations 5

Conclusion

In this literature review I introduced all the topics linked to the main objectives of my thesis work. In chapter one and two I focused on the DA signalling and in the basal ganglia. I intensively described the striatum and the different sub-populations of SPNs, pointing out the importance of considering the differences between the anatomical sub-territories (NAc, DS, patch/matrix) as well as its different populations of neurons to fully understand the functions that this region mediates. In Chapter 3 I reviewed the mechanisms and the substrates of action of the drug of abuse cocaine. I introduced some of the modifications induced by cocaine focusing on the striatum and the PFC. In particular, in the last part I described the transcriptional modifications induced by cocaine. I pointed out that the transcriptional modifications are likely responsible for the long-lasting modifications that are induced by cocaine intake. Although a lot of work has been already done on this topic, only few researches tried to distinguish the cocaine effect in pure populations of dopamine receptor expressing neurons. Although there is information on the cocaine induced transcriptional modifications on the different populations of SPNs, knowledge about their position in the striatum (NAc or DS) is still incomplete.

In the first part of chapter 4 I summarized the mechanisms of the homeostatic control of food intake. In the second part I focused on the reward-related control of the food intake and on the structural plasticity induced by highly palatable food. Compared to cocaine, the transcriptional regulation induced by the natural reward food, is much less described. Comprehensive information of the transcriptional effects induced by the palatability of the food in pure populations of neurons is still missing. Lastly, I introduced the different techniques available to distinguish discrete populations. Dopamine (DA) controls movement execution, action selection, and incentive learning by regulating the efficacy and plasticity of cortico/thalamo-striatal transmission. Long-term modifications require changes in gene transcription. Both natural rewards and drugs of abuse are able to induce structural changes in prefrontal cortex (PFC), nucleus accumbens (NAc) and dorsal striatum (DS) in the striatum (reviewed in Russo et al., 2010;Geugan T., 2012). The aim of the present work was to study the transcription profiles of selected population of neurons in PFC, NAc and DS in basal condition, following operant learning, or after mimicking the stimulation of the reward system with cocaine.

The striatum is composed of several cell types that exhibit different responses to drugs and mediate different features of "rewarding" stimuli. The largest neuronal population of the striatum is comprised of medium-sized spiny projection neurons (SPNs), which can be further differentiated in two population, based on the subtype of dopamine (DA) receptor they express, namely D1 or D2 receptors with few expressing both. Both D1 and D2 SPNs express their own subsets of markers and share many morphological characteristics and functional properties. They participate in distinct pathways that exert opposite effects on their target regions. Various functional studies showed that the NAc and the DS mediate distinct aspect of the striatal function. SPNs in those two striatal regions look similar in phenotype but they differ in their input/output connections and functions. Until today, this diversity was still not well understood and it is crucial to distinguish the response in the two populations of SNPs in the two different regions. So, in order do better characterize these cells, we used transgenic mice that express a tagged ribosomal protein (L10a-EGFP) under control of the D1 or D2 receptor promoter to isolate currently translated mRNA and nuclei from each population of SPNs, as well as from D1 pyramidal neurons of the PFC.

With this strategy we were able to tackle different questions and to facilitate their comprehension, the thesis work is subdivided in three parts with three main objectives.

Aim 1:

Characterization of the basal gene expression in the different neuronal populations expressing either D1 or D2 receptors in the NAc, and DS, or, for D1 in PFC. The first part of this study allowed for the first time profiling the translated (polysome-associated) mRNAs of D1 and D2 SPNs belonging to the ventral or dorsal striatum as well as a comparison between those cells and the D1 receptor-expressing neurons in the PFC. In the second part of this work we provided an in vivo validation of our analysis. Starting with an upstream analysis of the genes specifically expressed in the DS, we were able to modulate its normal function by using drugs known to have an effect on the expression of the genes enriched in this region.

Aim 2:

Analysis of the transcriptional changes following the active recruitment of the reward system (operant learning for food). In this part of the work we characterized for the first time the transcriptional changes induced by a natural reward -regular or highly palatable food -across the cortico-striatal system. This work provided an insight on different issues: 1) we profiled the genes regulated by the learning process and 2) we identified the regions, the neurons and the genes more responsive to the highly palatable food, 3) we compared the regulation of gene expression by a drug of abuse, cocaine, with that exerted by natural reward (highly palatable food). The in vivo manipulation of one of the genes differentially regulated by one of the treatment allowed the validation of part of the findings of our study.

Aim 3:

Characterization of the long-lasting modifications induced in each of these 5 neuronal populations by seven days of exposure to cocaine. In this part we provide a comprehensive study of the effects of cocaine administration on the two populations of neurons depending on their localization. Furthermore, we identify networks of coregulated genes associated that could be associated with features of drug addiction All these data allowed us a better comprehension of the neuronal typespecific gene expression in the main striatal regions and PFC, and its responsiveness to the stimulation of the reward system with a drug of abuse, cocaine, or natural reward.

Animals

For our experiments we used BAC (bacterial artificial chromosome) transgenic mice expressing the enhanced green fluorescent protein fused to the N-terminus of the large subunit ribosomal protein L10a under the control of dopamine D1a or D2 receptor promoter (Drd1a::EGFP-L10a or Drd2::EGFP-L10). These mice lines were generated by Heiman et al., 2008

Operant conditioning experiments

Drd1a::BACTRAP, or Drd2::BACTRAP, Ncdn FLOX/FLOX;iCRe (Ncdn-KO), Ncdn FLOX/FLOX and WT mice were trained in an operant conditioning paradigm.

Seven days before the beginning of the experiment all the animals were individually housed and maintained in an environment with controlled temperature and humidity with a 12:12-h reversed light dark cycle. All the experiments were carried out during the dark phase of the dark/light cycle. All the animals were randomly assigned to one of the following 4 groups: master highly palatable (mHP), master standard (mST), yoked highly palatable (yHP), yoked standard (yST). Five days before the starting conditioning all the mice were mildly food-deprived to maintain their weight to their original weight. The food restriction was maintained until the 9 th operant training session in order to facilitate the acquisition of the task. Mice then received ad libitum food from the 10 th session until the end of training. During the operant conditioning 6 -Material & Methods -102 sessions animals were presented with either 20 mg dustless precision standard pellets (TestDiet 5UTM #1811143) or highly palatable isocaloric pellets (TestDiet 5UTL #1811223). The standard diet was similar to the standard diet used to maintain the mice (TestDiet Purina 5053) in composition and taste (3.30 kcal/g, 24.1 % protein, 10.4 % fat, 65.5 % carbohydrates). The highly palatable diet was similar in calories content to the standard diet (3.48 kcal/g) but contained a higher level of sucrose among the carbohydrates (49%) and was modified by the addition of chocolate flavour. The training session started with a fixed ratio (FR)-1 reinforcing schedule.

During this period master animals had to poke once in the active hole to receive a pellet. Each poke in the active hole was followed by a 10-second time-out period, independently of the reinforcing schedule. The FR1 was followed by five days of FR5, during which mice were fasted at 90% of their original weight. The last phase consisted of 6 days of FR5 in which mice had ad libitum access to food between operant sessions. Only mice maintaining at least 75% responding on the active hole, a minimum of 10 reinforces per session and less than 20% deviation from the mean of the total number of reinforces earned in three consecutive days were allowed to continue the experiment, all the mice reach the criterion. Twenty four hours after the last training session the Ncnd-ko mice and their control were presented to a progressive ratio (PR) schedule in order to evaluate the relative reinforcement efficacy of the reward. During the PR the response requirements increased systematically within the session, after each reinforce. The PR schedule lasted for 1 h and respected the following series 1, 2, 3, 4, 5, 6, 7, 8, 10, 12, 14, 16, 18, 20, 22, 24, 28, 32, 36, 40, 44, 48, 52, 56, 64, 72, 80, 88, 96, 104, 112, 120, 128, 136, 144, 152, 160, 168, 176, 184, 192, 200, 208, 216, 224, 232, 240, 248, 256, 264, 272, 280, 288, 296, 304, 312, 320, 328, 336, 344, 352, 360, 368, 376, 384, 392, 400, 408, 416, 424, 432, 440, 448, 456, 464. The operant conditioning experiments were carried out in mouse Med Associate operant chamber (model ENV-307A-CT). Each chamber was composed by a grid floor (model EVV-414), 2 nose-poke holes, one randomly selected as active and the other as inactive, one house light, a food dispenser and a food magazine between the 2 nose-poke holes. The operant chambers were located in an isolation box equipped with fan. The beginning of the session was concomitant with the fan activation and the turning on of the house light for 3 seconds, and a pellet delivery. The session ended either after 60 minutes or after 100 pellets had been -Material & Methods -6 delivered. Poking in the active hole resulted in the delivery of one pellet concomitant with a 2-second poke-light. The pellet delivery was followed by 10 s of time out.

During this period, the pokes were inactive. Mice were sacrificed by decapitation 24 h after the last session, the brain was removed and prefrontal cortex, nucleus accumbens and dorsal striatum were rapidly dissected on ice. The tissues were homogenised and subjected to nuclear fractionation and RNA immunoprecipitation.

Pharmacological treatments

Mice received a single intraperitoneal injection (i.p.) of either 20 mg/kg cocaine•HCl (Sigma) or its saline vehicle for 7 consecutive days and were killed 24 h after the last injection.

mRNA extraction

Cell specific translated-mRNA purification, was performed as described in Heiman et al., 2008 with some modifications. Each sample consisted of a pool of 2-3 mice. BAC TRAP transgenic mice were sacrificed by decapitation. The brain was rapidly dissect out on ice and placed in a brain mouse brain matrix with 0.5 mm coronal section interval (Alto Stainless Steel Coronal 0.5 mm Brain Matrix). In the matrix, three blades were used do obtain two thick slices containing the PFC and the striatum. The first blade was used divide the olfactory bulb from the cortex, the second was placed at 2 mm from the first and defined the region containing the s PFC cortex, the third was placed at 3 mm from the second to define a slice containing the entire striatum. The prefrontal cortex was quickly dissected out and the nucleus accumbens and the dorsal striatum removed with small metal punches on ice. Each tissue were homogenised in 1 ml of cold lysis buffer (20 mM HEPES KOH (Avanti Polar lipids) to a final concentration of 30 mM. After mixing and 5-minute incubation on ice the lysate was centrifuged for 10 minutes at 20,000 x g to separate the supernatant from the insolubilized material (Heiman et al., 2008;[START_REF] Heiman | Molecular adaptations of striatal spiny projection neurons during levodopa-induced dyskinesia[END_REF]. Magnetic beads coated with anti-GFP antibody were prepared as follows: Then the RNA was purified according to the manufacturer's instructions, with an oncolumn DNAse-I digestion step. The quantity of RNA was determined by fluorimetry using the Quant-iT Ribogreen, and the quality was determined checked using the Bio-Analyzer Pico RNA kit before library preparation.

-Material & Methods -

106 then mapped to the genome of Mus musculus (UCSC mm10) using TopHat2 2.0.9 [START_REF] Kim | TopHat2: Accurate Alignment of Transcriptomes in the Presence of Insertions, Deletions and Gene Fusions[END_REF]], a splice junction mapper, with a set of matching gene annotation (genes.gtf downloaded from UCSC on December 8th 2015). Conservative options have been used to keep only reliable levels of expression for each gene such as the obligation to map only to one possible location for each read and by taking only into account the paired reads mapped in proper pairs. The gene counting step was then made with HTSeq-counts v0.6.0 (Anders et al. 2014). The exons were chosen as the mapping features and the reads of the same pair had to be mapped to the opposite strands of the gene. Before statistical analysis each library was checked using principal component analysis and also correlation matrix. Differentially expressed genes were identified with R using the Bioconductor package DESeq2 v1.10.1 (Love et al. 2014) taking advantage of its capacity to perform multi-factor analysis. Genes with adjusted p-value less than 5 % (with false discovery rate method from Benjamini and Hochberg) were declared differentially expressed. Gene ontology (GO) analysis was performed on a list of significantly differentially expressed genes (adj p<0.05) identified with DESeq2. The cluster Profiler v3.0.4 [START_REF] Yu | 1. Abbrev.: FC, fold-change, GPCR, G protein-coupled receptor, IC, ion channel, IUPHAR, International Union of Basic and Clinical Pharmacology, LGIC, ligand-gated ion channel, MGI, mouse genome informatics database symbol, NHR, nuclear hormone receptor, padj, adjusted[END_REF] package from Bioconductor was used. Overrepresented GO were identified using GSEA method. Conservative options were used to filter results (Bonferroni p value adjustment and 0.01 p-value threshold). We tested 3 sets of genes of interest for each comparison, the differentially expressed and the overexpressed for each condition.

Each set of genes was compared with all known genes present in the annotation. The GO categories were found in org.Mm.eg.db [Carlson] Bioconductor package based on official gene symbol

Total RNA purification and cDNA preparation

Each sample consisted of the tissue deriving from one mouse brain. Mice were sacrificed by cervical dislocation, the prefrontal cortex was quickly dissected out and mircodisks punched out from the nucleus accumbens and the dorsal striatum with a stainless steel cannula and placed on ice (slice thickness 3 mm). Each tissue sample was homogenized in TRIzol with loose and tight glass-glass 2 ml Dounce homogenizers. Total RNA was extracted with TRIzol Reagent (Life Technologies) according to manufacturer's instructions. The RNA was quantified by using the -Material & Methods -6 NanDrop 1000 spectrophotometer and its integrity checked with the Bionalyzer (agilent 2100 Bioanalyzer, Agilent RNA 6000 nano kit). Five hundred ng of mRNA from each sample were used for retro-transcription, performed with the Reverse Transcriptase II (Life Technologies) following the manufacturer's instructions.

Real-Time PCR

Quantitative real time PCR, was performed using SYBR Green PCR kit in 96well plates according to the manufacturer's instructions. Results are presented as normalised to the house-keeping gene and the delta-CT method was used to obtain a FC.

Western blots

The NAc, DS, and PFC were rapidly dissected on an ice-cooled dish and stored at -80°C. Each sample was sonicated in 150 uL of RIPA buffer. Protein content was estimated using BCA protein assay (Thermo scientific, prod #23235) following manufacturer's instructions and an equal amount of lysate was mixed with denaturing 3X STOP buffer. Fifteen µg of protein for each sample were separated in 4-20% SDS-polyacrylamide gel (BIO-RAD mini-protean-TGX) before electrophoretic transfer onto a nitrocellulose blotting membrane (Amersham, Lot No G9990998). Membranes were blocked 45 minutes in 30 g/L bovine serum albumin and 10 g/L non-fat dry milk in 0.1 M phosphate buffer saline (PBS). Membranes were then incubated overnight with the primary antibody Norbin (RU1002). The bound primary antibody was detected with a secondary luminescent antibody to mouse (IRDye 800 CW-conjugated, antimouse IgG, Rockland Immunochemical, Incubation for 2 × 5 min in a 2:1 (v/vo) isopropanol:EtOH mixture was followed by 1 × 5 min in pure isopropanol and 2 × 5 min in xylol. Finally, samples were mounted in mounting medium (Eukitt) and left for 24 h to settle.

Secondary dendrites from SPNs from the NAc and DS, and from pyramidal neurons of the layer 5 of the PFC were photographed using Z-stacks from 0.2-µm optical sections in bright field at × 100 resolution on a DM6000-2 microscope (Leica). A maximum of three dendrites per neuron and from at least 5 slices per animal were photographed. Files were analysed with the ImageJ software as follows: first, Z-stacks were summed using the plugin Bio-format importer, the scale was adjusted according to the pixel size of the images. The total number of spines was obtained using the cell counter tool. At least 40 dendrites per group, with at least eight mice per group were counted. For spines morphology, at least 3 dendrites per mouse were analysed. At least 20 spines on the dendrites that were clearly observed in the X-Y plane were analysed. An average of 480 spines per group were analysed for major head diameter as well as neck length. Two investigators performed acquisition and analysis. Results were subsequently pooled and showed minor differences in the counts from the two investigators. In the following section we present in the form of a preliminary manuscript the study of the transcriptional profiles characterizing the neurons expressing the DA receptors in the NAc, the DS and the PFC. This study includes the results from the animals that served as control in the operant training for food, the yoked-standard group, as they didn't go through any operant training neither were influenced by the palatability of the food

Regional specificity of gene expression in dopaminoceptive neurons

Enrica Montalban 1,2,3 , Jean-Pierre Roussarie 4 , Lieng Taing 1,2,3 , Albert Giralt 1,2,3 , Yuki Nakamura 1,2,3 , Benoit de Pins 1,2,3 , Assunta Pelosi 1,2,3 , Jack Zhang 4 , Paul Greengard 4 , Jean-Antoine Girault 1,2,3,* ((this is an early draft of the manuscript and additional authors may be included and the authors order is not definitive))

1-Inserm UMR-S 839, Paris, 75005, France;

ABSTRACT

Each cell type is defined by its pattern of gene expression. Forebrain dopaminoceptive neurons play a key role in movement, action selection, and motivation and are dysregulated in addiction, Parkinson's disease and many other conditions. To investigate similarities and differences between the main types of neurons sensitive to dopamine we compared the full complement of translated mRNAs in neurons expressing D1 or D2 dopamine receptors in the dorsal striatum and nucleus accumbens and in prefrontal cortex D1 receptor-expressing neurons. As large difference was observed between D1-positive prefrontal and striatal neurons and the differences in mRNA profiles between D1 and D2 striatal projection neurons were further characterized disclosing similarities and differences between the dorsal and ventral striatum. Intrastriatal comparisons revealed the important differences between these two regions, some being common to D1 and D2 neurons others being specifically found in one cell population. Further analysis allowed the identification of potential upstream regulators, with prostaglandin E2 being a potential regulator in the two neuronal populations of the dorsal striatum. Chronic stimulation with a prostaglandin agonist improved performance of mice in dorsal striatum-dependent behaviors, supporting the functional significance of this pathway. This study provides a powerful resource for the molecular studies of the striatum and new clues about its regional organization.

INTRODUCTION

In multicellular organisms, differentiation depends on the acquisition by each cell type of different patterns of gene expression. Although these transcriptional profiles are stable, work on induced pluripotent stem cells demonstrated that it is largely reversible [START_REF] Yamanaka | Nuclear reprogramming to a pluripotent state by three approaches[END_REF].

Identifying the characteristic complement of genes expressed in a cell population allows to better understand its functional properties as well as its vulnerability to pathological conditions. The nervous system represents a major challenge in terms of cells diversity and the definition and number of different cell types is still an open question [START_REF] Sharpee | Toward functional classification of neuronal types[END_REF]. Neuromodulators such as dopamine exert their effects on large brain regions containing many cell types. Major targets of dopamine (DA) are the striatum and the prefrontal cortex. The importance of this innervation in physiology and in a wide variety of pathological conditions has been abundantly documented. Tonic DA is necessary for the correct function of its target regions due to the existence of high affinity receptors, while phasic increase is triggered by errors in reward prediction and related salient stimuli, and is critical for incentive learning [START_REF] Schultz | Multiple dopamine functions at different time courses[END_REF]. The absence of DA results in Parkinson's disease and its repeated increase by drugs of abuse is a key element leading to addiction. DA is also involved in many other conditions ranging from attention deficit disorder to schizophrenia. Although there are 5 types of dopamine receptors expressed at various levels in many cell types, the D1 and D2 dopamine receptors (DRD1 and DRD2) are the most abundant in the striatum and are also expressed at a much lesser level in the cerebral cortex [START_REF] Beaulieu | The physiology, signaling, and pharmacology of dopamine receptors[END_REF]). In the striatum, the expression of these two receptors is largely segregated with few neurons expressing both [START_REF] Valjent | Looking BAC at Striatal Signaling: Cell-Specific Analysis in New Transgenic Mice[END_REF]. These neuronal populations have different functional properties although they function in an integrated manner to shape behavior [START_REF] Tecuapetla | Complementary Contributions of Striatal Projection Pathways to Action Initiation and Execution[END_REF]. In the dorsal striatum (DS), DRD1 is expressed by striatal projection neurons (SPNs) forming to the direct pathway which monosynaptically innervates the substantia nigra pars compacta and internal medial) globus pallidus, whereas DRD2 is expressed in SPNs which form the first step of the indirect pathway (Gerfen et al., 1990). DRD2 is also expressed, albeit at a lower level, in cholinergic interneurons (Bertran-Gonzalez et al., 2008a). In the ventral striatum corresponding to the nucleus accumbens (NAc), the correspondence between the type of expressed DA receptor and the projection pattern of SPNs is more complex [START_REF] Kupchik | Coding the direct/indirect pathways by D1 and D2 receptors is not valid for accumbens projections[END_REF]. D1R-and D2R-expressing SPNs have been known for many years to specifically express other genes, such as those coding for substance P and enkephalin for example (Gerfen et al., 1990). Recent studies have shown that a large number of genes are also differentially expressed between the two cell types (Heiman et al., 2008;[START_REF] Doll | Dopaminergic genes predict individual differences in susceptibility to confirmation bias[END_REF]. However there are several additional levels of anatomical and functional heterogeneity within the striatum which are less well characterized. This heterogeneity includes the distinction between dorsal and ventral regions, the NAc being itself a highly heterogenous structure, as well as the distinction between the patches/striosomes and the matrix (Voorn et al., 2004). There is therefore a need for further characterize the subpopulations within the SPNs.

A number of approaches have been used to address the challenge of identifying the transcriptional profile of specific cell types including single cell PCR and, more recently, single cell RNAs sequencing [START_REF] Gokce | Cellular Taxonomy of the Mouse Striatum as Revealed by Single-Cell RNA-Seq[END_REF], as well as various methods for cell labeling and microdissection or sorting (Lobo et al., 2006;[START_REF] Ena | FACS array profiling identifies Ecto-5' nucleotidase as a striatopallidal neuron-specific gene involved in striatal-dependent learning[END_REF]. A particularly powerful approach to investigate the patterns of genes expressed in a specific cell population is the BAC-TRAP (bacterial artificial chromosome-translated RNA affinity purification) strategy (Doyle et al., 2008;Heiman et al., 2008). In this method transgenic mice express an eGFP fused L10a ribosomal protein under the control of cell-type specific promoter. GFP-immunoprecipitation allows the isolation of cell type specific polysomes and thus access to currently translated mRNAs or "translatome". This method coupled to microarrays has been applied to D1 and D2 SPNs and showed the different profiles between the two cell types. Here, we use BAC-TRAP to study the translatome in D1R-and D2Rexpressing cells of the DS and the NAc and the D1R-cells in the prefrontal cortex. We show the existence of major differences in the mRNA profile in DS and NAc, some being common to the D1 and D2 populations, others being specific. Analysis of the differences allowed the identification of potential common upstream regulators and we demonstrate the functional role of one of them suggested to be active on both D1 and D2 neurons of the DS.

METHODS

Animals

BAC (bacterial artificial chromosome) transgenic mice expressing the enhanced green fluorescent protein fused to the N-terminus of the large subunit ribosomal protein L10a under the control of dopamine D1a or D2 receptor promoter (Drd1a::EGFP-L10a or Drd2::EGFP-L10) were previously described (Heiman et al., 2008). These mice lines were maintained as heterozygotes on a C57Bl/6J background. They express eGFP-L10a in the striatum and in striatofugal fibers (Figure 1A andB) with a pattern consistent with the previously described expression in D1 and D2 SPNs (Bertran-Gonzalez et al., 2008b;Heiman et al., 2008). In the PFC eGFP-L10a expression in Drd1a::eGFP-L10a mice was sufficient for further analysis whereas it was not the case for Drd2::eGFP-L10 mice (Figure 1A andB).

Male C57Bl/6 mice were purchased from Janvier (France) and used at 10-12 weeks. Mice were maintained on a 12 h light/dark cycle (light off 7:00 pm) and had, before the beginning the

mRNA extraction

Cell specific translated-mRNA purification, was performed as described (Heiman et al., 2008) with some modifications. Each sample consisted of a pool of 2-3 mice. BAC-TRAP transgenic mice were sacrificed by decapitation. The brain was quickly dissected out placed in cold buffer and then in an ice-cold brain form to cut thick slices from which the PFC was obtained and the NAc and the DS punched out using ice-cold stainless steel cannulas (Figure 1C). Each tissue piece was homogenized in 1 ml of lysis buffer (20 mM HEPES KOH [pH 7.4], 5 mM MgCl 2 , 150 mM KCl, 0.5mM dithiothreitol, 100 µg/ml CHX protease and RNAse inhibitors) with successively loose and tight glass-glass 2 ml Dounce homogenizers. Each homogenate was centrifuged at 2000 x g, at 4°C, for 10 min. The supernatant was separated from cell debris, and supplemented with NP-40 (EDM Biosciences) to a final concentration of 1% (vol/vol? 10 ml/l??) and DHPC (Avanti Polar lipids) to a final concentration of 30 mM. After mixing and a 5-minute incubation on ice, the lysate was centrifuged for 10 minutes at 20,000 x g to separate the supernatant from the insolubilized material. A mixture of streptavidincoated magnetic beads were incubated biotinylated protein L and then with GFP antibody was added to the supernatant and incubated ON at 4°C with gentle end-over rotation. After incubation, beads were collected with a magnetic rack and washed 5 times with high-salt washing buffer (20 mM HEPES-KOH [pH 7.4], 5 mM MgCl 2 , 150 µl 1M, 350 mM KCl, 1% NP-40) and immediately placed in "RTL plus" buffer (Qiagen). The mRNA was purified using the RNase micro KIT (Qiagen).

Libraries and sequencing

Five ng of RNA were used for reverse-transcription, performed with the Ovation RNA-Seq System V2 (Nugen). cDNA was quantified by fluorimetry, using the Quant-iT Picogreen reagent and ultrasonicated using a Covaris S2 sonicator with the following parameters: duty cycle 10%, intensity 5, 100 cycles/burst, 5 minutes. Two hundred ng of sonicated cDNA were then used for library construction using the Illumina TruSeq RNA sample prep kit, starting at the End-Repair step, and following the manufacturer's instructions. The libraries were quantified with the Bio-Analyzer high-sensitivity DNA kit, multiplexed and sequenced on an Illumina HiSeq 2500 instrument. At least 20 million 50-bp paired-end reads were collected for each sample. Reads were then aligned to the Genome Reference Consortium Mouse Build 38 GRCm38 / UCSC mm10 mouse genome assembly. The DEseq algorithm was used to test for differential expression of each gene in each pair-wise comparison.

Bioinformatic analysis

The quality of the raw data were assessed using FastQC [Andrews] from the Babraham Institute for common issues including low quality of base calling, presence of adaptors among the sequenced reads or any other overrepresented sequences, and abnormal per base nucleotide percentage. The different libraries were then mapped to the Mus musculus genome (UCSC mm10) using TopHat2 2.0.9 [START_REF] Kim | TopHat2: Accurate Alignment of Transcriptomes in the Presence of Insertions, Deletions and Gene Fusions[END_REF]], a splice junction mapper, with a set of matching gene annotation (genes.gtf downloaded from UCSC on December 8 th 2015). Conservative options were used to keep only reliable levels of expression for each gene including the obligation to map only to one possible location for each read and taking only into account the paired reads mapped in proper pairs. The gene counting step was then done with HTSeq-counts v0.6.0 [START_REF] Anders | HTSeq--a Python framework to work with high-throughput sequencing data[END_REF]. The exons were chosen as the mapping features and the reads of the same pair had to be mapped to the opposite strands of the gene. Before statistical analysis each library was checked using principal component analysis and correlation matrix. Differentially expressed genes were identified with R using the Bioconductor package DESeq2 v1.10.1 (Love et al., 2014) taking advantage of its capacity to perform multi-factor analysis. Genes with adjusted p-value less than 5 %, with false discovery rate [START_REF] Benjamini | Controlling the false discovery rate: a practical and powerful approach to multiple testing[END_REF] were declared differentially expressed. Gene ontology (GO) analysis was performed on a list of significantly differentially expressed genes (adj p<0.05) identified with DESeq2. The cluster Profiler v3.0.4 [START_REF] Yu | 1. Abbrev.: FC, fold-change, GPCR, G protein-coupled receptor, IC, ion channel, IUPHAR, International Union of Basic and Clinical Pharmacology, LGIC, ligand-gated ion channel, MGI, mouse genome informatics database symbol, NHR, nuclear hormone receptor, padj, adjusted[END_REF]package from Bioconductor was used. Overrepresented GO were identified using GSEA method. Conservative options were used to filter results (Bonferroni p value adjustment and 0.01 p-value threshold). We tested 3 sets of genes of interest for each comparison, the differentially expressed and the overexpressed for each condition. Each set of genes was compared with all known genes present in the annotation. The GO categories were found in org.Mm.eg.db [Carlson] Bioconductor package base on the gene reporter, official gene symbol.

Total RNA purification and cDNA preparation

Each sample consisted of the tissue deriving from one mouse brain prepared as described above.

Mice were sacrificed by cervical dislocation, the PFC was quickly dissected out and microdisks punched out from the NAc and the DS with a stainless steel cannula and placed on ice. Each tissue sample was homogenized in TRIzol with loose and tight glass-glass 2 ml Dounce homogenizers. Total RNA was extracted with TRIzol Reagent (Life Technologies) according to manufacturer's instructions.

The RNA was quantified by using the NanDrop 1000 spectrophotometer and its integrity checked with the Bionalyzer (agilent 2100 Bioanalyzer, Agilent RNA 6000 nano kit). Five hundred ng of mRNA from each sample were used for retro-transcription, performed with the Reverse Transcriptase II (Life Technologies) following the manufacturer's instructions.

Real-Time PCR

Quantitative real time PCR, was performed using SYBR Green PCR kit in 96-well plates according to the manufacturer's instructions. Results are presented as normalized to the indicated house-keeping genes and the delta-CT method was used to obtain a FC.

RESULTS

Comparison of polysomes-associated mRNA in D1 neurons of the prefrontal cortex and the striatum

The overall experimental approach is summarized in Figure 1C and D. Translated mRNA profiles were obtained from D1R-expressing neurons in the NAc, DS, and PFC, and from D2R-expressing neurons in the NAc and DS. Note that all the data were analyzed together and different comparisons are presented separately for clarity. We first compared the genes differentially expressed between D1 neurons of the PFC and those in the DS (Figure 2A) and in the NAc (Figure 2B, the full results are provided as Supplementary Table 1). Overall 2 942 genes were significantly more expressed in the PFC than in the DS and 2 872 in the DS than in the PFC (Figure 2C). For the NAc these numbers were 2 819 and 2 863 respectively (Figure 2D). Among all the genes more expressed in the DS or NAc than in the PFC, 1 787 were common (49%). Similarly, among those more expressed in the PFC than in the DS or NAc 2 182 were common (54%). This high degree of overlap between DS and NAc in the comparison is not surprising since both are GABAergic projection neurons whereas PFC D1Rexpressing neurons are for a large part, albeit not only, glutamatergic pyramidal neurons [START_REF] Smiley | D1 dopamine receptor immunoreactivity in human and monkey cerebral cortex: predominant and extrasynaptic localization in dendritic spines[END_REF]. However the differences suggested the existence of pronounced differences between the complement of genes expressed in the dorsal and ventral striatum (see below). The genes included in part or totality in the Drd1 BAC (Drd1, Sfxn1, and Hrh2) and in the Drd2 BAC (Drd2, GM4894, Ankk 1, and Ttc12) used for transgenesis were excluded from further analysis, since their expression levels did not reflect that of the endogenous gene. To obtain a first glimpse about the biological significance of these differences we selected the differentially expressed genes which match the IUPHAR list of genes (http://www.guidetopharmacology.org/download.jsp) including receptors, enzymes, channels, and transporters that are directly relevant for neuronal physiology or pharmacology (Tables 1 and2). Only genes with the strongest expression differences, i.e. four-fold or higher, are shown in these tables (the full list can be found in Supplementary Table 2). As expected mRNAs known to be enriched in SPNs as compared to glutamatergic neurons were identified (e.g., glutamic acid decarboxylase 2, GAD2, adenylyl-cyclase 5, Adcy5, adenosine receptor A2A, Adora2a, m4 muscarinic acetylcholine receptor, Chrm4). Examples of genes highly enriched in D1 neurons of the DS and/or NAc as compared to those of the PFC included several phosphodiesterases (Pde1b, Pde1c, Pde7b, Pdb10a), adenosine receptors (Adora2a and 2b), Drd3 dopamine receptor, several orphan receptors (Gpr6,55,83,88,139,and 149), retinoic acid receptors (Rarb, Rxrg), and the regulators of G protein signaling Rgs4 and 9. Interestingly some genes not usually associated with brain function and with relatively low expression were highly enriched in the striatum including interleukin receptors for IL-10 and IL-17 (about 20-fold enrichment) and the tyrosine kinase MuSK (>100-fold enrichment). Conversely, genes associated with glutamate transmission (e.g., vGluT1, slc17a7) were enriched in cortical neurons, as expected. Other highly cortically-enriched genes included those for GDNF and EGF receptors (Gfra2 and Egfr), adenosine receptors 1 (Adora1a and Adora1b), dopamine receptor 5 (Drd5), metabotropic glutamate receptor 2 (Grm2), nicotinic acetylcholine receptor α5 (Chrna5), as examples of potential pharmacologically relevant targets. The most significant over-represented gene ontologies (GO) included many related to synaptic and membrane regulation or function in the striatum and to neural development and axon extension in the PFC (Table 3, the full list of significantly over-represented GO are in Supplementary Table 3).

Comparison of polysomes-associated mRNA in D1 and D2 striatal neurons

Several publications have previously achieved global comparisons between genes expressed in striatal D1 and D2 neurons (Lobo et al., 2006;Heiman et al., 2008;[START_REF] Ena | FACS array profiling identifies Ecto-5' nucleotidase as a striatopallidal neuron-specific gene involved in striatal-dependent learning[END_REF][START_REF] Heiman | Molecular adaptations of striatal spiny projection neurons during levodopa-induced dyskinesia[END_REF]. Here we could examined the differences between D1 and D2 neurons separately in the DS and in the NAc (Figure 3A andB). In the DS 443 genes were significantly more expressed in D1 neurons and 308 in D2 neurons (Figure 3C, complete list for DS in Supplementary Table 4). The most significantly differently expressed genes in the DS are shown in Table 4 (higher in D1) and Table 5 (higher in D2). The differences were not as pronounced as between striatum and PFC both in terms of number of significant genes and fold-change. The significant differences in relation with IUPHAR identified genes are shown in Tables 6 and7 (complete list in Supplementary Table 5). Pronounced enrichments in D1 neurons were found for abundantly expressed genes such as Chrm4, and Tac1, as expected. Interestingly less expressed genes highly enriched in D1 neurons included homeobox protein Arx (10-fold enrichment in D1) and integrin-α8 (7-fold) which may have potential significance for their differentiation. The genes most enriched in D2 neurons in the DS included known markers of these neurons, such as Adora2a, 5'-ectonucleotidase (Nt5e), preproenkephalin (Penk). Other genes included a number with potential pharmacological interest such as purinergic receptor P2ry1, opiate receptor Oprd1, several orphan receptors (Gpr6 and Gpr52), and the receptor tyrosine phosphatase RPTPµ (Ptprm).

In the NAc 456 genes were significantly more expressed in D1 neurons and 640 in D2 neurons (Figure 3D, complete list for DS in Supplementary Table 6). The top significant enrichments in D1 and D2 neurons are indicated in Tables 8 and9 respectively. The most significantly highly expressed genes in D1 SPNs of the NAc included known markers of these neurons such as Pdyn, Drd1, Chrm4, and Tac1, as well as the phosphatase Eya1, the G protein subunit β4 (Gnb4). Other genes with lower mRNA levels but with predominant expression in D1 neurons of the NAc and potential pharmacological interest included GDNF receptor Gfra1, Drd3, the somatostatin receptor Ssrtr4, and the muscarinic acetylcholine receptor Chrm5. In D2 neurons of the NAc, a high enrichment of Drd2, P2ry1, Penk, Gpr52, Nt5e, Adora2a, Oprd1, and Ptprm was observed as in the DS. When comparing the D1 and D2 neurons, more genes appeared to display a high fold-increase in D2 than in D1 neurons, possibly reflecting a more homogenous phenotype among the D2 SPNs in the NAc. Overall comparison between DS and NAc indicated the existence of common differences between D1 and D2 neurons in the two regions but also pointed to many specificities, with 20-45% common genes identified (Figure 3E andF). These values are only indicative since the number of differences which reach the significance threshold depends on the size of the samples. Nevertheless they underline the differences that exist betwee the two man regions of the striatum. In this context it is noticeable that neither in the DS nor in the NAc did we find a pronounced enrichment of messengers coding for acetylcholine metabolism. Choline acetyl-transferase and choline transporter Slc5a7 were enriched 7-to 15-fold in D2 neurons but found at low levels (<150 reads and <270 reads, respectively). This indicated that with the BAC transgenic mice expressing L10a-eGFP directly under the control of the Drd2 receptor, the activity of the promoter is relatively low in cholinergic interneurons and together with the low abundance of these neurons as compared to SPNs, their contribution to the extracted mRNAs is negligible.

Comparison of polysomes-associated mRNAs in the dorsal and ventral striatum

The comparisons between PFC and DS and NAc, as well as the D1/D2 comparisons in the DS and NAc showed many similarities but also numerous differences, strongly indicating that the complement of genes expressed and translated in these two regions of the striatum are different, as anticipated from the abundant literature emphasizing the differences between dorsal and ventral striatum. We first carried out a global analysis using a multifactorial approach, taking onto consideration all striatal neurons analyzed to evaluate the transcriptional differences between the dorsal and ventral striatum (Tables 12 and13, Supplementary Table 8). This comparison indicated that 1838 mRNAs were found at significantly different levels between DS and NAc. Among these, 924 were higher in the DS and 914 in the NAc. We then conducted a separate analysis in D1 and D2 neurons (Figures 4A andB). A higher proportion of differentially expressed genes was found in the D1 neurons, possibly reflecting the lower size of the D2 sample (Figure 4C andD). In D1 neurons the cannabinoid receptor 1 (Cnr1) was among the most significantly highly expressed in the DS (Tables 14151617, Supplementary Table 9).

Other genes with a high degree of enrichment and fair expression level included in the DS neurogranin (Nrgn), sphingosine-1-P phosphatase (Sgpp2), an activin receptor (Acvr1c), and Gpr155 (Table 14 and16), and in the NAc Peg10, Stard5 Dlk1, and cGMP-dependent protein kinase 1 (Prkg1) (Table 15 and18). Integrin α8, which was found to be enriched in D1 (see above) was also more abundant in the DS as compared to the NAc (Table 16). In the D2 neurons, many genes were also differently expressed, including Acvr1c and Cnr1 (Tables 18192021). The genes relatively enriched in DS included Cnr1, Acvr1c, synaptopodin 2 (Synpo2), and reelin (Reln), while in the NAc the enriched genes included CART (Cartpt), diacylglycerol kappa (dgkk), and Kv channel interacting protein 1 (Kcnip1). Comparison between D1 and D2 neurons indicated that many of the differences between dorsal and ventral striatum were common in the two types of neurons (Figure 4E andF).

Validation of the observed regional differences and comparison with other approaches

To assess the validity of the differences observed with BAC-TRAP and RNA sequencing we selected a few genes and carried out reverse transcription followed by real time Q-PCR. Focusing first on genes enriched in the PFC as compared to the striatum we confirmed that Tbr1 which was highly enriched in the PFC with RNAseq (5.2-fold apparent enrichment as compared to DS) was indeed clearly more expressed in the PFC using RT-PCR (about 20-fold, Figure 5A). We similarly verified a few other differences with smaller fold-change differences, Ppp2r2b (1.4-fold according to RNAseq), Shank2

(1.7), or lower apparent expression, Tac2 (13.5-fold). All these were confirmed by RT-PCR (Figure 5A).

It should eb emphasized that the amplitude of the differences could differ between RNAseq from BAC-TRAP and RT-PCR for multiple reasons including the selection of cell specific mRNA and the focus on translated mRNAs in BAC-TRAP but not in our verification experiments. We then examined the corresponding in situ hybridization patterns available at the Allen Brain Institute (http://mouse.brainmap.org/). For the strong quantitative differences that we observed the hybridization differences were striking (e.g. Tbr1, Figure 5B), whereas for the less pronounced ones the RNAseq and RT-PCR were clearly more informative (Figure 5A andB).

We then investigated some typical genes that were different between the DS and NAc according to the BAC-TRAP experiments. We looked at some genes for which BAC-TRAP/RNAseq indicated a high degree of enrichment in the NAc as compared to the DS: Dlk1 (12.5-fold enrichment in NAc vs DS), Drd3 (4-fold), and Arhgap36 (3.8-fold). The differences observed with RT-PCR were consistently much larger (8-to 23-fold higher in NAc, Figure 5C). For genes with smaller apparent differences in RNAseq, Wfs1 (2.5-fold), Ahi1 (2.4-fold) or Gda (2.4-fold), RT-PCR also confirmed the enrichment with enrichment ranging from 2-to 4-fold (Figure 5C). Comparison with Allen Brain Atlas in situ hybridization showed that differences were visually detectable in all cases.

In the DS we investigated Hpca (hippocalcin, 2-fold enriched in DS), ATP2b1 (plasma membrane ATP-dependent Ca 2+ transporter 1, 2.5-fold), Slc24a2 (plasma membrane Na + /K + /Ca 2+ exchanger 2, 2.7-fold), and phosphodiesterase 10a (Pde10a, 1.9-fold). RT-PCR confirmed the mRNA enrichment in DS as compared to NAc (Figure 5E). The differences were also on in situ hybridization for Slc24a2 but not for the others, underlining the interest of the sequencing approaches.

Putative upstream regulators of dorso-ventral striatal gene expression

We next used the Ingenuity Pathway analysis (IPA) to identify putative upstream transcriptional regulators that could explain the observed differences in mRNAs between NAc and DS. We carried out this analysis in the comparison of the DS-NAc within both the D1 and D2 SPNs (Figure 6A andB).

This approach identifies pharmacological or toxic agents as well as endogenous compounds. For example the analysis suggested the possible role of transcription factors such as PR Domain 8 (PRMD8) in D2-SPNs and Forkhead box protein O1 (FOXO1) in D1-SPNs in DS (Figure 6 A andB). To provide a testable hypothesis for the gene regulatory networks possibly commonly involved in the two types of SPNs, we looked for common regulators between these two populations. Prostaglandin 2 (PGE2) appeared as a suitable candidate among the different regulators since it appeared to regulate a set of genes in both D1-and D2-SPNs in the DS (Figure 6C). Although few studies have investigated the role of prostaglandins in the striatum outside their involvement in inflammatory conditions, it was reported that PGE2 acting through prostaglandin 1 receptor (EP1) could amplify both D1 and D2 signaling [START_REF] Kitaoka | Prostaglandin E2 acts on EP1 receptor and amplifies both dopamine D1 and D2 receptor signaling in the striatum[END_REF]. We therefore decided to investigate further the role of this pathway.

Evaluation of the possible role of prostaglandins as a regulator of dorsal striatum function

To evaluate the possible effects of PGE2 as an upstream regulator we tested the behavioral effect of the chronic treatment with a non-selective agonist of PGE2 receptors that can be used parenterally misoprostol. Twelve week-old wild type mice were intra-peritoneally implanted with an osmotic mini-pump system to chronically administrate either misoprostol (50 μg/kg/day) or vehicle (Figure 7A). The mice were then subjected to several behavioral tests and were killed after 4 weeks for mRNA analysis. ((The mRNA studies are in progress at the time of the writing of this draft)).

Behavioral tests explored dorsal striatal functions.

In the open field mice showed a decreased locomotor activity after 6 days of misopristol treatment but no difference in the time spent in the center of the field (Figure 7B andC). Then, we tested motor coordination and learning using the rotarod task. Using successive trials of accelerating rotarod task there was no apparent differences in the ability of the two groups of mice to improve their performance (Figure 7 D). In contrast the use of two challenging fixed speeds once the task was acquired, 16 and 24 rpm, revealed differences between misopristol-and vehicle-treated mice. At 16 rpm the misoprostol group fell approximately twice less than the vehicle-treated mice but this difference did not reach the significance threshold. At 24 rpm the difference between the two groups was significant at 24 rpm indicating a better motor coordination in the misoprostol-treated group.

Finally, the two groups were subjected to a behavioral paradigm that evaluates striatum-dependent memory. The two groups of mice were first mildly food-deprived to 90 % of their weight and their ability to find the food-reinforced arm was evaluated in a Y maze during 10 consecutive sessions. In each group half of the mice were assigned to the right arm and the other half to the left arm. A score of 1 was assigned when the mice entered the reinforced arm and of 0 when they entered the nonreinforced arm. Results are plotted as averages of blocks of 10 trials. The mice reached about 80% of correct choices after 3 days of training, with no significant difference detected between the two groups (Figure 7G). Twenty-four hours after the training session, the mice were challenged in a reversal learning test. The habitual reinforced arm was systematically exchanged with the nonreinforced arm. The mice chronically treated with misoprostol were significantly faster in reversing the established memory and learning the new reward position (Figure 7G). These results indicated an improvement of misopristol-treated mice as compared to vehicle-treated controls in several tests that depend on dorsal striatum function.

DISCUSSION

This manuscript reports the first comprehensive study of the genome-wide study of translated mRNAs expressed in the main forebrain cellular populations expressing either D1 or D2 dopamine receptors, including a differentiation between the DS and the NAc. As expected the most striking differences were identified between PFC D1 receptor-expressing neurons and D1 SPNs. We provide a region-specific comparison of genes differentially expressed in D1 and D2 neurons, showing that there exist important variances between the DS and the NAc for the D1 D2 differences. We further characterize the important differences between these two striatal regions showing that they are comparable in amplitude to the differences between the D1 and D2 populations. We show that our data about the regional differences are supported by replication by RT-PCR experiments on total mRNA from wild type mice. Finally we identify PGE2 as a putative upstream regulator of genes expressed in the dorsal striatum, and we provide experimental functional evidence in support of this hypothesis.

The differences in gene expression between D1 and D2 SPNs have already been explored by BAC-TRAP and microarrays (Heiman et al., 2008;[START_REF] Heiman | Molecular adaptations of striatal spiny projection neurons during levodopa-induced dyskinesia[END_REF]. Our current study extends these findings in two ways. First the use of RNA-sequencing instead of microarrays increased the sensitivity of the approach, as indicated by the fact that it led to the identification of 2-6 times more significant differences depending on the region and population differences. Second it allowed to approach the comparison between the two types of SPNs on a regional basis, underlining the marked differences between the DS and the NAc. In a very recent paper [START_REF] Gokce | Cellular Taxonomy of the Mouse Striatum as Revealed by Single-Cell RNA-Seq[END_REF] the authors used single-cell RNA sequencing to study the whole striatal cell diversity. This paper confirmed most of the data already reported in literature, and showed that the D1 and the D2 neurons could be divided in 2 additional subpopulations that express a gradient of transcriptional states that could be related to the patch matrix organization of the striatum. Interestingly, the genes that the authors have chosen as defining the opposite gradient of expression in the two SPNs populations correspond to some of the genes that we identified as highly enriched in the NAc (Wfs1-Crym) or the DS (Cnr1). Further analysis will clearly be needed to determine whether the gradient observed in this paper correlates with the patch-matrix organization or the dorso-ventral gradient.

In relation with the anatomical organization of the inputs converging to the striatum and on the basis of multiple functional studies, the NAc has been associated to the motivation-related processes, while the DS is implicated in motor behavior, associative learning, and habits formation [START_REF] Corbit | Learning and Motivational Processes Contributing to Pavlovian-Instrumental Transfer and Their Neural Bases: Dopamine and Beyond[END_REF]. To evaluate whether the gene expression differences identified in the present study had some functional significance, we looked for potential upstream regulators common to several genes. Of course it is extremely unlikely that a single pathway could account for all the observed regional differences. Nevertheless we were intrigued by the potential role of PGE2, which was the only compound predicted to be a possible positive regulator of the genes expressed in both D1 and D2 neurons in the DS. Therefore, we chose it as possible target to study the effects of its manipulation on striatal function. Prostaglandins (PGs) are a family of lipid mediators involved in a plethora of processes including vascular homeostasis, inflammation, and reproduction (Narumiya et al., 1999). Although little is known about the role of PGs in general and of PGE2 in the striatum, it has been previously shown that PGE2 amplifies both the D1 and D2 signaling pathways [START_REF] Kitaoka | Prostaglandin E2 acts on EP1 receptor and amplifies both dopamine D1 and D2 receptor signaling in the striatum[END_REF]. Here we tested the effects of a chronic infusion of a non-selective agonist of PGE2 receptors that could be administered by osmopump. The treated mice displayed an improved performance in several behavioral tasks that are related with the dorsal striatum, including the time on rotarod and in reversal learning. This result supports the hypothesis that PGs, and possibly PGE2, are regulators of dorsal striatal function. Work in progress addresses the effects of misopristol on transcription of putative target genes and the pharmacological specificity of the effects.

In conclusion this work provides an extensive characterization of the translated mRNAs in the two main populations of striatal projection neurons, comparing them to D1-positive cortical neurons and unraveling the differences between the dorsal and ventral striatum. Our data should provide a useful resource for any further analyses of genes expressed in the striatum and should help the interpretation of results from other approaches, including single cell sequencing. In addition our results allowed the identification of PGE2 as an important regulator of dorsal striatum function. A number of genes were selected from the regional BAC-TRAP analysis for verification with independent methods. mRNA levels were studied by qRT-PCR in wild type mice. A. mRNAs analysis by qTR-PCR of gene products expressed at higher levels in the PFC than in the DS. The expression levels were calculated by the comparative DDCt method and expressed relative to the DS; β-actin was used as internal control. Data are means + SEM from 6 independent experiments. Statistical In conclusion, in the first part of my thesis we provide an overall characterization of the genes expressed, or more correctly the polysomes-associated mRNAs, in the D1 neurons of the prefrontal cortex and of the striatum. We then showed the differences between D1 and D2 SPNs separately in the dorsal striatum and the nucleus accumbens. Finally we characterized the important differences between the neurons of the dorsal and ventral striatum, in both the D1 and the D2 populations.

These results provide a thorough characterization of the "translatome" in D1 and D2

striatal neurons with the first investigation of their regional differences. They should provide a strong background for future studies and set the stage for the functional investigations in the next part of the thesis -Experimental results -7

The two foods are similar in shape and calories (ST=3.30 kcal/g, HP=3.48 kcal/g); however the highly palatable food is contains chocolate flavour and 50% of the carbohydrates are substituted by sucrose. During the 15 days of conditioning the mice pass trough two different phases: a learning phase, in which animals are kept under caloric restriction (9 days), and an ad libitum phase tin which the mice are fed ad libitum in their home cage (Figure 16-A). During this last phase we considered that mice had learned the task and we expected that only the mice motivated by the food palatability kept working in spite of the cessation of calories need. The behavioural results showed that mice working for the highly palatable food performed an increased number of positive pokes, obtained rewards, and consumed pellets already during the restriction phase. In these mice, the nose pokes in the active hole were maintained in the ad libitum phase and higher than the standard food group (Figure 16 B-D). As expected yoked mice did not show any learning, however, the amount of consumed pellets was comparable to the master group.

-Experimental results -7 Obesity is due to the accumulation of excess body fat occurring when energy intake exceeds that expended. This is a normal adaptive mechanism to variable food availability that allows storage in periods of abundance. Repeated exposure to palatable food can disrupt appetite regulation and it has been shown that the daily consumption of highly palatable and caloric food can become a habit that leads to develop obesity (Jarosz P.A., 2007). Due to the differences observed in operant training, we examined how mice exposed to the two types of food gained weight even though highly palatable food was almost isocaloric with the standard food. Two cohorts of wild type (WT) mice were randomly assigned to two groups. One group had access only to standard food while the other one had free access to both highly palatable and standard food. Mice weight was measured every 3 days over 30 days.

The mice having free access to highly palatable food ate more and gained a significantly more weight compared to the mice exposed to the standard food (Figure 17). This result suggests that the operant paradigm can provide information about the possible instauration of obesity with highly palatable, almost isocaloric food.

7 -Experimental results -120 7.2.3 Highly palatable food increases spines gain in PFC,

NAc and DS

The instauration and the maintenance of a learned behaviour are likely to modify the structural plasticity in the brain regions that serve as substrate of this behaviour.

Indeed, it has been already shown that a long period of access to both cocaine [START_REF] Lee | Cocaine-Induced Dendritic Spine Formation in D1 and D2 Dopamine Receptor-Containing Medium Spiny Neurons in Nucleus Accumbens[END_REF] and highly palatable food (Guegan T et al., 2012) has an effect on the structural plasticity of selected regions of the reward circuit. We measured the structural plasticity induced in the PFC and striatum by operant conditioning or noncontingent exposure to the two types of foods. WT mice were trained in the operant conditioning paradigm for 15 days and killed 24 h after the last training session. The brains were quickly dissected and stained with a Golgi-Cox solution. Spine density was measured in the layer five of PFC, NAc and DS in master and yoked mice trained with the two types of food. The analysis of spine density in both master and yoked group allowed separating the effect of conditioning from the effect of the simple exposure to the food (Figure 18). Our results show that the highly palatable food itself or the learning for the highly palatable food -but not regular or learning for regular food -had an effect on structural plasticity. Spine number was not changed in mice conditioned for standard food as compared to the yoked mice receiving the same food. In contrast, operant conditioning for highly palatable food increased the spine number in all the three regions analysed, whereas in yoked mice receiving highly palatable food this number was only increased in NAc. These results show that operant conditioning for highly palatable food specifically increases spine density in the PFC and DS, whereas the mere availability of highly palatable food is sufficient to increase the spine number in the NAc, independently of active conditioning. Different studies have shown that palatable food and associated cues lead to an increase in DA release in the NAc [START_REF] Hernandez | Food Reward and Cocaine Increase Extracellular Dopamine in the Nucleus Accumbens as Measured by Microdialysis[END_REF][START_REF] Hajnal | Accumbens Dopamine Mechanisms in Sucrose Intake[END_REF] and DS (Small DM, 2003).

-

Effect of a specific treatment across different regions

We took advantage of the different groups, regions, and neuronal populations to analyse the effect of a specific treatment in regulating the transcription of the same set of genes in different regions. Concerning the learning for HP food, we found that a large set of genes is regulated in the D2 neurons of both the NAc and DS (420 genes, nominal p-value <0.05, FC>1.3 or <0.7, >30 reads at least in one condition).

Interestingly, among the 420 genes, only 29 genes are regulated in the same direction in the 2 regions, all the rest being regulated in a different fashion. To gain insight into the function of this opposite regulation, we performed a GO analysis of the different groups of genes. Among the oppositely regulated genes we found an overrepresentation of mRNAs linked to neuronal spines (Adj p=0.008), postsynapse (Adj p=2.88^10-6), postsynaptic membrane (Adj p=0.00073), synaptic membranes (Adj p=0.0010) axon (Adj p=0.001), and GABA-A receptor complex (Adj p=0.009).

Of note, we observed the completely opposite result when comparing the effects of learning for the ST food in the D2 neurons in the NAc and DS. Only 15 regulated genes were common between the 2 regions and all were regulated in the same direction. Interestingly, the gene ontology on this group of genes showed an overrepresentation of mRNAs potentially implicated in the "transcriptional activator activity" (Adj p=0.007). In the D1 neurons, we found only two genes commonly regulated by the HP food conditioning in the DS and NAc: the actin nucleator Pkib, and G protein-coupled receptor 107, both up-regulated by learning (i.e. they were not changed in yoked mice). According to our cut-off, conditioning for standard food regulated only 1 gene in the D1 neurons of the DS and NAc: the Ext 2 exostosin glycosyltransferase. This gene encodes one of two glycosyltransferases involved in the chain elongation step of heparan sulfate biosynthesis. Interestingly, we found 3 mRNAs commonly up-regulated by conditioning for ST food in the D1 neurons of the NAc, DS and PFC: the intellectual disability-associated hivep2 (human immunodeficiency virus type I enhancer binding protein 2), Kcnv1 (potassium channel, subfamily V, member 1), and the brain specific protein Ank2 (ankyrin 2). In some of the regions, the increase in mRNA expression was only around 20% (FC=1.20). Two of these genes are already known to be regulated in relation with the reward system: Hivep2 has been already shown to be regulated by cocaine selfadministration (Reynolds et al. 2006) and there is evidence supporting the 7 -Experimental results -126 involvement of Ank2 in cocaine-induced locomotion (Scotland P., 1998). Finally two genes regulated by conditioning for HP food were common between the 3 regions: Pkib, up-regulated in the 3 regions and Syndigin1L (synapse differentiation inducing-1-like), down-regulated in DS and NAc and up-regulated in PFC.

This preliminary analysis of our results shows that the conditioning for HP food and for ST food exerts different effects on transcription depending on the neuronal type and the regional location of the neurons. In D2 neurons, the HP foodrelated learning induces a major opposite regulation of the transcriptional profiles of the NAc and DS. In D1 neurons, in agreement with the lower amount of changes induced by the operant training, we found much less common genes in the different region. When in common the genes were always regulated in the same fashion in the NAc and DS. Importantly, we also identified 3 genes commonly regulated by learning for the ST food in the NAc, DS and PFC, while only 2 genes were found in common in the 3 regions after learning for the HP food.

Effect of a specific treatment across different regions

We reasoned that by comparing the effect of conditioning for ST and HP food in the same region, we could gain insight on the genes that may be generally regulated by operant conditioning in the specific region and type of neurons. This analysis is not fully completed yet. However, we already have some information. In the D2 NAc, conditioning for HP and ST food had an effect on the transcription of 80 common genes. Among those, only 19 were regulated in the same direction, while the majority were regulated in different fashion by the 2 types of food. 34 mRNAs HP food compared to the yoked control group (Figure 20C). In addition, we measured norbin protein levels in the DS of WT mice trained to obtain highly palatable food. The levels of norbin were increased in the DS of mice conditioned for HP food when compared to the yoked group (Figure 20D). These results confirmed the results of RNA-seq in the DS and showed that mRNA alterations had consequences on protein levels.

-Experimental results -7 a progressive ratio schedule to obtain food. Our results showed that the deletion of the Ncdn exert differential regulation of learning depending on the different phases of the paradigm (Fig. 7B). During the food restricted learning phase, the mice lacking Ncdn learned faster to obtain HP food as compared to their Cre-negative controls, as demonstrated by the increase in positive pokes and consumed pellets (Fig. 7B). Interestingly, this difference was inverted when the mice were switched to the ad libitum phase. During this last phase the norbin mutant mice showed a more pronounced decrease in the positive pokes and consumed pellets as compared to their WT control (Fig. 21B andC).

We then examined the behavioural consequences of the mutation after the conditioning period using a progressive ratio schedule (Fig. 21C). The norbin mutant mice were willing to work significantly less to obtain HP pellets, as compared to their WT littermates. These results indicate that in the absence of norbin, the persistently increased apparent motivation to obtain HP food was not acquired.

-Experimental results -7

In conclusion, in the second part of this work we used a combination of behavioural and genome-wide approach to study the effects of the operant learning for standard and highly palatable food in D1 and D2 SPNs of the NAc and DS and in D1 pyramidal neurons of the PFC. The preliminary analysis of our results allowed to define the regions (NAc and DS), the neurons (D2 SPNs) and the genes more responsive to the highly palatable food. The in vivo manipulation of one of the genes (Ncdn) differentially regulated by the training for the highly palatable food allowed a first validation of part of the findings of our study. However, a lot should still be done with the results we obtained. A validation by independent techniques of some target genes that we have found as regulated in the different conditions is in progress.

In parallel, a systems biology approach to explore the transcriptional synchrony induced by food conditioning would give a reasonable indication of the overall mechanisms that are taking place during conditioning for highly palatable food, as well as of the targets that could be fundamental to the loss of the eating control. The clusterization of the genes would allow measuring if the connectivity of certain clusters is affected by a certain treatment compared to the control, as well as to find the hub of cluster, and modify its expression.

Aim

PFC

Early investigations established that a robust form of neuronal plasticity associated with repeated psychostimulant administration is a long-lasting restructuring of dendritic spines in components of the brain reward circuits [START_REF] Robinson | Persistent Structural Modifications in Nucleus Accumbens and Prefrontal Cortex Neurons Produced by Previous Experience with Amphetamine[END_REF]. We checked whether our protocol of chronic cocaine treatment was able to induce structural plasticity by Golgi staining in NAc, DS, and PFC of mice i.p. with cocaine or vehicle for 7 days, and sacrificed 24 h after the last injection. This chronic cocaine regimen induced different types of morphological alterations in three different regions. In the NAc, chronic cocaine increased spine density and spine head area, without altering neck length. (Fig. 22A-D). In the DS chronic cocaine increased spine neck length, without altering spine density or head area (Fig. 23A-D). In the PFC, repeated cocaine did not significantly change the spine density or spine head surface (Fig. 24A-C). It tended to increase spine neck length, without reaching the statistical significance (Fig. 24D).

-Experimental results -7 developmental processes, possibly linked withcocaine in the plasticity. The GO on the totality of the genes altered by cocaine (i.e. positively or negatively) showed an enrichment in genes linked to "axon", "neuron projection", "dendrite-membrane", and " actin skeleton" indicating an involvement of genes possibly linked with plasticity (Fig. 26B).

In NAc D2 SPNs, the number of genes changing in response to cocaine -72was smaller than the D1 SPNs. However, the regulation exerted by the cocaine administration was apparently stronger when comparing the fold of expression. As for the D1 neurons we found genes already reported as associated to cocaine. Among others, we found the kinase domain-containing 1, Ankk1, a gene closely linked to the Drd2 gene, that has been already connected to the susceptibility to addiction (Bao Zhu- [START_REF] Yang | Haplotypic Variants in DRD2, ANKK1, TTC12, and NCAM1 Are Associated with Comorbid Alcohol and Drug Dependence[END_REF] and to the subjective modulation of the acute effects of cocaine in humans (C.J. Soellicy; 2004). Interestingly, we found two genes coregulated regulated by cocaine in D1 and D2 NAc: Ret (ret proto-oncogene), down regulated in both neuronal populations, and Lpl (lipoprotein lipase) up in D2 and down in D1 (Fig. 26D).

-Experimental results -7

Interestingly, with our p-value cut-off (adj p-value <0.05), we did not find any cocaine-induced changes in the DS. By moving the cut-off to a less stringent p-value criteria (nominal p-value<0.05) (Figure 27) we observed that cocaine tended to increase the expression of 422 genes and to decrease the expression 531 in D1 SPNs PFC of cocaine abusers showed that cocaine triggers the activation of several genes in this region (Lehrmann E., 2003). However a clear information about the genes regulated in the DA-expressing receptors neurons in the PFC is still missing. Using BAC-TRAP to identify translated mRNA from D1R-expressing neurons in the PFC, we identified 48 genes differentially regulated by cocaine (Fig. 28A). There were 46 up-regulatd genes and 29 down-regulated ones. The GO analysis on the genes differentially regulated by cocaine showed enrichment in genes involved in "postsynapse", "postsynaptic density" and "postsynaptic specialization" (Fig. 28B).

By crossing this data set with the data obtained in NAc D1 and D2 SPNs, we found only one gene commonly regulated by cocaine between PFC and NAc D1, Stx1a

(coding for Syntaxin 1A, Fig. 28C), and 2 genes commonly regulated by cocaine in NAc-D2 and PFC (Pcid2, PCI domain containing 2, and Ntng1, netrin G1, Fig. 28D).

Coexpression analysis identifies modules of genes clustering in response to the cocaine treatment

Several studies suggest that gene coexpression analysis is useful for identifying transcriptional alterations in disorders whose phenotype is characterized by the orchestrated alteration of numerous small changes rather than from isolated single gene effects [START_REF] Ghazalpour | Integrating Genetic and Network Analysis to Characterize Genes Related to Mouse Weight[END_REF][START_REF] Gaiteri | Beyond Modules and Hubs: The Potential of Gene Coexpression Networks for Investigating Molecular Mechanisms of Complex Brain Disorders[END_REF]. Having identified the broad patterns of transcriptome-wide changes in D1 and D2 neurons across several brain regions, we then thought to identify specific gene coexpression networks that could be critical in determining the exact response of D1 and D2 SPNs and D1 cortical neurons to the cocaine administration. We used a weighted gene coexpression network analysis (WCGNA) approach to construct a gene coexpression network integrating expression data across brain regions and specific neurons -D1 pyramidal neurons in PFC and D1 SPNs in NAc -and treatment -cocaine and saline -to identify module of co-expressed genes that could underlie the response to chronic cocaine treatment. WCGNA Is a systems biology method for describing the correlation patterns among genes across microarray and RNA-seq samples. It can be used for finding cmodules of highly correlated genes, for summarizing such clusters using the module eigengene or an intramodular hub gene, for relating modules to one another and to external sample traits (using eigengene network methodology), and for calculating module membership measures. For both D1 neurons in NAc and PFC we found four different modules in which the genes differentially regulated by cocaine are highly intra-correlated (Fig. 29A and30A). The two networks are completely independent and while the modules names and the arbitrary (blue, yellow, brown, turquoise) colours on the figures are the same, there is no common gene implicated in the two neuronal populations, as showed by the fact that cocaine induced a common regulation of only 2 genes in NAc and PFC. For each module we calculated its overall correlation with cocaine treatment and we performed a GO analysis to get an insight about the biological relevance of the identified coexpression modules. In NAc D1 SPNs, the GO of the cocaine-associated genes clusters, indicated an enrichment in genes implicated in the synaptic and structural plasticity, axogenesis, modulation of synaptic transmission, regulation of synaptic plasticity, dendrites development, and synaptic vesicles localization among the most 7 -Experimental results -144 represented pathways among the GO terms (Fig. 31A). Interestingly, the same analysis performed on the D1 pyramidal neurons showed a major enrichment in genes involved in the epigenetic modifications such as histone methylation (Fig. 31b). To gain insight into the biology of the cocaine related modules, we identified the most interesting modules for further study. In particular we used STRING (Search Tool for the Retrieval of Interacting Genes/Proteins), a database predicting the protein-protein interaction , to reconstruct the network structure of genes within each of the modules mostly related to presence or the absence of cocaine for each type of D1 neurons and identified so-called key-drivers or hub genes. A key-driver gene is a gene that has the highest probability to connect with the highest number of genes. Our analysis showed a network-type organization in the module D1-SPNs-NAc that mainly clusters in response to the presence of cocaine (blue module) (Fig.

32

) in which we observed several potential hub-genes, such as Tuba4a (Tubulin Alpha 4a), Phlpp1 (PH Domain And Leucine Rich Repeat Protein or phosphatase 1)

or Prkaca (Protein Kinase CAMP-Activated Catalytic Subunit Alpha) The other module in which we could resolve a network organization was in the D1 pyramidal neurons of the PFC, specifically in the module that mostly anti-correlates with the cocaine stimulation (Fig. 33). The network analysis of this module clearly revealed 4 hubs or key drivers genes Acly, Srsf1, Adrkb1, Crebbp. and3). On the one hand we identified the changes induced by 1) active stimulation of the reward system by operant conditioning for highly palatable food (HP) (mHP) 2) active stimulation of the reward system induced by operant conditioning for standard food (ST) (mST) 3) passive stimulation of the reward system by non-contingent presentation of HP food, (yHP), as compared to yoked controls which received non contingently ST food, (yST). On the other hand we identified changes induced by chronic cocaine injections (cCoc) as compared to saline-treated controls (saline) (Figure 2). Then we compared qualitatively the changes observed in the two types of conditions. Since the batches of animals were different and the experiments were not done at the same time, we did not compare the two control groups to each other.

In the NAc we didn't observe any changes in spines number in mice conditioned for ST food as compared to yST. In contrast both operant conditioning for highly palatable food and the non-contingent presentation of HP increased the number of spines (Figure 34 this section and figure 18, results section 2). The NAc was also the only region in which we found an increase in spines density in the cCoc group.

In the DS (Figure 34 this section and figure 19, results section 2), only the operant training for highly palatable food increased spines density. This suggests that the operant conditioning for HP food by itself played a critical role in the morphological change in this brain region.

Lastly, the comparison between the effect of food and cocaine on the PFC showed that spines density was increased only by conditioning for the highly of our data set showed that cocaine and highly palatable food, when administered non-contingently, regulated in an opposite fashion a common set of genes (among the 41 common genes only 5 are co-regulated). Interestingly, the same was not true when we considered the conditioning effect, since the mHP group and the cCoc group showed a set of commonly co-regulated genes. In this comparison, we found 336 common genes and 318 regulated in the same fashion. We also identified a number of genes that were regulated in the same direction (81 over 93) in mST and cCoc groups, as compared to their respective controls. When keeping the most stringent p-value (adj-p value < 0.05) we could only compare the mHP-cCOC in the D2 SPNs of the NAc. By this comparison we obtain only 2 common genes in mHP and cCOC: Rangap1 (RAN GTPase activating protein 1) and Tuba1b (tubulin, alpha 1B). Both genes are down-regulated.

To gain insight into the biological significance of this correlated regulation of gene expression we performed a gene ontology analysis on the genes in common between the different treatments. We found an enrichment in genes connected to "microtubules" (Adj p-value 0.0044) and "axones" (Adj p-value 0.00054) among those commonly regulated in cCoc and mHP indicating their possible contribution to the spine changes induced by both treatments. Our behavioural results showed that only the mHP mice continued to work for pellets when the caloric restriction was over. Our results indicate that cocaine and conditioning for HP food share some commonly regulated genes in the D2 SPNs in the NAc.

The comparison of the other regions-treatment-types of neurons showed a number of genes in common between the different treatments. However, it did not show any precise scheme of co-regulation or anti-regulation of the common genes, except for the PFC in which we found the tendency of anti-regulation of the gene expression over the comparisons cocaine-yoked and co-regulation in the comparison cocainelearning for standard food.

-Experimental results -7

Genes decreased in the NAc D1 neurons

mHP-yHP yHP-yST mST-yST 3110035E14Rik,4930451G09Ri k 5330434G04Rik,Aff3, Atp6v1g2 Bmi1,Cck, Cobl, Dok5, Dusp14, Etl4, Fam124a, Fezf2, Fmnl1 Gal3st3, Grem2, Grik3, Lrp3, Lypd6b, Mfsd9, Nkain1
Nlrp6, Nrep,Nrgn,Preb ,Ptpn3

Rab26, Rhou, Runx2

In the first part of the introduction of this thesis I reviewed the anatomy of the basal ganglia (BG), a large collection of subcortical nuclei interconnected with the cerebral cortex, thalamus, and brainstem. The striatum is the major entry structure of BG. The striatum is characterized by a complex intrinsic organization related to the segregation of its neurons in two different populations expressing either of the two types of DA receptors, D1 and D2. The D1 and D2 SPNs closely correspond to the direct and indirect projection pathways in the DS, although this correlation is much less straightforward in the NAc, especially in its shell part. In addition other subdivisions of the striatum are well described and correspond to important anatomical and functional differences, including the patch/matrix organization of the striatum and the multiple subdivisions of the NAc shell. Several studies have already addressed some of the differences in transcriptional profiles that underlie this anatomical complexity. For example it has been shown that D1 and D2 neurons have strikingly different transcriptional profiles [START_REF] Lobo | Genetic Control of Instrumental Conditioning by Striatopallidal Neuron-Specific S1P Receptor Gpr6[END_REF][START_REF] Guez-Barber | FACS Identifies Unique Cocaine-Induced Gene Regulation in Selectively Activated Adult Striatal Neurons[END_REF][START_REF] Guez-Barber | FACS purification of immunolabeled cell types from adult rat brain[END_REF]Heiman et al., 2008). In addition, some of the differences between striosomes and matrix have been identified, including 60 genes specifically enriched in one or the other compartment (reviewed in Crittenden and Graybel 2011).

In the first part of this thesis we used the BAC-TRAP system to isolate currently translated mRNA from identified D1 and D2 cell populations (Heiman et al. 2008) in the DA and NAc and D1 cells in the PFC.

A first point concerned the comparison between the D1 SPNs and the D1 PFC neurons which share the expression of D1 receptors. Our analysis showed that 5481 transcripts are differentially enriched in D1 SPNs and D1 pyramidal neurons. This result is not surprising if we consider that D1 SPNs and D1 pyramidal neurons are respectively GABAergic and glutamatergic neurons.

We then confirmed that D1 and D2 SPNs are characterized by different transcriptional profiles. We found more than 700 hundreds transcripts being differentially expressed between D1 and D2 in the DS and between D1 and D2 in NAc (adj p-value < 0.05, reads > 30). Most of these genes are expressed in the two populations of neurons but are more expressed in one population or the other, and very few are significantly expressed only in D1 or in D2 neurons.

For the first time we specifically compared the genes expressed in D1 and D2 neurons in the DS and in the NAc, and reciprocally the differences between the NAc 8 Discussion 166 and the DS in D1 and D2 neurons. Interestingly we observed that the differences between the DS and the NAc, within D1 or D2 neurons, are as big as the difference that we could find between D1 and D2. By crossing our data sets we also identified a group of genes common to D1 and D2 and enriched in the NAc or the DS. In absence of anatomical borders, the only criterion used for distinguishing NAc and DS is the regional distribution of the inputs coming from the afferent regions. Here we provided a novel criterion to distinguish these two regions and a novel level of description of the two types of neurons.

In a very recent paper Gokce and coll. [START_REF] Gokce | Cellular Taxonomy of the Mouse Striatum as Revealed by Single-Cell RNA-Seq[END_REF]2016) used FACS-based single-cell RNA sequencing to study the whole striatal cell diversity. This paper confirmed most of the data already reported in literature, and showed that the D1 and the D2 neurons could be divided in 2 additional subpopulations that express a gradient of transcriptional states that could explain the patch matrix organization of the striatum. Interestingly, the genes that the authors have chosen as defining the opposite gradient of expression in the two SPNs populations correspond to some of the genes that we identified as highly enriched in the NAc (Wfs1-Crym) or the DS (Cnr1). Further analysis will be needed to determine whether the gradient observed in this paper correlates with the patch matrix organization and the dorsoventral gradient.

In relation with the anatomical organization of the inputs converging to the striatum and on the basis of multiple functional studies, the NAc has been associated

to the motivation-related processes [START_REF] Lobo | The Striatal Balancing Act in Drug Addiction: Distinct Roles of Direct and Indirect Pathway Medium Spiny Neurons[END_REF], while the DS is implicated in motor behaviour, associative learning, and habits formation [START_REF] Albin | The Functional Anatomy of Basal Ganglia Disorders[END_REF][START_REF] Chang | Neuronal Responses in the Frontal Cortico-Basal Ganglia System during Delayed Matching-to-Sample Task: Ensemble Recording in Freely Moving Rats[END_REF][START_REF] Balleine | The Role of the Dorsal Striatum in Reward and Decision-Making[END_REF]Graybiel et al., 2008). As a prelude to testing the biological relevance of the genes that we found to be enriched in D1 and D2 neurons in the DS and in the NAc, we performed an upstream analysis. The analysis showed that different chemicals or transcription factors are predicted to regulate specific sets of genes in the D1 or the D2 neurons depending on their location in the NAc or the DS. Among these different upstream regulators, we found that prostaglandin E2 (PGE2) was the only compound predicted to be a possible positive regulator of the genes expressed in both D1 and D2 neurons in the DS.

Therefore, we chose it as a possible target to study the effects of its manipulation on striatal function. Prostaglandins (PGs) are a family of lipid mediators involved in a 8 Discussion

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NAc and DS and in D1 pyramidal neurons of the PFC. Furthermore the presence of yoked controls allowed the analysis of the effects of the same food availability without operant conditioning but all other conditions (i.e. food deprivation, exposure to operant cages, manipulations…) being identical. The rewarding effects of a stimulus can be measured by the willingness of the subject to work in order to gain the access to it. In this framework, it has been known for more than a century that the responses to stimuli that produce positive effects are likely to be repeated again (E.L.

Thorndike 1898). The idea that behavioural responses can be a direct measure of the rewarding properties of a certain object is the basis of the theory of reinforcement initially proposed by Skinner in 1938. A form of operant training was also used in the studies of [START_REF] Olds | Positive Reinforcement Produced by Electrical Stimulation of Septal Area and Other Regions of Rat Brain[END_REF] to investigate the responses to intracranial self-stimulation, leading to the discovery of brain reward systems. Operant training is still one of the paradigms mainly used in measuring the rewarding properties of an object. In agreement with previous studies (Balleine and[START_REF] Balleine | Effects of Ibotenic Acid Lesions of the Nucleus Accumbens on Instrumental Action[END_REF]Guegan T et AL., 2012), we observed that food palatability strongly increases the positive pokes in the operant behaviour. However, while most of the previous studies used a longer training paradigm, we obtained a stable operant response after only 2 weeks of training in order to get access to relatively early molecular brain alterations.

Indeed the mice trained with highly palatable food worked sensibly more than those trained for standard food (figure 16, results section 2).

Different studies have already shown that striatum and PFC play a central in mediating the effects of reward. The DA released by the DA neurons of the VTA modulates the D1 and D2 SPNs activity in the striatum, and the D1 pyramidal neurons in the PFC. A DA increase has been reported in the NAc after both drugs (Di Chiara G., 1992) and natural reward [START_REF] Hernandez | Food Reward and Cocaine Increase Extracellular Dopamine in the Nucleus Accumbens as Measured by Microdialysis[END_REF][START_REF] Hajnal | Accumbens Dopamine Mechanisms in Sucrose Intake[END_REF]. We reasoned that -as for the drugs of abuse -the increase of DA induced by highly palatable food could regulate the structural plasticity in the areas targeted by the VTA projection. Therefore we measured the spine density induced by our training paradigm in PFC, DS and NAc. From this series of experiment we could study both the effect of the training and of the food on spine formation. Concerning the training, we obtained an increase of spines only in the mice trained with highly palatable food.

This is in agreement with the previous results reported in literature (Guegan T et al., 2012). However here we report that the training for highly palatable food increases the spine number in PFC, NAc and DS, while the non-contingent presentation of highly palatable food has the same effect only in the NAc. The spine increase in the DS agrees with humans imaging studies in which this region has been reported to be activated in response to food (Small DM, 2003). The increase in spines in NAc seems related more to the highly palatable food itself rather than to the learning for the highly palatable food. Hence, we obtain an increase of the spine density also in the control mice that have been show to do not learn during the operant training. This result is in agreement with the increased spine density in the yoked mice reported in the mesocorticolimbic system in response to a psychostimulant [START_REF] Maze | Essential role of the histone methyltransferase G9a in cocaine-induced plasticity[END_REF].

In particular, the increase of spines in the yoked mice in the NAc is not surprising in given the fact that the NAc is the region traditionally considered associated with the primary reinforcing effect of drugs (Di [START_REF] Chiara | Dopamine and Drug Addiction: The Nucleus Accumbens Shell Connection[END_REF][START_REF] Wise | Dopamine, Learning and Motivation[END_REF][START_REF] Ikemoto | The Functional Divide for Primary Reinforcement of D-Amphetamine Lies between the Medial and Lateral Ventral Striatum: Is the Division of the Accumbens Core, Shell, and Olfactory Tubercle Valid?[END_REF][START_REF] Wise | Dopamine and Reward: The Anhedonia Hypothesis 30 Years on[END_REF]). It is important to mention that at the same time our results are in contrast with those of Guegan and collaborators (2012). In their work, no spine induction was observed in yoked mice. This discrepancy might be due to the different length of the operant training (15 days vs 41 days), as well as to the fact that we did not use any progressive ratio at the end of the experiment and that we waited 24 h before sacrificing mice after the last training session. Concerning the PFC the increase of dendritic spines in the master highly palatable food group is in agreement with recent work that demonstrated with completely different approaches a role for D1-type dopamine receptor-expressing neurons in the medial prefrontal cortex (mPFC) in the regulation of feeding (Land BB 2014). Food intake increased the activity of D1 neurons of the mPFC in mice, and optogenetic stimulation of D1 neurons increased feeding. Conversely, inhibition of D1 neurons decreases intake (Land BB 2014). The analysis of the structural plasticity induced by highly palatable food is still in progress, in particular the morphology of the spines. Additional experiments would be useful to test some of the points discussed above. For instance, an interesting experiment would be to distinguish between D1 and D2 by using a transgenic line expressing GFP under the control of the promoter of DA receptors and perform spine quantification with an appropriate technique such as gene gun labelling with DiI. This would allow to define which cells are responsive to highly palatable food conditioning, and test whether the increase in spine density in the DS is due to a neuroadaptation of the D2 neurons as in the case of the drugs of abuse [START_REF] Porrino | The Expanding Effects of Cocaine: Studies in a Nonhuman Primate Model of Cocaine Self-Administration[END_REF][START_REF] Porrino | The Expanding Effects of Cocaine: Studies in a Nonhuman Primate Model of Cocaine Self-Administration[END_REF]. This type of experiment would also allow to better correlate the structural plasticity that we observed with the data that we obtained from sequencing (see below). Another potentially interesting experiment would be to analyse the dendritic spines of the mice in the 3 regions just after the learning phase. This would allow to see which differences exist between the synaptic structures induced by the 2 different phases of the training, and to check if, as for cocaine self-administration, the NAc is the most involved structure during the first phase of training.

Several studies have already shown that drugs of abuse are able to induce long-lasting modifications in gene expression. Such modifications might underlie some aspects of addictive behaviour and require changes in gene transcription. We investigated whether highly palatable food could also induce long-lasting modifications in gene expression. We found that transcriptional changes induced by highly palatable food conditioning were mainly taking place in the D2 neurons, in the DS and to a lesser extent in the NAc. This result might seem surprising, as D1 neurons appear to be primary targets for rewarding stimuli such as cocaine (e.g.

Pascoli&Luscher 2012

). However it is in agreement with several other work using completely different approaches and readouts, which showed the importance of the D2 neurons in both obesity and drug addiction (ref). Indeed, a clear common feature between drugs of abuse and obesity is the lower availability of the D2 receptors within the striatum. Human imaging studies have established that less D2 receptor are available in the striatum of obese relative to lean individuals [START_REF] Stice | Relation between Obesity and Blunted Striatal Response to Food Is Moderated by TaqIA A1 Allele[END_REF]Wang GJ, 2001, Barnard ND.;2009) as well as in addicted individual compared to controls (Asensio S, 2010;Volkow ND., 1993). Importantly, in both the obese [START_REF] Stice | Relation between Obesity and Blunted Striatal Response to Food Is Moderated by TaqIA A1 Allele[END_REF]Wang GJ, 2001, Barnard ND;2009) and drug-dependent population, (Noble EP, 2000;Lawford BR, 2000) an over representation of individuals harbouring the TaqIA A1 allele has been observed, which results in ~30-40 % reduction in striatal D2Rs (Stice E., 2010;Jönsson EG., 1999). In rodents, a lot of work has already been done for the elucidation of the role of D2 neurons in the context of drugs of abuse.

For instance, it has been showed that impulsivity is associated to the prediction of cocaine intake and to the lower availability of D2/3 DA receptors in the ventral striatum [START_REF] Dalley | Nucleus Accumbens D2/3 Receptors Predict Trait Impulsivity and Cocaine Reinforcement[END_REF]. It has been proposed that drug exposure would be able to induce plasticity in D2 neurons, possibly diminishing their activity and facilitating inflexible, compulsive-like drug-taking behaviour, which in rodents can be measured by the presentation of a progressive ratio schedule. Consistently Alvarez and coworkers have shown that the synaptic strengthening in the D2 SPNs in the NAc was inversely correlated with the emergence of compulsive-like cocaine responding in mice with a history of self-administration (Bock R., 2013). Moreover, DREADDmediated inhibition of D2 SPNs increases the compulsive like response to cocaine, while their optical stimulation decreases it (Bock R., 2013). These observations suggest a clear link between the D2 neurons and the appearance of the compulsivity.

It raises on the other hand the question of whether the D2 neurons have a similar role in the consumption of highly palatable food. Indeed, the knockdown of striatal D2receptor, using a lentiviral vector, accelerates the emergence of compulsive-like consumption of calorically dense, palatable but not standard food (Johnson PM., 2010). These results suggest that the activation of the D2 neurons during the training for highly palatable food could have similar functional consequences as their activation in response to cocaine. Compulsivity could perhaps arise from the sensitization of the DA fibres that have been already excited by the reward.

Sensitization has been linked to a faster formation of habits, which according to the work of Everitt's group, would be associated with a greater activation of the DS [START_REF] Johnson | Dopamine D2 Receptors in Addiction-like Reward Dysfunction and Compulsive Eating in Obese Rats[END_REF]. In this perspective, the identification of the neurons in which synaptic density is altered by highly palatable food would be useful to determine if, as for drugs, the structural plasticity is taking place mainly in the D2 neurons. A deep analysis of the transcriptional profile that we obtained after the training for the highly palatable food is still in progress. The ensemble of results that we report provides a strong base for a deeper analysis of the response of the DA SPNs in to the highly palatable food.

We started an in vivo validation of our results by genetically knocking down Importantly, it is possible to contextualize these results with the reported norbin functions, and with the different inputs and outputs of the NAc and DS. Norbin or neurochondrin is a 75 kDa protein that interacts with 3 important modulators of the striatal function that could be potentially involved in the behavioural effects we observed in the norbin KO mice: GluR5 (Wang H. et al., 2009), MCHR1 (melanin concentrating hormone) (Francke F., 2006) and CaMKII (Dateki M, et al., 2005).

Norbin has been shown to inhibit MCHR-induced G protein activation and downstream calcium influx. By binding to MCHR1, norbin sterically competes with the binding of G-proteins to the receptor and thereby inhibits G protein-coupled transduction (Francke F., 2006). The NAc is the only region of the striatum innervated by the melanin-concentrating hormone (MCH) producing neurons. As discussed in the introduction of this thesis, MCH neurons have been implicated in the homeostatic regulation of feeding: intracerebroventricular injections of the peptide increase feeding and body weight in rodents, and MCH mRNA levels are increased by food deprivation [START_REF] Qu | A Role for Melanin-Concentrating Hormone in the Central Regulation of Feeding Behaviour[END_REF]; mice lacking MCH neurons (Alon, T. et al., 2006) or the MCH gene [START_REF] Shimada | Mice Lacking Melanin-Concentrating Hormone Are Hypophagic and Lean[END_REF]) are hypophagic and lean. On the other hand the overexpression of MCH results in hyperphagia, resistance to insulin and obesity (Ludwig, D.S., 2001). We found that norbin is down-regulated specifically by highly palatable food in the NAc during the first phase of conditioning. We could speculate that this down-regulation is a homeostatic response to HP food, since norbin is a negative regulator of the MCHR. The down-regulation of norbin would lead to an increased MCHR activity facilitating the feeding behaviour. The same idea could explain the effects of the norbin KO during this phase. The combination of the normal overproduction of MCH in fasted mice, with the increased activity of the MCHR due to the loss of the negative regulation exerted by norbin could explain the facilitation of the feeding behaviour that we obtained with the norbin-ko mice.

As mentioned above, mGluR5 is another important partner interacting with norbin. In 2009, Wang and co-workers showed that norbin increases the synaptic surface localization of mGluR5 and positively regulates mGluR5 signalling.

Furthermore, in the hippocampus, the genetic deletion of norbin attenuates mGluR5dependent stable changes in synaptic function measured as long-term depression (LTD) or long-term potentiation (LTP) of synaptic transmission. In the striatum, excitatory synaptic inputs from cortical neurons can undergo mGluR5-dependent LTD (reviewed in Lüscher C, Huber KM, 2010). Such cortico-striatal afferents impinge on both D1 and D2 SPNs. Both D1 and D2 are able to express forms of mGluR5-LTD [START_REF] Calabresi | Role of Dopamine Receptors in the Short-and Long-Term Regulation of Corticostriatal Transmission[END_REF]; however a striking difference between the 2 types of neurons consists in the fact that while the LTD in the D1 is blocked by the activation of the D1 receptor, in the D2 neurons it is induced by the D2 activation [START_REF] Kreitzer | Endocannabinoid-Mediated Rescue of Striatal LTD and Motor Deficits in Parkinson's Disease Models[END_REF][START_REF] Shen | Adenosine A(2A) Receptors in Psychopharmacology: Modulators of Behavior, Mood and Cognition[END_REF]. In the D2 neurons of the indirect pathway of the DS, LTD is initiated by a high frequency stimulation, which leads to postsynaptic activation [START_REF] Choi | Decreased Frequency but Not Amplitude of Quantal Synaptic Responses Associated with Expression of Corticostriatal Long-Term Depression[END_REF]. The postsynaptic activation triggers the production of an endocannabinoid retrograde messenger -most probably anandamide -that binds to the presynaptic cannabinoid 1 receptor (CB1) and triggers the presynaptic expression of LTD (Genderman et al., 2002). This phenomenon is referred to as mGluRs-LTD. Importantly, it has been demonstrated that this mechanism takes place only when the D2 activation is induced [START_REF] Kreitzer | Endocannabinoid-Mediated Rescue of Striatal LTD and Motor Deficits in Parkinson's Disease Models[END_REF]); thus, logically in normal conditions mGluR-LTD will be induced in the D2 neurons of the DS only in the case of a concomitant activation of both the glutamatergic afferences from the prefrontal cortex and the DA fibres arising from the midbrain. SPNs have been shown to express a higher-level of mGluR5 compared to mGluR1, however, a genetic model that clearly links this form of LTD to mGluR1 or mGlur5 is not yet available. Given this background, we could speculate that the absence of norbin from the DS would correlate with a decreased expression of mGluR5 at synapses. This down-regulation of mGluR5 would prevent the production of the endocannabinoid messenger, the activation of the CB1R, and lastly the presynaptic plasticity. In support to this hypothesis CBR1-KO mice showed the same phenotype as we observed with the norbin-KO mice when presented to a progressive ratio schedule for obtaining highly palatable food (Guegan T et al, 2012).

Discussion

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It has been published that norbin-ko mice show a lower preference for sucrose (Wang H. et al., 2015). However the long term implication of the loss of sucrose preference has not been explored. Because we already proved that it is possible to induce obesity by using our highly palatable isocaloric pellets, we are currently performing an obesity experiment with the norbin-ko mice, to check if Ncdn is implicated in the obesity development.

In the third part of my thesis we used a chronic regimen of cocaine injections to analyze the structural plasticity induced by cocaine administration as well as the influence of cocaine on the transcriptional profiles in identified neuronal populations in the NAc, DS, and PFC.

We found that in our conditions, chronic cocaine administration triggered an increase of dendritic spines in the NAc as compared to vehicle-treated controls. This result confirms previous studies reporting that cocaine and other drugs of abuse produce persistent changes in the structure of dendrites and dendritic spines in D1 and D2 SPNs in the NAc [START_REF] Lee | Cocaine-Induced Dendritic Spine Formation in D1 and D2 Dopamine Receptor-Containing Medium Spiny Neurons in Nucleus Accumbens[END_REF][START_REF] Li | Cocaine-Induced Dendritic Remodeling Occurs in Both D1 and D2 Dopamine Receptor-Expressing Neurons in the Nucleus Accumbens[END_REF][START_REF] Guez-Barber | FACS purification of immunolabeled cell types from adult rat brain[END_REF]. We also confirmed [START_REF] Dobi | Cocaine-Induced Plasticity in the Nucleus Accumbens Is Cell Specific and Develops without Prolonged Withdrawal[END_REF] that in the NAc, chronic cocaine administration triggered an increase of the spine head area, suggesting a general increase of the area available for the synaptic contact after cocaine administration.

Correlating with this persistent increase of spines in the NAc our sequencing data showed that for both D1 and D2 SPNs, the NAc is the region in which the largest number of genes was altered by cocaine administration. Interestingly, the GO analysis on the genes regulated by cocaine in the NAc showed a major enrichment in genes related to synaptic and structural plasticity, and to actin cytoskeleton remodelling. For example, chronic cocaine administration induced an up-regulation of the cordon-bleu WH2 repeat gene, Cobl. WH2 Cobl binds to actin to promote actin filament formation. It therefore has a role in neuronal morphogenesis, dendrite formation, and dendritic arborisation (Ahuja R. et al., 2007). This effect is in line with the observation that chronic cocaine administration reduces the activity of Rac1, leading to the intensification of the polymerization rate of filamentous actin in the NAc (Dietz MD et al., 2012).

Interestingly we did not observe any cocaine-mediated induction of spines in the DS and PFC. This observation could be explained by the fact that the PFC and DS are more associated to habit formation (reviewed in [START_REF] Everitt | Neural Systems of Reinforcement for Drug Addiction: From Actions to Habits to Compulsion[END_REF]. Since in our protocol we passively stimulate the reward system, not implying any learning or habit formation, we might not be sufficient to engage a major involvement of the DS and a de novo synthesis of spine formation. Accordingly with this result, we were not able to observe any significant change in gene expression neither in D1 or D2 SPNs in the DS. However, it is important to note that we could observe changes in the spine morphology. An increase in the neck length in the DS and in minor extend in the PFC have been observed in mice injected with cocaine compared to their vehicle-injected controls. Although the mechanism through which spine shape affect its function is not fully understood yet, morphology does play a role, as long and thin necks prevent the diffusion of calcium, whereas shorter and thicker necks allow for better diffusional coupling with the dendrite [START_REF] Majewska | Regulation of Spine Calcium Dynamics by Rapid Spine Motility[END_REF][START_REF] Holthoff | Calcium Dynamics in Spines: Link to Synaptic Plasticity[END_REF][START_REF] Sabatini | The Life Cycle of Ca(2+) Ions in Dendritic Spines[END_REF][START_REF] Korkotian | Dynamic Regulation of Spine-Dendrite Coupling in Cultured Hippocampal Neurons[END_REF][START_REF] Noguchi | Spine-Neck Geometry Determines NMDA Receptor-Dependent Ca2+ Signaling in Dendrites[END_REF].

Furthermore It was shown that reduction of the spine neck length after synaptic potentiation mediates enhanced electric coupling between the spine and the dendrite, thereby increasing the influence of the potentiated spine on the dendritic and somatic membrane potential [START_REF] Araya | Activity-Dependent Dendritic Spine Neck Changes Are Correlated with Synaptic Strength[END_REF][START_REF] Tønnesen | Spine Neck Plasticity Regulates Compartmentalization of Synapses[END_REF]. However, the morphological changes that are associated with synaptic modulation could just be a secondary effect of altered actin dynamics required to more directly modulate synapse functioning or actin-based transport.

The cell population specific RNA sequencing in the NAc, DS and PFC, showed that several transcript are differentially regulated by repeated cocaine administration.

Within the striatum, the majority of the changes were taking place in the NAc for both the D1 and the D2 SPNs in which 136 and 72 transcripts were differentially regulated, respectively. Importantly, among these transcripts we identified some genes that have been already described as regulated by cocaine. Although it is often complicated to make a direct comparison of data across drug administration experiments from published studies due to the differences in strain background, cocaine doses and assay sensitivity, we were able to identify various genes that have been already reported to be affected by cocaine administration. Importantly, our experimental approach provides information in which neurons and which sub-region of the striatum those genes are expressed. Examples of those genes are Crtac1, Etv5, Tbr1, Dusp3, Plcg1 Txndc13, Epdr1, Snapc3 (Heiman et al., 2008) (up in NAc D1),

Nfib [START_REF] Feng | Chronic Cocaine-Regulated Epigenomic Changes in Mouse Nucleus Accumbens[END_REF]2014) (up in D1 NAc). Unc5b, Gna12 and Ttbk1 (Heiman et al., 2008) (up in NAc D2). GO analysis in the NAc -D1 neurons showed an enrichment of genes implicated in the regulation of synaptic and structural plasticity. Indeed, we found some genes that have been already implicated with those processes. One interesting gene is homer2, up regulated in D1 SNPs of the NAc. The homers isoforms interact with the mGluR1 receptor to induce LTD and have already been implicated in synaptic plasticity induced by cocaine stimulation. Disrupting the interaction between homer and mGluR1 has opposite effects depending on the location of the manipulation. The homer-mGlur1 connection disruption in the VTA, induces a plasticity response to a single injection of cocaine in the NAc comparable to the synaptic adaptations that are normally obtained by chronic cocaine injections.

Interestingly, the opposite is true in the NAc, where mGluR1 is a positive modulator of synaptic plasticity [START_REF] Knoflach | Positive Allosteric Modulators of Metabotropic Glutamate 1 Receptor: Characterization, Mechanism of Action, and Binding Site[END_REF][START_REF] Mameli | Cocaine-Evoked Synaptic Plasticity: Persistence in the VTA Triggers Adaptations in the NAc[END_REF]. Indeed, the upregulation of Homer2 in the NAc could contribute to the synaptic plasticity linked to the mGluR1 receptor.

Another interesting example of a regulated gene is Grin3a, which was downregulated after 7 days in the D1 neurons of the NAc. Grin3A is a gene coding for the semi-Ca2+-impermeable NMDARs subunit GluN3A. As Homer, GluN3A has a central role in the plasticity induced by cocaine. In the VTA a single cocaine injection is able to induce a considerable potentiation of the excitatory synaptic transmission in the DA neurons of the VTA, due to the increase of the AMPAR/NMDAR ratio [START_REF] Ungless | Single Cocaine Exposure in Vivo Induces Long-Term Potentiation in Dopamine Neurons[END_REF]. It has been shown that the increase in the ratio is due to an increase of the AMPAR-dependent currents, related to the insertion of the Ca2+-permeable Glu2A subunit and to the reduction of the NMDAR-dependent currents, related to the insertion of the semi-Ca2+-impermeable NMDARs containing GluN3A (Mameli et al., 2011[START_REF] Yuan | Expression of Cocaine-Evoked Synaptic Plasticity by GluN3A-Containing NMDA Receptors[END_REF]. Unlike in the VTA, multiple non-contingent doses of cocaine administration are required to elicit synaptic plasticity in excitatory synapses in the NAc [START_REF] Thomas | Long-Term Depression in the Nucleus Accumbens: A Neural Correlate of Behavioral Sensitization to Cocaine[END_REF] and to reduce the AMPA/NMDRA ration [START_REF] Thomas | Long-Term Depression in the Nucleus Accumbens: A Neural Correlate of Behavioral Sensitization to Cocaine[END_REF][START_REF] Beurrier | Enhanced Inhibition of Synaptic Transmission by Dopamine in the Nucleus Accumbens during Behavioral Sensitization to Cocaine[END_REF]Thomas et al., 2008). It could be argued that the down-regulation of the expression of Grin3A in the NAc could be related to an increased permeability of NMDA receptors to Ca2+ in the D1 neurons, contributing to the decrease of the AMPAR/NMDAR ratio and to the cocaine-induced synaptic plasticity in the NAc.

Our cell-specific RNA sequencing confirmed that the PFC is a region clearly receptive to cocaine stimulation. We identified a number of genes regulated by chronic cocaine in the D1 pyramidal neurons of the PFC.

We (WCGNA) approach to construct a gene coexpression network of the D1 neurons in NAc and PFC in response to cocaine, to identify candidate genes more involved in the instauration of the modification induced by chronic cocaine exposure.

The GO on the modules more related to the cocaine treatment showed an enrichment of genes involved in the structural plasticity for the NAc and in the epigenetic regulation of the DNA for the PFC. Different studies suggest that the PFC underlies 180 Lastly, we compared the effects of operant conditioning and those of cocaine administration on the structural plasticity and the transcription.

The NAc is the only region in which we found an increase of spine density after both non-contingent presentation of HP food and chronic cocaine administration.

The increase in spine number in both cocaine and yHP is in line with the finding that both sweets [START_REF] Hajnal | Accumbens Dopamine Mechanisms in Sucrose Intake[END_REF] and cocaine [START_REF] Wise | Brain Dopamine and Reward[END_REF]) induce an activation of the NAc, especially in the shell subregion [START_REF] Imperato | Effects of Locally Applied D-1 and D-2 Receptor Agonists and Antagonists Studied with Brain Dialysis[END_REF]). In particular it has been demonstrated that DA release in the NAc increases in response to unexpected food [START_REF] Norgren | Gustatory Reward and the Nucleus Accumbens[END_REF]). Moreover, it is important to observe that those results correlate well with our gene expression data in which we observed that both cocaine and learning for highly palatable food induce an alteration of the gene expression within the NAc. Of note, only the learning for the highly palatable food has an effect on the gene expression within the DS.

We identified common sets of regulated genes in all the comparisons, however we mainly focused on the D2 neurons of the NAc, being the only population in which significant changes were occurring in both operant training for highly palatable food and cocaine treatment. Depending on the type of conditioning, some genes were regulated in the same direction compared to cocaine exposure (mHP vs cCOC), and some in the opposite direction (yHP vs cCOC and mST vs cCOC). All the comparisons were obtained by setting the threshold to a nominal p-value of 0.05. By setting the threshold to an adj p-value of 0.05, we could find only 2 genes in common between cocaine and training for highly palatable food in the NAc D2 SPNs:

Rangap1 (RAN GTPase activating protein 1) and Tuba1b (tubulin, alpha 1B). Both genes are commonly down regulated by cocaine and training for highly palatable food. The common regulation of the Tuba1b codes for the tubulin alpha 1B; tubulin is the major component of the microtubules. Rangap1 is a protein that associates with the nuclear pore complex and participates in the regulation of nuclear transport by interacting with the Ras-related nuclear protein 1 (RAN) and by regulating the guanosine triphosphate (GTP)-binding and exchange. When binding to RAN, Rangap1 induces its conversion to the putatively inactive GDP-bound state.

Although this protein has never been directly investigated in response to cocaine or food reward, it has been found associated to other types of addiction such as alcohol [START_REF] Marballi | Alcohol Consumption Induces Global Gene Expression Changes in VTA Dopaminergic Neurons[END_REF][START_REF] Sikela | DNA Microarray and Proteomic Strategies for Understanding Alcohol Action[END_REF]. This comparison thus provides interesting Genes are ranked by FC. In blue the genes up-regulated and in orange the genes down-regulated.

A total of 71 genes is differentially regulated in this comparison.

Introduction

In the brain, long-lasting alterations that underlie learning and memory require modifications in gene expression. Specific signalling pathways triggered by synaptic activity converge on the nucleus where they can modify gene expression and other nuclear functions [START_REF] Jordan | Nucleocytoplasmic protein shuttling: the direct route in synapse-to-nucleus signaling[END_REF][START_REF] Matamales | Signaling from the cytoplasm to the nucleus in striatal medium-sized spiny neurons[END_REF]. Differentiated cell phenotypes result from specific patterns of gene expression, while long-lasting changes in cellular properties involve gene transcription regulation. In addition to the interplay of numerous transcription factors, epigenetic marks, including histone and DNA modifications, as well as non-coding RNAs, play a crucial role in transcription regulation [START_REF] Borrelli | Decoding the epigenetic language of neuronal plasticity[END_REF][START_REF] Meaney | Epigenetic regulation of the neural transcriptome: the meaning of the marks[END_REF]. Epigenetic marks are characteristic of cell types and their modifications may provide further traces of the cell history. In neurons, it has been proposed that epigenetic modifications contribute to long-lasting alterations reflecting environmental stimuli [START_REF] Zhang | Epigenetics and the environmental regulation of the genome and its function[END_REF]. All these processes can be altered in neurological and psychiatric disorders [START_REF] Telese | Seq-ing" insights into the epigenetics of neuronal gene regulation[END_REF]. Tissue heterogeneity, however, makes the study of nuclear signalling particularly challenging, as relevant changes occur only in a fraction of specific cells. Therefore it is critical to design methods to selectively study nuclei of interest. Laser capture allows microdissection of specific cell types, including, in principle, their nuclei [START_REF] Cheng | Laserassisted microdissection in translational research: theory, technical considerations, and future applications[END_REF], but generally yields low amounts of material. Cell-sorting has been used to recover specific neuronal or glial populations from brain tissue (Lobo et al., 2006;[START_REF] Guez-Barber | FACS purification of immunolabeled cell types from adult rat brain[END_REF], but this approach is limited by the difficulty and time required to dissociate adult brain, with the risk of biochemical reactions altering observed responses. A more promising approach is the direct purification of nuclei. Nuclei can be purified from post-fixed brain tissues [START_REF] Herculano-Houzel | Isotropic fractionator: a simple, rapid method for the quantification of total cell and neuron numbers in the brain[END_REF][START_REF] Jiang | Isolation of neuronal chromatin from brain tissue[END_REF][START_REF] Collins | A rapid and reliable method of counting neurons and other cells in brain tissue: a comparison of flow cytometry and manual counting methods[END_REF][START_REF] Okada | Flow cytometric sorting of neuronal and glial nuclei from central nervous system tissue[END_REF][START_REF] Young | Use of flow cytometry for high-throughput cell population estimates in brain tissue[END_REF]. These methods do not prevent alterations before fixation, a problem particularly serious with labile modifications such as protein phosphorylation and contamination with cellular debris is very high. As commonly used fluorescent proteins can leak out of nuclei before fixation, elegant approaches using specific tagging of nuclear proteins that allows affinity-purification of nuclei [START_REF] Deal | The INTACT method for cell type-specific gene expression and chromatin profiling in Arabidopsis thaliana[END_REF][START_REF] Steiner | Cell-type-specific nuclei purification from whole animals for genome-wide expression and chromatin profiling[END_REF] or their fluorescence-activated sorting [START_REF] Jiang | Isolation of neuronal chromatin from brain tissue[END_REF]Kriaucionis & Heintz, 2009) have been recently developed. However, these methods require the production of specific transgenic lines and the proposed protocols do not fully preserve labile modifications.

To overcome these difficulties we have developed a methodology (fluorescence-activated sorting of fixed nuclei; FAST-FIN) to prepare, stain, and analyse or sort by flow cytometry specific nuclei from fixed tissue. Although we have set up this method using mouse striatum as a model tissue, it should have general applicability.

Materials and methods

Animals

We used C57Bl/6J (Janvier, Le Genest Saint Isle, France) and mutant adult male (unless otherwise indicated) mice. Drd1::EGFP (enhanced green fluorescent protein under the control of dopamine D1a receptor promoter) and drd2::EGFP transgenic mice were generated by GENSAT (Gene Expression Nervous System Atlas; [START_REF] Gong | A gene expression atlas of the central nervous system based on bacterial artificial chromosomes[END_REF] and drd1a::tdTomato, produced by N. Calakos, Duke University Medical Center (Shuen et al., 2008) were obtained from the Jackson lab. In these latter mice the transgene is inserted into the X chromosome and in hemizigous females tdTomato is expressed in about half of the D1R-positive cells due to X chromosome inactivation. In contrast, in males all the D1R-positive cells express tdTomato. Pvalb::Cre mice, expressing the Cre recombinase under the control of the parvalbumin promoter [START_REF] Hippenmeyer | A developmental switch in the response of DRG neurons to ETS transcription factor signaling[END_REF], crossed with RCE:LoxP reporter mice expressing EGFP under the control of promoter sequences of the Rosa locus [START_REF] Sousa | Characterization of Nk6-2-derived neocortical interneuron lineages[END_REF] were provided by Jean-Christophe Poncer, Institut du Fer a Moulin, Paris. Animals were housed in a 12-h light-dark cycle, in stable conditions of temperature, with food and water ad libitum. All the experiments were in accordance with the European Communities Council Directive of 24 November 1986 (86/609/ EEC) and approved by the Comit ed ' ethique pour l'exp erimentation animale Charles Darwin (Paris, France).

Drugs

Cocaine hydrochloride (Cooper, Melun, France) was dissolved in 0.9% (wt/vol) NaCl solution (saline) at 2 mg/mL and injected i.p. (10 mL/kg).

Antibodies

The primary antibodies used with their corresponding final concentrations were the following: acH4K 5 rabbit IgG (Merck-Millipore, Billerica MA, USA; ref. 156 (Abcam;ref. ab37415). The secondary antibodies and their corresponding dilutions were the following: anti-rabbit allophycocyanin (APC)-conjugated (Abcam, ref. ab130805, 1 : 500) and anti-mouse phycoerythrin (PE)-conjugated (Abcam,ref. ab7003,1 : 400). For drd1a::EGFP mice that express less EGFP, we incubated the nuclei with a fluorescein isothiocyanate (FITC)-conjugated anti-GFP antibody (Abcam; ref. ab6662; 625 ng/mL).

Fixation

Animals were quickly and deeply anesthetised with 500 mg/kg pentobarbital (Sanofi-Aventis, France) i.p. and perfused transcardially with 4% (wt/vol) formaldehyde in PBS (pH 7.4) at 20 mL/min at room temperature for precisely 5 min. The brain regions of interest were dissected before being dipped into the homogenisation solution which contained a formaldehyde-neutralising chemical NH 4 + (50 mM NH 4 Cl; see below), exactly 9 min after the perfusion had begun. Excess of fixation would induce more nuclei loss during homogenisation especially from neurons (which have bigger nuclei) whereas insufficient fixation would result in more protein leakage. For quantitative studies it is really critical to respect identical fixation duration for each sample.

Tissue dissociation

Cross-linked tissue was homogenised in 2-mL Dounce homogeniser (Dominique Dutscher, Brumath, France) containing 1 mL of solution (in mM: sucrose, 50; KCl, 25; MgCl 2 , 5; NH 4 Cl,50;and Tris,pH 7.4,120). Twenty-five strokes of pestle A (clearance 76-127 lm) followed by 25 strokes of pestle B (clearance 12-63 lm) were applied gently to avoid damage to nuclei.

Nuclei purification

The considerable amount of debris from a cross-linked dissociated tissue makes nuclei purification essential. Previous work used filtration and myelin removal beads [START_REF] Collins | A rapid and reliable method of counting neurons and other cells in brain tissue: a comparison of flow cytometry and manual counting methods[END_REF][START_REF] Bonn | Cell type-specific chromatin immunoprecipitation from multicellular complex samples using BiTS-ChIP[END_REF], but this resulted in a substantial loss of material. We opted for an iodixanol (Optiprep TM ; Sigma Aldrich) discontinuous density gradient, which yielded a very good purity nuclear fraction with little nuclei loss. For optimisation, Hoechst-stained fractions with various iodixanol concentrations were verified by observation with a fluorescence microscope. As fixed glial nuclei had a slightly lower density than fixed neuronal nuclei, any variation in the gradient could lead to a relative enrichment of one cell type vs. the other. The iodixanol gradient solutions were prepared as follows: five volumes of Optiprep containing 60% (wt/vol) iodixanol were mixed with one volume of 150 mM KCl, 30 mM MgCl 2 and 120 mM Tris, pH 7.4. This 50% iodixanol solution was then further diluted to make two solutions of different densities (containing 22% and 43% of iodixanol, respectively) using 250 mM sucrose, 25 mM KCl, 5 mM MgCl 2 and 20 mM Tris, pH 7.4, as a diluent.

After homogenisation, the solutions containing brain extracts were transferred into Eppendorf tubes to be centrifuged at 2000 g for 5 min. The supernatant was completely replaced by 1 mL of the 22% iodixanol solution and the pellet was resuspended by pipetting up and down 15 times to ensure proper dissociation of the material. After that, a 43%-22% iodixanol gradient was prepared in centrifuge tubes compatible with swinging buckets (rotor TI-SW60; Beckman Coulter, Villepinte, France), 22% iodixanol was carefully layered onto 500 lL of the 43% iodixanol solution, and the homogenate was added on top. This preparation was centrifuged at 10 000 g for 30 min at 4 °C. Given their density the nuclei accumulate between the 22% and the 43% iodixanol layers. This interface was collected and diluted 1 : 2 with the resuspension solution [in mM: sucrose, 250; KCl, 25; MgCl 2 , 5; and Tris, 20; with 1% (wt/vol) BSA, pH 7.4], the nuclei were collected by centrifugation at 10 000 g for 10 min and resuspended in the same solution to remove iodixanol.

Nuclear preparation for flow cytometry

For all the following steps, nuclei were kept in the resuspension solution described above, with the indicated supplements. Every time the solution had to be changed, the nuclei were collected by a 5-min centrifugation at 2000 g and resuspended thereafter (except for consecutive washes). DNA was labelled using 0.2 lg/mL Hoechst 33258 and incubated for 20 min at 4 °C. Subsequent wash was necessary as we observed that a too-strong Hoechst labelling can blur the GFP signal. For immunolabelling, nuclei were permeabilised with 0.1% (vol/vol) Triton X-100 for 10 min and the nuclear suspensions were aliquoted for incubation with different antibodies. A minimum of 5000 nuclei of the population of interest per aliquot was generally required to obtain a reliable signal. Nuclei were incubated with primary antibodies overnight at 4 °C and then washed twice before incubation with the secondary antibody (1 h) and washed twice again.

Antibody titration and isotype controls

For every sample labelling, a control experiment was carried out in parallel with a nonspecific antibody of the same isotype at the corresponding final concentration and with the secondary antibody, to reveal background fluorescence. For each primary antibody, several dilutions were tested (serial two-fold dilutions) and the final fluorescence intensity was compared to the labelling with the nonspecific isotype control. The highest specific signal with the best signal-tonoise ratio was determined and used to choose the optimal dilution.

Flow cytometry analysis

All the acquisitions were done using a MACSQuant VYB (Miltenyi Biotec, Bergisch Gladbach, Germany) flow cytometer analyser. The Hoechst fluorescence was collected with a 450/50 nm filter (405 nm laser), GFP/FITC with a 525/50 nm filter (488 nm laser), PE with a 586/15 nm filter (561 nm laser), tdTomato with a 615/20 nm filter (561 nm laser) and APC with a 661/20 nm filter (561 nm laser). For quantitative analysis of the signal, the population of interest was gated, then the mean and median intensity of fluorescence of each channel were given by the MACSQuant software (Miltenyi Biotec, Germany). The background fluorescence, given by the isotype control, was subtracted for each labelling.

Fluorescence-activated nuclei sorting

Sorting was done at the Cell Imaging and Flow Cytometry facility of the IFR83 (Paris, France). We used a Moflo XDP (Beckman Coulter) flow cytometry sorter, equipped with a 70-lm nozzle. We collected Hoechst fluorescence with a 355-nm laser and 450/60-nm filter, and GFP fluorescence with a 488-nm laser and 530/40-nm filter.

Microscopy

A DM600 fluorescence microscope (Leica) was used for image acquisition, with a 10 9 objective. It was carried out at the Institut du Fer a Moulin Imaging Facility.

Results

Preparation quality and gating strategy Most of our experiments were performed using mouse striatum as starting material. The general procedure used is summarised in Fig. 1. We first examined a scatter plot of the particles in the nuclear fraction (Fig. 2A) in which each 'event' (debris or nucleus) is plotted as a single dot whose coordinates (forward scatter and side scatter) are respectively correlated with the volume of the particle and its inner complexity. To distinguish the isolated nuclei from the various debris and aggregated nuclei we plotted the intensity of the side scatter as a function of the Hoechst DNA labelling (Fig. 2B). On this plot the population of nuclei singlets can be easily distinguished from the debris which contain no DNA and the doublets that have twice the Hoechst labelling intensity. The cloud for singlet nuclei was clearly the most visible and contained the majority of the events (Fig. 2B). Large quantities of myelin debris are generated when brain tissue is dissociated, making flow cytometry analysis difficult [START_REF] Young | Use of flow cytometry for high-throughput cell population estimates in brain tissue[END_REF]. This contamination could have been particularly problematic in the striatum which is crossed by numerous myelinated fibre tracts responsible for its striate appearance. Our iodixanol gradient protocol, however, allowed removal of most of the debris and clean nuclear preparations were obtained (Fig. 2B). We then plotted the area vs. the height of the peak of Hoechst signal for all the events containing DNA (Fig. 2C). This plot allowed an accurate selection of nuclei singlets. As the tissue was fixed with formaldehyde before dissociation, it could have been expected to deliver a larger proportion of nuclei multiplets, yet between 85 and 95% of the nuclei events were singlets in our protocol, as compared to ~95% in the absence of fixation (data not shown). On the Hoechst plot (Fig. 2C), some events had twice the area and twice the height as singlets, corresponding to what is commonly considered to be cells in G2M phase, when analysing entire cells. However, sorting and observation under the microscope of these events only revealed nuclei doublets (data not shown). We then selected by gating only the singlet nuclei (Fig. 2D). We obtained ~1.2 million singlets from the two striata of a mouse. Comparison of the raw (Fig. 2A) and gated (Fig. 2D) plots of the same analysis shows our protocol yielded a very clean nuclei preparation. The gating strategy allowed us to perform further analysis without interference from debris or multiplets and was used for all subsequent analyses.

Neuronal and glial nuclei differ by size

When singlet nuclei from mouse striatum were shown in a scatter plot, we observed a broad distribution that could contain several populations of nuclei (Fig. 3A). To determine the position of the nuclei from various cell types in this cloud we used Neuronal Nuclei (NeuN) antibodies that react with a nuclear epitope present in most neuronal populations [START_REF] Mullen | NeuN, a neuronal specific nuclear protein in vertebrates[END_REF], later identified as the splicing factor Fox-3 [START_REF] Kim | Identification of neuronal nuclei (NeuN) as Fox-3, a new member of the Fox-1 gene family of splicing factors[END_REF]. NeuN labelling (Fig. 3B) revealed that non-neuronal (presumably mostly glial) cells were not more numerous than neurons in the mouse striatum, contrary to a common belief but in accordance with previous studies [START_REF] Herculano-Houzel | Isotropic fractionator: a simple, rapid method for the quantification of total cell and neuron numbers in the brain[END_REF][START_REF] Matamales | Signaling from the cytoplasm to the nucleus in striatal medium-sized spiny neurons[END_REF][START_REF] Jordi | Differential effects of cocaine on histone posttranslational modifications in identified populations of striatal neurons[END_REF]. Actually, in our experiments the proportion of neuronal to non-neuronal nuclei in the mouse striatum was close to a 1 : 1 ratio with slightly more neuronal nuclei (Fig. 3C). FAST-FIN does not favour either neurons or glia, as NeuN staining performed on nuclei preparations without fixation led to similar ratios (52% of neurons; data not shown). Moreover, we observed that the population with higher scatter values consisted of neurons (NeuN+), whereas the other population was NeuN-negative (Fig. 3D). This finding was in agreement with histochemical studies showing that nuclei of striatal neurons are larger than those of glial cells [START_REF] Matamales | Striatal medium-sized spiny neurons: identification by nuclear staining and study of neuronal subpopulations in BAC transgenic mice[END_REF]. Furthermore, as background fluorescence was consistently lower in glial nuclei it was possible to distinguish neuronal nuclei from glial nuclei without any labelling, simply by combining scatter and background fluorescence plots (Fig. 3E andF), providing a simple means of identifying these two populations in our experimental conditions, without specific labelling.

Fixation preserved size and prevented soluble proteins from leaking out of the nucleus FAST-FIN includes an early formaldehyde fixation by intracardiac perfusion and we optimised this essential step to prevent proteins Fluorescence-activated sorting of fixed nuclei 1237 from leaking out of the nucleus without preventing tissue dissociation and nuclear preparation. For example, GFP, a very commonly used protein, is present in both the nucleus and the cytoplasm when not fused to any other proteins. To test whether some GFP would remain in the nucleus without fixation and evaluate the importance of the fixation step, we carried out the same protocol with or without formaldehyde fixation, using transgenic mice which express GFP under the control of the D2R promoter (drd2::EGFP). In these mice > 40% of neurons are expected to be GFP-positive [START_REF] Matamales | Striatal medium-sized spiny neurons: identification by nuclear staining and study of neuronal subpopulations in BAC transgenic mice[END_REF]. Without fixation we could not distinguish any GFP-positive population (Fig. 4A), whereas with fixation (Fig. 4B) we could discriminate three peaks, the most intense corresponding to GFP-positive neurons and the two others to glia and GFP-negative neurons (as identified by their background fluorescence, as shown above; see Fig. 3E). Moreover, the difference in nuclear size between neurons and glia was lost without fixation (Fig. 4C andD). Thus, unlike other approaches in which the material is not crosslinked or is cross-linked at a later stage after tissue dissociation or nuclei separation, FAST-FIN prevents leakage out of the nucleus of proteins that are not anchored to the nuclear matrix or DNA and preserves nuclear size.

Estimate of the abundance of specific subpopulations among glial or neuronal cells

We then investigated various cell populations among glia and neurons using immunolabelling. We first used 2′,3 ′-cyclic nucleotide 3′-phosphodiesterase (CNPase) to identify oligodendrocytes [START_REF] Braun | 2′,3′-cyclic nucleotide 3′-phosphodiesterase in the nervous system. Electrophoretic properties and developmental studies[END_REF][START_REF] Sheedlo | The localization of 2′:3′-cyclic nucleotide 3′-phosphodiesterase in bovine cerebrum by immunofluorescence[END_REF]. A control with a nonimmune antibody of the same isotype (isotype control) allowed in each experiment evaluation of the non-specific background fluorescence (Fig. 5A). CNPase-positive singlet nuclei displayed a low side scatter (Fig. 5B), in accordance with the above observation that glial nuclei have a lower side scatter than neurons (Fig. 3). Interestingly, the CNPase-positive events had the largest side scatter among those ascribed to non-neuronal nuclei (Fig. 5B). Based on parallel NeuN labelling we found that, in the mouse striatum, more than half of the glial nuclei were CNPase-positive and thus were derived from oligodendrocytes (Fig. 5C).

The vast majority of striatal neurons are medium-sized spiny neurons (MSNs) that are GABAergic efferent neurons (Kreitzer, 2009). To identify these neurons we used antibodies to dopamineand cAMP-regulated phosphoprotein of 32 kDa [DARPP-32, protein phosphatase 1, regulatory (inhibitor) subunit 1B, PPP1R1B], a protein highly enriched in MSNs [START_REF] Ouimet | DARPP-32, a dopamine-and adenosine 3′:5′-monophosphate-regulated phosphoprotein enriched in dopamine-innervated brain regions. III Immunocytochemical localization[END_REF] that shuttles between nucleus and cytoplasm [START_REF] Stipanovich | A phosphatase cascade by which rewarding stimuli control nucleosomal response[END_REF]. As compared to isotype control, the DARPP-32 labelling gave a specific signal (Fig. 5D andE). DARPP-32-positive singlets had a relatively large side scatter (Fig. 5E) and were included in the large nuclei population (see Fig. 3). As DARPP-32 leaks out of the nucleus in the absence of fixation as readily as GFP (our unpublished observations) the good labelling observed in our conditions Fluorescence-activated sorting of fixed nuclei 1239 demonstrates the efficacy of our fixation protocol. In our analysis 85% of the neuronal nuclei were DARPP-32-positive (Fig. 5F). Comparison of the proportion of DARPP-32-positive neuronal nuclei with FAST-FIN is compatible with the percentage observed by immunohistochemistry taking into account perikarya, as not all MSNs contain nuclear DARPP-32 [START_REF] Ouimet | Quantitative immunocytochemistry of DARPP-32-expressing neurons in the rat caudatoputamen[END_REF][START_REF] Matamales | Striatal medium-sized spiny neurons: identification by nuclear staining and study of neuronal subpopulations in BAC transgenic mice[END_REF].

AB CD

Selectively expressed GFP and Tomato fluorescent proteins allow distinguishing nuclei from the two types of MSNs

The dorsolateral and ventromedial regions of the striatum receive dopaminergic inputs from the substantia nigra pars compacta and the ventral tegmental area, respectively (Voorn et al., 2004). MSNs are divided into two populations according to their projections and the type of dopamine receptors they express (Gerfen et al., 1990). Striatonigral MSNs express dopamine D1 receptors (D1R), whereas striatopallidal MSNs express D2R. Fluorescent proteins such as GFP or Tomato are present in nuclei but in the absence of fixation leak out during nuclear preparation, as they are not fused to any resident nuclear protein. We took advantage of our protocol in which the nuclear localisation of proteins is preserved by fixation to identify the two populations of MSNs with these markers. We used transgenic mice carrying a bacterial artificial chromosome expressing EGFP under the control of either the D1R promoter (Drd1a::EGFP) or the D2R promoter (Drd2::EGFP; [START_REF] Gong | A gene expression atlas of the central nervous system based on bacterial artificial chromosomes[END_REF] or tdTomato under the control of the D1R promoter (Drd1a::tdTomato; Shuen et al., 2008), as well as double transgenic mice (Drd2::EGFP 9 Drd1a::tdTomato). In wild-type mice a dot plot using emission fluorescence for EGFP (525/50 nm) and tdTomato (615/20 nm) showed the background level of fluorescence (Fig. 6A). All events were close to the diagonal, with two dot clouds corresponding to glial cells and neurons (Fig. 6A), as identified on the scatter plot (not shown). In Drd2::EGFP mice, a population of events with a strong signal at 525/50 nm was detected (Fig. 6B), whereas in Drd1a::tdTomato mice a population with a strong signal at 615/20 nm was apparent (Fig. 6C). The tdTomato-positive (Fig. 6C) and EGFP-positive (Fig. 6B) nuclei were very well separated and in double-mutant mice, carrying the two types of transgenes, the two populations could easily be identified simultaneously (Fig. 6D). Using Drd1a::EGFP and Drd2:: EGFP transgenic mice, the proportions of D1R-and D2R-positive nuclei were 46% and 42% of the neuronal nuclei, respectively (Fig. 6E), identified on side scatter vs. background fluorescence plots.

Comparison of nuclear signalling events between glial and neuronal nuclei

One of the aims of FAST-FIN being to study nuclear signalling events in specific nuclear populations, we first examined the differences between nuclei of neuronal and glial origin for a number of markers of interest. We examined two histone post-translational modifications which have been previously reported to be important in the striatum, acetylH4K5 and tri-methylH3K9, respectively associated with active and silent chromatin regions [START_REF] Brami-Cherrier | Parsing molecular and behavioral effects of cocaine in mitogen-and stress-activated protein kinase-1-deficient mice[END_REF][START_REF] Maze | Essential role of the histone methyltransferase G9a in cocaine-induced plasticity[END_REF][START_REF] Jordi | Differential effects of cocaine on histone posttranslational modifications in identified populations of striatal neurons[END_REF]. These two modifications were more abundant in neuronal than in non-neuronal nuclei, with an eight-fold and a 100-fold enrichment, respectively (Fig. 7). We also examined the nuclear enrichment of two proteins, MeCP2 and MBD2, which interact with methylated DNA [START_REF] Klose | Genomic DNA methylation: the mark and its mediators[END_REF]. The immunoreactivity for these proteins was ~10-fold higher in neuronal nuclei than in non-neuronal nuclei (Fig. 7). Finally we examined two phosphoproteins that have been shown to be important in nuclear signalling in striatal neurons, pERK and pMSK1 [START_REF] Brami-Cherrier | Parsing molecular and behavioral effects of cocaine in mitogen-and stress-activated protein kinase-1-deficient mice[END_REF]Bertran-Gonzalez et al., 2008). The signals for these phosphoproteins were six-and 10-fold higher in neuronal than in non-neuronal nuclei, respectively (Fig. 7). In contrast, immunolabelling for CNPase was highly enriched in non-neuronal cells (Fig. 7). These results show that nuclear sorting allows a good preservation of post-translational modifications and a clear distinction between cell types.

Detection of signalling responses in nuclei of a specific MSN population

We then examined whether a drug-induced post-translational modification could be preserved in our experimental protocol. MSK1 is a nuclear protein kinase phosphorylated and activated by ERK in striatal nuclei following a single injection of cocaine, which mediates some of the long-lasting effects of this drug [START_REF] Brami-Cherrier | Parsing molecular and behavioral effects of cocaine in mitogen-and stress-activated protein kinase-1-deficient mice[END_REF]Bertran-Gonzalez et al., 2008). We treated two groups of drd1a::GFP mice with either vehicle or 20 mg/kg cocaine 5 min before killing. The intensity of labelled nuclei in the ventral part of the striatum (the nucleus accumbens) was approximately three-fold increased in GFP-positive nuclei from cocaine-treated mice as compared to vehicle-treated controls, whereas no difference was observed in the rest of the neurons (Fig. 8). This result confirmed the applicability of FAST-FIN for studying acute signalling responses in subpopulations of nuclei.

Isolation of nuclei from a 'rare' cell population

In the striatum as in other brain regions interneurons are less numerous than principal cells and are divided in a variety of subpopulations.

In spite of their relatively low number they play critical functional roles yet they are poorly studied from a biochemical or signalling standpoint due to the difficulty of isolating these cell types in sufficient numbers. We took advantage of the FAST-FIN protocol to select a subtype of nuclei from parvalbumin-expressing interneurons. We used transgenic mice that express EGFP in cells in which the parvalbumin promoter is active and purified nuclei from the striatum and hippocampus of these mice. Before sorting, only a few EGFP-positive Fluorescence-activated sorting of fixed nuclei 1241 nuclei were detected among an overwhelming majority of negative nuclei (Fig. 9A). The GFP-positive nuclei corresponded to 0.6% and 1.2% of the total number of nuclei in these brain regions, respectively.

By fluorescence-activated sorting we were able to isolate ~5000 GFP-positive nuclei from the whole striata and ~10 000 from the hippocampi of one mouse. The obtained fraction contained GFP-positive nuclei singlets which appeared intact (Fig. 9B) and had a 97% purity, as determined by flow cytometry reanalysis. Thus FAST-FIN efficiently allowed purification of a rare population of neuronal nuclei from adult brain tissue.

Discussion

The FAST-FIN method proposed here allows immunolabelling and sorting of nuclei from fixed adult brain tissue. The characterisation of the obtained fractions shows that the contamination by debris is very low and that most nuclei are isolated (singlets), not attached to other nuclei (multiplets). This is very important when considering sorting by flow cytometry. The fast but not excessive fixation preserves the distinct characteristics of the nuclei, including shape, size and protein content. It is noteworthy that size and background intensity, although they are not specific by themselves, when combined allow a reliable distinction between nuclei from neuronal and non-neuronal cells with a flow cytometer, even without any labelling. Moreover, we designed the fixation and labelling steps of the FAST-FIN protocol as very similar to standard protocols extensively used in many laboratories for immunohistochemistry, which have been validated and applied to the study of numerous post-translational modifications. We were actually able to recover strong differences in histone modifications or protein phosphorylation between neurons and glia. We could also readily detect a pre- et al., 2008). We were able to detect not only post-translational modifications of histones that are tightly linked to the chromatin but, importantly, also of proteins that are transiently present in the nucleus (DARPP-32, phospho-ERK1/2). Thus, FAST-FIN allows the quantitative study of very dynamic labile post-translational modifications of presumably any nuclear protein. All these characteristics distinguish the FAST-FIN protocol from previously used approaches that either did not preserve the initial state of the purified nuclei (i.e. preservation of labile proteins or post-translational modifications) and/or were too strongly fixed and highly contaminated with debris [START_REF] Herculano-Houzel | Isotropic fractionator: a simple, rapid method for the quantification of total cell and neuron numbers in the brain[END_REF][START_REF] Collins | A rapid and reliable method of counting neurons and other cells in brain tissue: a comparison of flow cytometry and manual counting methods[END_REF][START_REF] Okada | Flow cytometric sorting of neuronal and glial nuclei from central nervous system tissue[END_REF][START_REF] Young | Use of flow cytometry for high-throughput cell population estimates in brain tissue[END_REF]. The quality of the FAST-FIN protocol in adult mammalian brain is similar to that obtained with a different protocol adapted to drosophila embryos [START_REF] Bonn | Cell type-specific chromatin immunoprecipitation from multicellular complex samples using BiTS-ChIP[END_REF], which are, among other differences, devoid of myelin.

Recently transgenic mice have been developed that express EGFP fused to a ribosomal protein, L10a, which transiently accumulates in nucleoles before ribosomes are exported to the cytoplasm (Doyle et al., 2008;Heiman et al., 2008). These mice provide powerful tools for studying DNA methylation or hydroxy-methylation (Kriaucionis & Heintz, 2009), or histone post-translational modifications in specific nuclei populations [START_REF] Jordi | Differential effects of cocaine on histone posttranslational modifications in identified populations of striatal neurons[END_REF]. Similarly, EGFPtagged histone allowed chromatin immunoprecipitation from purified nuclei [START_REF] Jiang | Isolation of neuronal chromatin from brain tissue[END_REF]. A limitation to these approaches is that they require the availability of the relevant transgenic mouse line. Using FAST-FIN, fluorescent proteins do not need to be fused to any resident nuclear protein and can be maintained in the nuclei during the whole process. This is a considerable advantage as it allows analysis or sorting of nuclei with the most commonly used reporter genes such as EGFP or tdTomato. We applied it to transgenic mice expressing EGFP or tdTomato under the control of promoters specific for either of the two populations of striatal MSNs. The resolution between negative and positive populations was high and resulted in proportions similar to those obtained by tissue section studies (Bertran-Gonzalez et al., 2008;[START_REF] Matamales | Striatal medium-sized spiny neurons: identification by nuclear staining and study of neuronal subpopulations in BAC transgenic mice[END_REF]. For quantitative studies, FAST-FIN allows the automatic study of all recovered nuclei in which fluorescence intensity can be quantitatively assessed, in contrast with histochemistry studies in which an arbitrary threshold is used and the number of cells counted is necessarily limited. Thus FAST-FIN quantification of a positive cell population can be faster, more accurate and more objective than microscopy. Combining fluorescent proteins with antibody labelling, we were able to separate D1R-and D2R-expressing MSNs and examine a signalling response specifically in one of these populations. We were also able to purify a rare population of neurons, parvalbumin-positive interneurons from the striatum or the hippocampus. This opens the possibility for molecular or epigenetic studies in low-abundance brain cell populations, provided a good marker is available.

A particularly interesting prospect for FAST-FIN is its possible application not only to histone post-translational modifications but also to DNA modifications which can be readily studied in fixed material. Therefore FAST-FIN is a simple method that should be useful for studying nuclear molecular markers, signalling events and epigenetic marks in specific neuronal populations, in response to physiological or chemical stimuli or in pathological conditions. In addition it should be applicable to other types of material in which it is important to identify specific nuclei populations, including in cancer. Sorting cells on simple gating parameters can already be useful but much broader applications can be envisaged based on the use of transgenic reporters and immunolabelling.

FIGURE 1 :

 1 FIGURE 1: SCHEMATIC REPRESENTATION OF THE NIGROSTRIATAL, MESOCORTICOLIMBIC AND TUBEROINFUNDIBULAR CIRCUITS. -------------FIGURE 2: DA SIGNALLING THROUGH D1 AND D2 STRIATAL RECEPTOR.

-FIGURE 3 :

 3 FIGURE 3: SCHEMATIC REPRESENTATION OF THE MESOCORTICOLIMBIC AND NIGROSTRIATAL DOPAMINE PROJECTIONS IN THE MOUSE BRAIN. -FIGURE 4: SCHEMATIC WIRING DIAGRAM OF THE BG. -----------------------FIGURE 5: STRIATUM ------------------------------------------------------------------

FIGURE 7 :

 7 FIGURE 7: THE DIFFERENT RELEASE OF DA IN THE PATCH AND MATRIX COMPARTMENTS OF THE STRIATUM.-----------------------------------------------

-FIGURE 8 :

 8 FIGURE 8: DIFFERENT POPULATIONS OF STRIATAL NEURONS. -----------FIGURE 9: MEDIUM-SIZED SPINY PROJECTION NEURONS (SPNS) ---------FIGURE 10: MECHANISM OF ACTION OF COCAINE ON SYNAPTIC TERMINALS OF DA NEURONS.----------------------------------------------------------FIGURE 11: COCAINE-INDUCED STRUCTURAL PLASTICITY -----------------FIGURE 12: SCHEMATIC REPRESENTATION OF THE HOMEOSTATIC MECHANISMS OF THE REGULATION OF FOOD INTAKE -------------------------FIGURE 13: INTERACTION BETWEEN HEDONIC AND HOMEOSTATIC CIRCUIT REGULATING FOOD INTAKE. -----------------------------------------------

  FIGURE 14: SCHEMATIC REPRESENTATION OF THE TISSUE SELECTION. FIGURE 15 FIGURE 1: EXPERIMENTAL DESIGN. ------------------------------FIGURE 16: HIGHLY PALATABLE FOOD INCREASES THE OPERANT TRAINING AND LEADS TO OBESITY. ------------------------------------------------

-FIGURE 17 :

 17 FIGURE 17: HIGHLY PALATABLE FOOD LEADS TO OBESITY. -------------FIGURE 18: EFFECT OF OPERANT CONDITIONING AND FOOD TYPE ON DENDRITIC SPINES.----------------------------------------------------------------------

-FIGURE 19 :

 19 FIGURE 19: OVERVIEW THE DIFFERENTIAL GENE EXPRESSION INDUCED BY THE LEARNING FOR HP FOOD IN D2 SPNS OF THE NAC AND THE DS.

FIGURE 20 :

 20 FIGURE 20: REGULATION OF NORBIN (NCDN) EXPRESSION AFTER TRAINING FOR HP FOOD. --------------------------------------------------------------

  FIGURE 21: THE CONDITIONAL DELETION OF NCDN IN FOREBRAIN NEURONS DIFFERENTIALLY ALTERS THE TWO PHASES OF OPERANT LEARNING. ----------------------------------------------------------------------------------

-FIGURE 22 :

 22 FIGURE 22: EFFECT CHRONIC COCAINE ON SPINES IN THE NAC ---------FIGURE 23: EFFECT CHRONIC COCAINE ON SPINES IN THE DS -----------FIGURE 24: EFFECT CHRONIC COCAINE ON SPINES IN THE DS -----------FIGURE 25: EXPERIMENTAL DESIGN. --------------------------------------------

-FIGURE 26 :

 26 FIGURE 25: EXPERIMENTAL DESIGN. --------------------------------------------FIGURE 26: OVERVIEW THE DIFFERENTIAL GENE EXPRESSION INDUCED BY COCAINE IN D1 AND D2 SPNS OF THE NAC ------------------------------------

-FIGURE 27 :

 27 FIGURE 27: DIFFERENTIAL GENE EXPRESSION INDUCED BY COCAINE IN D1 NEURONS OF THE PFC. --------------------------------------------------------------

  FIGURE 28: DIFFERENTIAL GENE EXPRESSION INDUCED BY COCAINE IN D1 NEURONS OF THE PFC. --------------------------------------------------------------

  FIGURE 29: MODULE ANALYSIS ON D1-NAC-SPNS REVEALS CLUSTERS OF GENES THAT CO-CLUSTER IN RESPONSE TO COCAINE TREATMENT ------FIGURE 30: MODULE ANALYSIS ON D1-PFC-SPNS REVEALS CLUSTERS OF GENES THAT CO-CLUSTER IN RESPONSE TO COCAINE TREATMENT ------FIGURE 31: GOS ANALYSIS ON THE MODULE MORE ASSOCIATED TO THE COCAINE PRESENCE OR ABSENCE. ---------------------------------------------------

-FIGURE 32 :

 32 FIGURE 32: GENES COEXPRESSION NETWORKS OF BLUE MODULE IN NAC-D1-SPNS -------------------------------------------------------------------------------

-FIGURE 33 :

 33 FIGURE 33: GENES COEXPRESSION NETWORKS OF BLUE MODULE IN PFC-D1-SPNS -------------------------------------------------------------------------------

  FIGURE 34 SUMMARY OF THE EFFECTS OF OPERANT CONDITIONING FOR FOOD AND CHRONIC COCAINE TREATMENT ON DENDRITIC SPINES.

  FIGURE 35: COMPARISON OF THE EFFECTS OF OPERANT CONDITIONING, FOOD TYPE AND CHRONIC COCAINE TREATMENT ON THE TRANSCRIPTIONAL PROFILES OF D2-NAC-SPNS. --------------------------------

-FIGURE 39

 39 FIGURE 38 GRAPHICAL CONCLUSION AIM-3 -----------------------------------

  ) is a molecule that belongs to the catecholamine family and plays

Figure 8 :

 8 Figure 8: Different populations of striatal neurons. Different types of striatal cells described by Ramón y Cajal (Ramón y Cajal, 1911).

  upregulated in D2 after acute treatment and in both D1 and D2 after chronic treatment Vamp2 and Kcnd2 up-regulated by chronic cocaine treatment in D1 and already described in McClung and Nestler, 2003 and Zfp64 up-regulated striatopallidal neurons after acute cocaine treatment and down-regulated in striatopallidal following the chronic treatment (McClung & Nestler 2003) Although really instructive, this work still presents two major limitations: first the mRNA study has been performed by microarray, that present as prior limitation the fact that only the genes on the chip can be investigated; second the study has been conducted in the full striatum. In the light of the different connections and responses in the NAc and the DS a profiling of D1 and D2 neurons taking into account the different location of the neurons in the striatum would give a more comprehensive view of the changes occurring in the striatum after the stimulation of the reward system.

Figure 12 :

 12 Figure 12: Schematic representation of the homeostatic mechanisms of the regulation of food intake In the arcuate nucleus, nneuropeptides AGRP and NPY stimulate food intake, whereas αMSH and CART inhibit food intake. Insulin and leptin are produced by the adipose stores. They inhibit AGRP/NPY neurons and stimulate adjacent POMC/CART neurons. The circulating peptide ghrelin is secreted from the stomach and can activate AGRP/NPY neurons and stimulate appetite.

  system is characterized by large cell heterogeneity as well as remarkable flexibility in gene expression that modulates the correct action in response to a wide variety of environmental cues. Multicellularity represents a serious limit to study the alterations in gene regulation within individual cell types. The very first representation of the cell composition and diversity of the central nervous system dates from the end of the XIX th century, when Ramon y Cajal used Golgi staining to show that neurons are one main component of the mammalian central nervous system (CNS) and to illustrate their diverse appearance. Ever since Santiago Ramon y Cajal discovered the varied structure of neurons, scientists have attempted to classify them into discrete groups. The problem of distinguishing different subpopulations of neurons is particularly evident in the striatum. D1 and D2 SPNs are mostly indistinguishable in shape, size and number but they are highly segregated in two inhibitory outputs, and form two main efferent pathways -the direct and indirect pathway -that respectively activate or inactivate their end targets. Considering this heterogeneity, it is crucial to distinguish the two different populations. Analysing lysates of pooled neuronal populations averages the responses, masks low signals and changes in opposite directions in the two populations can cancel each other.

  In 2011 Sharp BM and collaborators, used the LCM technology to purify GABAergic neurons projecting from NAc to ventral pallidum, and compared by microarray the gene expression in GABAergic neurons projecting from NAc to ventral pallidum in inbred Lewis and Fisher 344 rats. This work allowed the identification of a group of genes (Mint-1, Cask, CamkII , Ncam1, Vsnl1, Hpcal1, and Car8) possibly involved in the higher susceptibility of the Lewis strand to selfadminister cocaine. The same technique, followed by RT-PCR, has been used in 2006 by Perez-Manso et al. in order to investigate the changes in gene expression of the vGLUT2 thalamostriatal pathway in unilaterally 6-OHDA lesioned rats. In 2003 it has been

  Flow cytometry, is a laser-based technology that measures and analyses the optical properties of single particles such as cells, nuclei, bacteria, passing in a single file through a focused laser beam. Physical properties, such as size (represented by forward angle light scatter) and internal complexity (represented by right angle scatter) can resolve certain cell populations. The laser can also excite fluorophores used to mark various molecules. The use of fluorophores with different fluorescence characteristics, multiple lasers and multiple photo-detectors allows flow cytometers to measure many characteristics of each particle simultaneously. An important feature of this technique is that with flow cytometer thousands of particles per second are analysed. This allows a strong statistically significant picture of a specimen's physical and biochemical make-up. FACS (fluorescence-activated sorting) allows the physical separation of particles of interest from a heterogeneous population in addition to collection of data. Particles are held into a stream of fluid, which breaks up into droplets, each of them containing one particle. Droplets are introduced into the laser beam for interrogation.

  and maintained as heterozygotes on a C57Bl/6J background. Male and female mice were maintained on a 12 h light/dark cycle (light off 19:00 hours) and had, before the beginning the experiment, free access to water and food. Animal protocols were performed in accordance with the National institutes of Health Guide for the Care and Use of Laboratory Animals, and approved by Rockefeller University's Institutional Animal Care and Use Committee; or in accordance with the guidelines of the French Agriculture and Forestry Ministry for handling animals (decree 87-848) under the approval of the "Direction Départementale de la Protection des Populations de Paris" (Authorization number C-75-828, license B75-05-22)

[

  pH 7.4], 5 mM MgCl 2, 150 mM KCl, 0.5mM DTT, 100 µg/ml CHX protease and the RNAse inhibitors Superasin (final concentration 200 U/mL, Life Technologies) and Rnasin (final concentration 400 U/mL, Promega) with loose and tight glass-glass 2 ml Dounce homogenizers. Each homogenate was centrifuged at 2000 x g, at 4°C, 6 -Material & Methods -104 for 10 minutes. The supernatant was separated from cell debris, and supplemented with NP-40 (EDM Bioscences) (final concentration of 1% vol/vol) and DHPC

  300 µL of Streptavidin MyOne T1 Dynabeads (Invitrogen) per sample were washed in PBS, incubated 35 min at room temperature with 120 µg of biotinylated protein L in PBS, washed 5 times with PBS Bovine Serum Albumine 3% (wt/vol), incubated 1h at room temperature with 100 µg of monoclonal anti-GFP antibodies (50 µg of clone 19F7 + 50 µg of clone 19C8, Memorial Sloan-Kettering Monoclonal Antibody Facility, New York) in the homogenization buffer containing 1% (vol/vol) NP-40, washed 3 times and finally ressuspended in 200 µL of homogenization buffer complemented with 1% (vol/vol) NP-40. The mixture of magnetic beads coated with anti-GFP antibody was added to the homogenates. After addition of Superasin (final concentration 200 U/mL, Life Technologies) and Rnasin (final concentration 400 U/mL, Promega), the samples underwent 18 h incubation at 4°C under gentle endover-end rotation. After 4 washes with homogenization buffer complemented with 1% (vol/vol) NP-40 and 200 mM KCl (total concentration KCl 350mM), the RNA was eluted with RLT Plus buffer from the RNeasy micro plus kit (Qiagen) and 10 µL/mL β-mercaptoethanol (10 min incubation at room temperature and vortex).

  dilution 1:5000) and visualized with Odyssey-LI-COR infrared fluorescence 6 -Material & Methods -108 detection system (LI-COR). The optical density after acquisition was assessed using the GELpro32 software. Results were normalized to the detection of β-tubulin in the same sample and plotted as percentage of the control treatment. 6 -Materials & Methods -1096.10 Golgi-cox staining and analysis of spine density and morphologyThe Golgi staining was performed as in Marco S. et al., 2013. Briefly, 24 h after the last training session or the last cocaine injection mice were sacrificed by cervical dislocation and their brain was quickly dissected out. One mouse brain hemisphere was subsequently dipped in the Golgi dye filtered solution (1% potassium dichromate, 1% mercury chloride and 0.8% potassium chromate. After 21 days incubation in dark, each hemisphere was washed 3 x 2 min in distilled water, and EtOH 90 % (v/v) for 30 minutes. The brains were next cut with a vibratome (Leica) in a 200 microns slices in a solution of 70 % (v/v) EtOH. Slice, were next washed for 5 minutes in distilled water. The slices were then reduced in 16 % (v/v) ammonia for 60 minutes, washed 2 minutes in distillate water for 2 minutes, and mounted on coverlips (ThermoFisher). As next dehydration of the slices was performed by placing the coverslips for 3 min in 50% (v/v), then 70, 80 and 100% (v/v) EtOH.

7. 1

 1 Aim 1: Characterization of the basal gene expression in the different neuronal populations expressing either D1 or D2 receptors in the NAc, and DS, or, for D1 in PFC.

  2-Sorbonne Universités, UPMC, Université Paris 06, Paris, 75005, France; 3-Institut du Fer à Moulin, 17 rue du Fer à Moulin, Paris, 75005, France; 4-Laboratory of Molecular and Cellular Neuroscience, the Rockefeller University, 1230 York Avenue, New York, NY 10065, USA * To whom correspondence should be sent: Dr Jean-Antoine Girault Inserm UPMC UMR-S 839 Institut du Fer à Moulin 17, rue du Fer à Moulin 75005 Paris, France. Contact: jean-antoine.girault@inserm.fr Running Title: Gene expression in dopaminoceptive neurons

  experiment, free access to water and food. Animal protocols were performed in accordance with the National institutes of Health Guide for the Care and Use of Laboratory Animals, and approved by Rockefeller University's Institutional Animal Care and Use Committee or in accordance with the guidelines of the French Agriculture and Forestry Ministry for handling animals (decree 87-848) under the approval of the "Direction Départementale de la Protection des Populations de Paris" (Authorization number C-75-828, license B75-05-22 ).

Figure 1 :

 1 Figure 1: D1 and D2 BAC-TRAP mice and experimental design.

Figure 2 :

 2 Figure 2: Differential gene expression in the PFC and striatum of D1 BAC-TRAP mice.

Figure 3 :

 3 Figure 3: Differential gene expression in D1 and D2 BAC-TRAP mice.

Figure 4 :

 4 Figure 4: Differential gene expression between the DS and the NAc.

Figure 5 :

 5 Figure 5: Expression analysis of selected mRNAs enriched in DS, NAc or PFC.

Figure 6 :

 6 Figure 6: Ingenuity pathway analysis of upstream regulators of genes differentially expressed in the DS and NAc.

Figure 7 :

 7 Figure 7: Behavioral effects of a prostaglandin agonist on dorsal striatum function.

Figure 16 :

 16 Figure 16: Highly palatable food increases the operant training and leads to obesity. A. Outline of the operant training schedule. Mice had to nose poke to obtain food pellets with the indicated fixed ratios (FR). B. Daily number of positive pokes in mice working for highly palatable (master, mHP, blue) and standard (master, mST, red) food, and in their respective yoked controls (yHP in green, yST in purple). C. Daily number of obtained pellets in the same mice as in B (same color code). D. Daily number of consumed pellets in the same mice as in B. in B-D, data are expressed as means +/-SEM, n = 36-38. Two-way ANOVA, followed by Bonferroni Post-hoc test mS vs mHP *** p<0.00

  the transcriptional modifications induced by operant training in D1 and D2 neurons of NAc, DS, and PFC

  Continuous consumption of high caloric food produces neuroadaptive changes in the brain reward systems that may drive the development of compulsive eating (P. M.[START_REF] Johnson | Dopamine D2 Receptors in Addiction-like Reward Dysfunction and Compulsive Eating in Obese Rats[END_REF]. Although much work has been done on natural rewards, a clear description of the corresponding transcriptional profiles is still missing. Thus we aimed to identify the transcriptional profiles in D1 and D2 SPNs in the NAc, DS and D1 pyramidal neurons of the PFC in mice working in an operant training paradigm to obtain either standard or HP food and in yoked control mice that received the same foods passively. To do so we used transgenic mice that express a tagged ribosomal protein (L10a-EGFP) under the control of the D1 or D2 receptor promoter to isolate currently translated mRNA(Heiman et al. 2008) from each population of SPNs , as well as from D1 pyramidal neurons of the PFC. mRNAs were studied 24 h after the last training session. For each region and each population of neurons we performed three different types of comparisons: 1) the master groups versus their respective control (mHP vs yHP and mST vs yST), 2) the mice working for highly palatable food versus the mice working for standard food (mHP vs mST), and 3) the mice non-contingently receiving highly palatable or standard food (yHP vs yST). Those comparisons were done to gain insight on two different aspects of the regulation of the gene expression: the regulation induced by the learning for each food (master vs yoked and master vs master) and the regulation induced by the simple exposure to the food yoked vs yoked. The largest changes were driven by the learning for the highly palatable food in the D2 SPNs (101 up regulated genes, 135 down regulated) of both NAc and DS (622 up regulated genes, 417 down regulated genes) (adj pv <0.05 FC>1.3 FC<0.7 reads number>30).Furthermore, the comparison of the transcriptional profile in the yoked mice showed that in the D2 neurons of the DS, a large number of genes is regulated by the consumption of the HP food. Together these results suggest that the D2 neurons undergo a more stable modification of the gene expression following the stimulation of the reward system by HP food (Figure19) To further characterize the observed -Experimental results -7 123 changes in gene expression we observed the population-region-treatment-gene ontologies (GOs). In the D2 SPNs of the DS we found an enrichment in genes involved in the GABA transmission (GABA receptor adj pvalue= 0.01, GABA-A receptor adj pvalue=0.008) In the D2 SPNs we found enrichment in genes involved in the regulation of the chromatin (e.g. nucleosome DNA binding adj p-value=0.00008, chromatin binding, adj p-value=0.0002, transcription activity adj p-value=0.001).

  were up-regulated by conditioning for ST food and down-regulated by conditioning for HP food, 29 are down-regulated by conditioning for ST food and up-regulated by the learning for the HP food. The opposite tendency can be observed in the DS, where among the 10 genes in common between the 2 types of conditioning, only 3 were regulated in opposite fashion. Interestingly, among those 3 genes Fos was down-regulated in the D2 neurons of the DS of mice working for HP food. The D1 neurons share much less common genes. Only one gene was common to HP and ST conditioning in both the NAc and DS, which is the nuclear factor of kappa light -Experimental results -7 polypeptide gene enhancer in B cells inhibitor, epsilon (Nfkbie). The same types of comparisons are in progress for the neurons in PFC, DS and NAc. 7.2.7 Probing a candidate gene in operant learning for highly palatable food: the possible role of norbin To validate our analysis of the effects of the operant training on the regulation of gene expression, we tested the consequence of the genetic manipulation of one of the genes differentially regulated by conditioning for HP food. We focused on Ncdn, a gene coding for the protein norbin (also known as neurochondrin) as our analysis showed that conditioning for HP food exerted an opposite regulation on Ncdn transcription in the D2-SPNs of the NAc and the DS (Figure20 A & B). We hypothesized that if this regulation is functionally relevant, the deletion of Ncdn in neurons should have a specific effect on the behavioural consequences of training for HP food. We first used two different techniques, in two independent experiments, to confirm the positive regulation on the Ncdn gene expression exerted by the operant training for highly palatable food. RT-PCR on D2-mRNA, purified by immunoprecipitation with anti-GFP antibodies from drd2::L10a-GFP transgenic mice, confirmed the up-regulation of the Ncdn mRNA in the DS of mice trained with

  induced structural plasticity in NAc, DS, and

  with two genes inversely regulated in the Nac and the DS (upregulated in the NAc and own regulated in the DS): Bdnf and Lypd6b (LY6/PLAUR Domain Containing 6B). In D2 SPNs cocaine increases increase 177 genes, and decrease 246 genes in D2 SPNs (Fig. 6A and B), with only one gene in common with the D2-NAc: MAM domain containing glycosylphosphatidylinositol anchor 1 (Madga1) up regulated in the DS-D2 and down regulated in the D2-NAc. -induced gene expression changes in D1 pyramidal neurons of the PFC Several lines of studies have already shown that the PFC plays a key role in the development of addiction to cocaine. Human studies of post mortem dorsolateral

  of the effects on dendritic spines induced by highly palatable food and chronic cocaine injections First, we compared the structural plasticity induced in the NAc, the DS and PFC in the various treated groups as compared to their respective controls (See experimental results aim 2

  one of the genes regulated in the D2 neurons in both NAc and DS. Norbin (encoded by the Ncdn gene) was up-regulated by the learning for HP food in the DS while it was down-regulated in the NAc. In line with the general hypothesis that a certain regulation of the gene is related to the facilitation of a certain behavioural output, we observed two different behavioural effects of the norbin-KO that correlated with the differential regulation of the expression of this gene in NAc and DS. As already explained our training paradigm encompassed two different phases: a learning phase in which animals are under food restriction, and an ad libitum phase in the established. According to the view of "dorsalization" of the striatal function in the paradigms in which a reward is associated to a cue, we might speculate that the first phase of operant training would be more associated to the activation of the nucleus accumbens, while the second phase would be more associated to the activation of the DS. The norbin-ko mice showed an amelioration of the behaviour during the learning phase and an impairment during the ad libitum phase, in which mice worked at the same rate for HP food as those trained with standard food. This result was particularly clear when mice were presented with a PR schedule (figure21B-D).

Fig. 1 .

 1 Fig. 1. Outline of the FAST-FIN procedure.

Fig. 2 .

 2 Fig. 2. Flow cytometry characterisation of purified nuclei and gating strategy. (A) Scatter dot plot (Forward Scatter, Side Scatter) of all recorded events from a nuclear preparation made with the FAST-FIN protocol. (B) Dot plot of the side scatter as a function of the Hoechst fluorescence intensity (area of the fluorescence peak), for all the events. The blue rectangle defines the events considered as Hoechst-positive (in this case 56% of total events), including nuclei singlets and multiplets. (C) Dot plot of the height of the fluorescence peak as a function of the area of the peak, for each event from the previously selected population (nuclei). Nuclei singlets are circled in red and represent in this example 86% of the total Hoechst-positive events. (D) Scatter dot plot (Forward Scatter, Side Scatter) of the nuclei singlets only.

Fig. 3 .

 3 Fig. 3. Neuronal and non-neuronal nuclei differ by simple flow cytometry parameters. (A) Scatter density plot of all the singlet nuclei. Blue dots correspond to sparse events whereas green and yellow dots indicate increasingly higher densities of events. (B) Histogram of the frequencies of measured fluorescence intensity values. NeuN immunolabelling was revealed using a phycoerythrin-conjugated secondary antibody. (C) Estimate of the proportion of NeuN-positive nuclei. Data are means + SEM (n = 4 mice). (D) Scatter dot plot of the nuclei singlets, as in (A), in which NeuN-positive events are coloured in red whereas NeuNnegative events are in blue. This staining allows clear the identification of the two populations in (A). (E) Density plot of the background fluorescence (a channel with no labelling, here with a 525/50 nm filter and a 488 nm laser) as a function of the side scatter. The population with the lowest Side Scatter and background fluorescence is circled in green and the other population is circled in orange. (F) Superimposition of histograms of frequencies of measured fluorescence intensity values. NeuN labelling was revealed with a phycoerythrin-conjugated secondary antibody (as in B). All the nuclei singlets are plotted in black, whereas the two populations selected in (E) are plotted in green and orange, respectively.

Fig. 4 .

 4 Fig. 4. Fixation preserves size and prevents soluble proteins from leaking out of the nucleus. Nuclei preparations from drd2::EGFP mice, without (A and C) or with (B and D) formaldehyde fixation by intracardiac perfusion. (A and B) Histograms of the frequencies of measured flurorescence intensity values (525/ 50 nm), normalised to the area under the curve. (C and D) Dot plots of nuclei singlets according to their side scatter and their fluorescence intensity (525/ 50 nm). In nuclei preparation from fixed brain tissue (B and D) it is easy to recognise distinct nuclear populations which were identified as 'glial' and 'neuronal' nuclei as in Fig. 3.

Fig. 5 .

 5 Fig. 5. Selective analysis of glial and neuronal subtypes nuclei. Dot plots of nuclei singlet events according to their side scatter and their fluorescence intensity at (A and B) 586/15 nm or (D and E) 661/20 nm. In all plots non-neuronal and neuronal nuclei were identified according to their side scatter and background fluorescence as in Fig. 3E and plotted in blue and red, respectively. (A) Labelling with a mouse IgG1 primary isotype control and a PE-conjugated secondary antibody. (B) Labelling of nuclei singlets from the same animal with a CNPase primary antibody. (C) Estimate of the proportion of CNPase-positive nuclei among non-neuronal nuclei, mean + SEM (n = 4 mice). (D) Labelling with a rabbit IgG primary isotype control and an APC-conjugated secondary antibody. (E) Labelling of nuclei singlets from the same animal, with a DARPP-32 primary antibody. (F) Estimate of the proportion of DARPP-32-positive nuclei among neuronal nuclei; mean + SEM (n = 4 mice).

FluorescenceFig. 6 .

 6 Fig. 6. Selective analysis of medium-sized spiny neuron subtypes in transgenic mice expressing EGFP and tdTomato fluorescent proteins. Density plots using emission fluorescence for EGFP (525/50 nm) and tdTomato (615/20 nm). (A) Nuclei singlets from a wild-type animal (background fluorescence). Neuronal and non-neuronal nuclei were identified as in Fig. 3E and were clearly distinguished by their background fluorescence. (B) Nuclei singlets from a drd2::EGFP mouse. (C) Nuclei singlets from a drd1a::tdTomato female mouse (see Materials and Methods for explanation of male-female differences). (D) Nuclei singlets from a drd1a::tdTomato drd2::EGFP double transgenic male mouse. (E) Proportion of EGFP-positive nuclei among neuronal nuclei from drd1a::EGFP mice (n = 8) and drd2::EGFP mice (n = 4). Data are means AE SEM.

Fig. 7 .

 7 Fig.7. Comparison of signalling markers between non-neuronal and neuronal nuclei. Flow cytometry analysis of nuclei immunostained for H4 acetyl-Lys5 (acH4K5), H3 trimethyl-Lys9 (me 3 H3K9), MeCP2, MBD2, phospho-ERK1/2 (pERK) and phospho-Thr581-MSK1 (pMSK1). CNPase labelling was used as a control to show that labelling can also be stronger in nonneuronal than in neuronal nuclei. The amount of nuclear immunoreactivity was estimated as the median of the immunofluorescence intensity minus background. Neuronal and non-neuronal nuclei were identified according to their scatter plot as in Fig.3E. Data are means + SEM plotted on a log 2 scale (n = 8 mice).

Fig. 8 .BFig. 9 .

 89 Fig.8. Acute cocaine administration increased MSK1 phosphorylation specifically in D1R neurons of the nucleus accumbens. drd1a::GFP mice were injected with 20 mg/kg cocaine or vehicle, anesthetised with pentobarbital after 5 min and perfused with formaldehyde when reflexes were abolished (~2 min). Nucleus accumbens was dissected and nuclei preparations were labelled with phospho-Thr581-MSK1 and anti-GFP antibody. The amount of nuclear phosphoMSK1 was estimated as the geometric mean of the immunofluorescence intensity (661/20 nm, minus background) in GFP-positive and GFP-negative neuronal nuclei. Data are means + SEM of results from four saline-and three cocaine-treated mice. Two-way ANOVA: drug effect, F (1,10) = 135.5, P < 0.0001; cell-type effect, F (1,10) = 92.9, P < 0.0001; interaction, F (1,10) = 92.9, P < 0.0001). Bonferoni post hoc test, ***P < 0.001; n.s., non-significant.

Table 3 : Examples of highly significantly overrepresented GOs in DS or NAc as compared to PFC

 3

	ID	Description

Table 4 : Top 30 most significant genes with mRNA enriched in D1 as compared to D2 neurons of the DS

 4

	Gene	Gene Description	log2 FC	padj	D1 mean D2 mean
	Arx	aristaless related homeobox	-3.215	2.70E-27	675
	Chrm4	cholinergic receptor, muscarinic 4	-2.957	2.70E-27	2 110
	Fxyd7	FXYD domain-containing ion transport regulator 7	-2.147	2.43E-17	1 036
	Tac1	tachykinin 1	-2.560	3.48E-17	19 058	1
	Hpcal1	hippocalcin-like 1	-2.202	4.50E-16	833
	Cntnap3	contactin associated protein-like 3	-2.619	6.30E-15	583
	Gnb4	guanine nucleotide binding protein (G protein), beta 4	-2.035	1.61E-13	2 038
	Dgkz	diacylglycerol kinase zeta	-1.739	5.89E-13	5 524	1
	Stmn2	stathmin-like 2	-1.805	7.62E-13	6 869	1
	Tns1	tensin 1	-1.648	6.40E-12	2 059
	Nrxn1	neurexin I	-1.318	1.62E-11	9 549	4
	Rasgrf2	RAS -specific guanine nucleotide-releasing factor 2	-2.078	1.67E-11	2 405
	Itga8	integrin alpha 8	-2.604	2.19E-11	101
	Myh3	myosin, heavy polypeptide 3, skeletal muscle, embryonic	-2.565	2.24E-11	206
	Isl1	ISL1 transcription factor, LIM/homeodomain	-2.448	2.44E-11	1 347
	Cpeb1	cytoplasmic polyadenylation element binding protein 1	-1.520	3.14E-10	1 160
	Hs3st2	heparan sulfate (glucosamine) 3-O-sulfotransferase 2	-1.944	6.81E-10	563
	Cplx3	complexin 3	-2.240	7.38E-10	991
	Gng2	guanine nucleotide binding protein (G protein), gamma 2	-1.885	7.51E-10	7 636	1
	Tmem178 transmembrane protein 178	-1.962	2.80E-09	620
	Ube2ql1	ubiquitin-conjugating enzyme E2Q family-like 1	-1.778	3.01E-09	2 757
	Otof	otoferlin	-1.665	4.13E-09	604
	Dlk1	delta-like 1 homolog (Drosophila)	-2.227	4.26E-09	373
	Scn1b	sodium channel, voltage-gated, type I, beta	-1.413	5.47E-09	3 740	1
	Pitpnm3	PITPNM family member 3	-1.691	6.43E-09	2 562
	Rassf3	Ras association (RalGDS/AF-6) domain family member 3	-1.595	8.62E-09	1 590
	Ikzf1	IKAROS family zinc finger 1	-1.993	1.16E-08	378
	Cpne9	copine family member IX	-1.486	1.28E-08	934
	Lingo2	leucine rich repeat and Ig domain containing 2	-2.174	1.29E-08	980
	Lrpap1	LDL receptor-related protein associated protein 1	-0.988	1.67E-08	4 596	2

Table 5 : Top 30 most significant genes with mRNA enriched in D2 as compared to D1 neurons of the DS

 5

	Gene	Gene Description	log2 FC	padj	D1 mean D2 mean
	Adora2a	adenosine A2a receptor	4.865	2.20E-69	327	19
	Gpr52	G protein-coupled receptor 52	3.396	1.04E-61	191	2
	Nt5e	5' nucleotidase, ecto	3.692	2.63E-57	72	1
	Penk	preproenkephalin	4.176	1.26E-51	4 970	189
	P2ry1	purinergic receptor P2Y, G-protein coupled 1	3.721	9.78E-48	84	2
	Oprd1	opioid receptor, delta 1	3.962	3.73E-43	77	2
	Sp9	trans-acting transcription factor 9	3.539	5.98E-41	127	2
	Fig4	FIG4 phosphoinositide 5-phosphatase	2.292	1.62E-39	460	2
	Gpr6	G protein-coupled receptor 6	3.711	6.78E-31	58	1
	Upb1	ureidopropionase, beta	3.332	1.48E-19	2
	Gucy1a3 guanylate cyclase 1, soluble, alpha 3	1.671	1.59E-16	7 095	30
	Necab1	N-terminal EF-hand calcium binding protein 1	1.912	3.04E-16	3 705	18
	Grik3	glutamate receptor, ionotropic, kainate 3	2.577	3.15E-16	936	9
	Sema3e	semaphorin) 3E	1.446	3.66E-14	1 014	3
	Prkd1	protein kinase D1	1.694	7.99E-12	250	1
	Ptprm	protein tyrosine phosphatase, receptor type, M	2.181	4.69E-11	367	2
	Gpr88	G-protein coupled receptor 88	1.180	1.16E-10	20 501	60
	Gprin3	GPRIN family member 3	0.976	2.93E-10	2 588	5
	Fnip2	folliculin interacting protein 2	1.519	2.02E-09	445	1
	Nell1	NEL-like 1	1.857	2.89E-09	177
	Kctd12	K+ channel tetramerisation domain containing 12	1.024	6.67E-09	2 648	6
	Adk	adenosine kinase	1.759	1.29E-08	244	1
	Wnt7a	wingless-type MMTV integration site family, 7A	1.800	2.29E-08	85
	Mro	maestro	1.844	2.89E-08	209	1
	Thpo	thrombopoietin	2.091	3.06E-08	28
	Sox11	SRY (sex determining region Y)-box 11	1.380	3.68E-08	608	2
	Trim62	tripartite motif-containing 62	1.493	4.01E-08	605	2
	Galnt13	UDP-N-ac-αD-galactosamine:polypept. transferase 13	1.057	5.43E-08	3 019	7
	Tacr1	tachykinin receptor 1	1.500	6.41E-08	128
	Kcnk2	potassium channel, subfamily K, member 2	1.067	6.86E-08	3 940	9

Table 6 : IUPHAR data base-selected genes with mRNA enriched in D1 as compared to D2 neurons in the DS*

 6

	Type	Family name	MGI symbol	Mean	log2 FC	padj
		GDNF receptor family	Gfra1 Gfra2	238 428	-1.69 -0.98	2.82E-05 9.90E-03
		IL-17 receptor family	Il17rc	83	-1.16	5.68E-03
	Catalytic	Integrins	Itga8	52	-2.60	2.19E-11
	receptors		Ptprt	2 190	-0.99	1.26E-03
		Receptor tyrosine phosphatases (RTP)	Ptprf	653	-0.80	1.02E-02
			Ptprn	7 117	-0.55	4.09E-02
		Type I RTKs: ErbB (EGF) receptor family	Erbb4	253	-1.64	4.13E-07

* Translated mRNAs were isolated from the dorsal striatum of Drd1::BAC-TRAP and Drd2::BAC-TRAP mice. Only identified in the IUPHAR nomenclature, with adjusted p value <0.05 and expression levels > 30 reads are included. Fold change D2/D1. Abbrev.: FC, fold-change, GPCR, G protein-coupled receptor, IC, ion channel, IUPHAR, International Union of Basic and Clinical Pharmacology, LGIC, ligand-gated ion channel, MGI, mouse genome informatics database symbol, NHR, nuclear hormone receptor, padj, adjusted p value, VGIC, voltage-gated ion channel. http://www.guidetopharmacology.org/download.jsp

Table 7 : Selected IUAPHAR genes whose mRNA is enriched in D2 as compared to D1 neurons in the DS*

 7

	Type	Family name	MGI symbol	Mean	log2 FC	padj
		Receptor tyrosine phosphatases (RTP)	Ptpro Ptprm	1	1.09 2.18	1.79E-05 4.69E-11
	Catalytic receptors	Type II receptor serine/threonine kinases Type II RTKs: Insulin receptor family	Acvr2a Tgfbr2 Igf1r	1 2	0.80 1.87 0.68	1.14E-02 2.64E-06 2.13E-02
		Type IV RTKs: VEGF receptor family	Flt4		1.50	2.89E-04
		Type XIX RTKs: LTK receptor family	Alk		1.46	1.09E-03
		1.14.11.-Histone demethylases	Kdm6b	1	1.02	2.01E-03
		2.1.1.43 Histone methyltransferases (HMTs)	Setd8 Kmt2e	4 23	0.56 0.62	2.16E-02 1.54E-02
			Kat6a	6	0.48	1.00E-02
		2.3.1.48 Histone acetyltransferases (HATs)	Ncoa2	10	0.58	2.44E-02
			Kat2b		0.67	3.60E-02
		2.4.2.30 poly(ADP-ribose)polymerases	Parp3		1.38	3.70E-04
		Acetylcholine turnover	Chat		1.69	6.85E-05
		Adenosine turnover	Adk Nt5e		1.76 3.69	1.29E-08 2.63E-57
		Arginine:glycine amidinotransferase	Gatm		0.77	3.32E-02
		CAMK1 family	Camk4	47	0.46	2.86E-02
		CLK family	Clk1		0.75	3.32E-04
	Enzymes	Lipid phosphate phosphatases M13: Neprilysin	Pten Mme	18 7	0.72 0.93	4.83E-05 2.75E-04
		MLK subfamily	Zak		1.29	9.51E-07
		Phosphatidylinositol kinases	Pik3c2a Pik3r5	1	0.73 0.98	3.73E-03 2.96E-02
		Phosphatidylinositol-4-phosphate 3-kinase	Pik3c2a	1	0.73	3.73E-03
		Phosphodiesterases, 3',5'-cyclic nucleotide	Pde4b Pde3a	17	0.73 1.20	1.11E-04 1.25E-02
		Protein kinase D (PKD) family	Prkd3 Prkd1		0.80 1.69	3.78E-02 7.99E-12
		RCK family	Ick		0.78	1.14E-02
		Soluble guanylyl cyclase	Gucy1b3 Gucy1a3	8 18	0.66 1.67	1.84E-03 1.59E-16
		Trbl family	Trib2	2	0.73	5.52E-04
		YANK family	Stk32a	2	0.53	1.22E-02
		5-Hydroxytryptamine receptors	Htr2c	1	1.20	1.37E-04
		Acetylcholine receptors (muscarinic)	Chrm3		1.02	3.64E-02
		Adenosine receptors	Adora2a	10	4.86	2.20E-69
		Calcitonin receptors	Calcrl		1.11	3.07E-02
			Gpr88	40	1.18	1.16E-10
	GPCR	Class A Orphans	Gpr149 Gpr52 Gpr6	1	1.38 3.40 3.71	9.35E-07 1.04E-61 6.78E-31
		Histamine receptors	Hrh3	9	0.94	3.47E-05
		Opioid receptors	Oprm1		0.82	1.79E-02
		Opioid receptors	Oprd1	1	3.96	3.73E-43
		P2Y receptors	P2ry1	1	3.72	9.78E-48
		Prostanoid receptors	Ptger2		1.73	5.92E-05
		Tachykinin receptors	Tacr1		1.50	6.41E-08
	LGIC	GABA-A receptors Ionotropic glutamate receptors	Gabra2 Gabrg3 Grik3	1 1 5	0.87 1.21 2.58	2.72E-03 7.75E-06 3.15E-16
		Nicotinic acetylcholine receptors	Chrna4	1	0.93	3.98E-02

Abbrev.: FC, fold-change, GPCR, G protein-coupled receptor, IC, ion channel, IUPHAR, International Union of Basic and Clinical Pharmacology, LGIC, ligand-gated ion channel, MGI, mouse genome informatics database symbol, NHR, nuclear hormone receptor, padj, adjusted p value, VGIC, voltage-gated ion channel. http://www.guidetopharmacology.org/download.jsp

Table 8 : Top 30 most significant genes with mRNA enriched in D1 as compared to D2 neurons of the NAc

 8

	Gene	Gene Description	log2 FC	padj	D1 mean	D2 mean
	Pdyn	prodynorphin	-3.57	9.23E-42	7 308
	Eya1	EYA 1	-4.14	2.85E-36	19 975
	Isl1	ISL1 transcription factor, LIM/homeodomain	-2.93	3.16E-24	1 427
	Arhgap36 Rho GTPase activating protein 36	-3.18	2.80E-22	960
	Chrm4	cholinergic receptor, muscarinic 4	-2.28	3.50E-20	1 170
	Ebf1	early B cell factor 1	-2.29	2.93E-15	1 280
	Gnb4	G protein, beta 4	-1.83	9.71E-15	3 508
	Gfra1	GDNF family receptor alpha 1	-2.25	1.04E-13	1 149
	Ngb	neuroglobin	-2.83	4.68E-13	282
	Asic4	proton-gated ion channel family member 4	-2.06	3.68E-12	1 258
	Dlk1	delta-like 1 homolog (Drosophila)	-1.62	7.81E-12	6 218	1
	Irak3	interleukin-1 receptor-associated kinase 3	-2.55	3.46E-11	337
	Cntnap3	contactin associated protein-like 3	-2.13	1.00E-10	573
	Gpr101	G protein-coupled receptor 101	-1.64	1.20E-10	1 392
	Cyb5r3	cytochrome b5 reductase 3	-1.41	1.23E-09	6 271	2
	Tac1	tachykinin 1	-1.81	1.57E-08	12 231	3
	Chrm5	cholinergic receptor, muscarinic 5	-2.60	4.25E-08	97
	Agbl2	ATP/GTP binding protein-like 2	-1.88	9.80E-08	668
	Arl4a	ADP-ribosylation factor-like 4A	-1.09	3.77E-07	6 173	2
	Ano7	anoctamin 7	-2.22	1.97E-06	252
	Pde1a	phosphodiesterase 1A, calmodulin-dependent	-1.17	2.98E-06	18 873	7
	Calb2	calbindin 2	-1.70	1.06E-05	585
	Peg10	paternally expressed 10	-1.24	1.32E-05	17 193	5
	Rapgef1	Rap guanine nucleotide exch. factor (GEF) 1	-0.96	1.35E-05	4 387	2
	Tns1	tensin 1	-1.48	1.39E-05	1 313
	Tspyl2	TSPY-like 2	-0.96	1.44E-05	9 126	4
	Sstr4	somatostatin receptor 4	-2.06	2.33E-05	218
	Camk1g	CaMK1 gamma	-1.15	2.35E-05	2 101
	Mapk8ip3 MAP kinase 8 interacting protein 3	-0.94	2.46E-05	9 315	4

Table 9 : Top 30 most significant genes with mRNA enriched in D2 as compared to D1 neurons of the NAc

 9

	Gene	Gene Description	log2 FC	padj	D1 mean D2 mean
	P2ry1	purinergic receptor P2Y, G-protein coupled 1	3.76	1.83E-36	402	8
	Penk	preproenkephalin	3.18	2.39E-26	11 608	145
	Gpr52	G protein-coupled receptor 52	2.75	2.83E-16	236	2
	Fst	follistatin	3.01	2.68E-14	45
	Nt5e	5' nucleotidase, ecto	2.34	2.70E-14	181	1
	Sp9	trans-acting transcription factor 9	1.91	1.30E-12	362	1
	Adk	adenosine kinase	1.91	1.36E-12	313	1
	Upb1	ureidopropionase, beta	2.96	2.21E-11	35
	Adora2a	adenosine A2a receptor	2.71	2.51E-11	783	9
	Gucy1a3	guanylate cyclase 1, soluble, alpha 3	1.60	4.55E-11	12 146	38
	Mro	maestro	2.21	1.03E-10	110
	Malat1	Metastasis-assoc. lung adenocarc. transcript 1	1.94	1.23E-09	8 524	42
	Oprd1	opioid receptor, delta 1	2.45	2.33E-09	137	1
	Gpr88	G-protein coupled receptor 88	1.44	1.38E-08	9 662	25
	Stc1	stanniocalcin 1	2.13	3.11E-08	364	2
	Skap2	src family associated phosphoprotein 2	1.78	6.99E-08	312	1
	Marcks	myristoylated alanine rich PKC substrate	1.18	9.80E-08	5 411	10
	Htr2a	5-hydroxytryptamine (serotonin) receptor 2A	1.43	1.40E-07	652	1
	Fgfr2	fibroblast growth factor receptor 2	1.92	2.93E-07	104
	2900097C17Rik RIKEN cDNA 2900097C17 gene	1.41	4.56E-07	25 855	80
	Gse1	genetic suppressor element 1	1.40	1.17E-06	3 096	9
	Klf3	Kruppel-like factor 3 (basic)	1.32	1.28E-06	607	1
	Plp1	proteolipid protein (myelin) 1	1.49	1.31E-06	943	2
	Yeats2	YEATS domain containing 2	1.72	1.74E-06	1 434	6
	4930465K10Rik RIKEN cDNA 4930465K10 gene	2.38	2.00E-06	2
	Fam101b	family with sequence similarity 101, member B	1.36	2.01E-06	586	1
	Bcor	BCL6 interacting corepressor	1.89	3.51E-06	1 804	9
	Uqcrb	ubiquinol-cyt c reductase binding protein	1.40	3.86E-06	2 357	7
	C330007P06Rik RIKEN cDNA C330007P06 gene	1,13	5,12E-06	3 795	7
	Cldn11	claudin 11	2.16	5.18E-06	28
	Qk	quaking	1.30	5.40E-06	2 532	6

Table 10 : IUPHAR data base-selected genes with mRNA enriched in D1 as compared to D2 neurons in the NAc*

 10

	Type	Family name	MGI symbol	Mean	log2 FC	padj
		GDNF receptor family	Gfra1		-2.25	1.04E-13
		IL-10 receptor family	Il20ra		-1.90	4.48E-04
	Catalytic	NOD-like receptor family	Nlrp10		-1.16	4.15E-02
	Receptors	Prolactin receptor family	Epor		-1.10	9.04E-03
		Receptor tyrosine phosphatases (RTP)	Ptprn	6	-0.57	4.02E-02
		Type I RTKs: ErbB (EGF) receptor family	Erbb4		-1.35	1.77E-03

* Translated mRNAs were isolated from the dorsal striatum of Drd1::BAC-TRAP and Drd2::BAC-TRAP mice. Only identified in the IUPHAR nomenclature, with adjusted p value <0.05 and expression levels > 30 reads are included. Fold change D2/D1. Abbrev.: FC, fold-change, GPCR, G protein-coupled receptor, IC, ion channel, IUPHAR, International Union of Basic and Clinical Pharmacology, LGIC, ligand-gated ion channel, MGI, mouse genome informatics database symbol, NHR, nuclear hormone receptor, padj, adjusted p value, VGIC, voltage-gated ion channel. http://www.guidetopharmacology.org/download.jsp

Table 11 : IUPHAR data base-selected genes with mRNA enriched in D2 as compared to D1 neurons in the NAc*

 11

	Type	Family name	MGI symbol	Mean	log2 FC	padj
		Integrins	Itgb1		1.15	6.75E-03
		Receptor tyrosine phosphatases (RTP)	Ptprm	1	0.91	5.92E-03
		TNF receptor family	Tnfrsf11a		1.02	5.00E-02
	Catalytic	Type I receptor serine/threonine kinases	Bmpr1a	1	0.84	1.12E-02
	receptors	Type II receptor serine/threonine kinases	Acvr2a Tgfbr2	2	0.74 2.01	2.68E-02 1.31E-04
		Type V RTKs: FGF receptor family	Fgfr2		1.92	2.93E-07
		Type XIII RTKs: Ephrin receptor family	Epha7	3	1.00	4.15E-02

Table 12 : Top 30 most significant genes with mRNA enriched in DS as compared to NAc (D1 and D2 combined)

 12

	Gene	GeneDescription	log2 FC	padj	Nac mean	DS mean
	Entpd1	ectonucleoside triphos. diphosphohydrolase 1	-2.65	6.74E-41	108
	Sgpp2	sphingosine-1-phosphate phosphotase 2	-2.62	1.50E-38	174	1
	Synpo2	synaptopodin 2	-2.77	3.99E-36	163	1
	Coch	cochlin		-2.91	5.53E-33	303	3
	Reln	reelin		-1.95	3.13E-32	1 094	4
	Ace	ACE (peptidyl-dipeptidase A) 1	-3.10	8.31E-32	56
	C030013G03Rik RIKEN cDNA C030013G03 gene	-3.01	8.64E-31	34
	Acvr1c	activin A receptor, type IC	-1.67	8.64E-31	923	3
	Dab2ip	disabled 2 interacting protein	-1.25	4.76E-27	2 046	5
	Sema7a	semaphorin 7A	-1.97	1.82E-26	671	2
	Cnr1	cannabinoid receptor 1	-2.39	5.87E-25	1 935	12
	Cyp2s1	cytP450, family 2, subfamily s, polypeptide 1	-2.25	8.59E-25	163	1
	Itga9	integrin alpha 9	-1.89	1.29E-24	278	1
	Pld5	phospholipase D family, member 5	-1.76	5.06E-24	531	2
	Clspn	claspin		-2.56	4.10E-23	189	1
	Gpr155	G protein-coupled receptor 155	-1.95	9.04E-23	1 821	10
	Atp2b1	ATPase, Ca	++ transporting, plasma memb. 1	-1.29	3.22E-22	20 880	57
	Lpcat4	lysophosphatidylcholine acyltransferase 4	-1.74	1.40E-21	2 426	8
	Gcnt2	Glucosaminyl transfer. 2, I-branching enzyme	-1.53	6.88E-21	1 849	6
	Vmp1	vacuole membrane protein 1	-1.31	1.73E-20	1 045	2
	Scn4b	sodium channel, type IV, beta	-1.39	2.46E-20	8 286	23
	AI593442	expressed sequence AI593442	-1.30	1.62E-19	13 929	45
	Nrgn	neurogranin	-1.70	4.64E-19	11 747	40
	Ctsb	cathepsin B	-1.11	6.20E-19	5 737	12
	Tpm2	tropomyosin 2, beta	-1.94	3.49E-18	414	1
	Prima1	proline rich membrane anchor 1	-1.95	1.54E-17	120
	Fam84b	family with sequence similarity 84, member B	-1.43	1.54E-17	312	1
	Slitrk2	SLIT and NTRK-like family, member 2	-1.27	1.64E-17	780	2
	Slc24a2	solute carrier family 24 (Na/K/Ca exchanger) 2	-1.43	2.13E-17	9 454	31
	Kcnt1	potassium channel, subfamily T, member 1	-1.00	2.73E-17	1 301	2

Table 13 : Top 30 most significant genes with mRNA enriched in NAc as compared to DS (D1 and D2 combined)

 13

	Gene	GeneDescription	log2 FC	padj	Nac mean	DS mean
	Fam196b	family with sequence similarity 196, member B	2.50	6.23E-43	702
	Dgkk	diacylglycerol kinase kappa	2.96	6.23E-43	1 126
	Crym	crystallin, mu	1.98	6.23E-43	4 766	1
	Dlk1	delta-like 1 homolog (Drosophila)	3.64	5.27E-37	4 307
	Peg10	paternally expressed 10	2.43	7.60E-29	12 310	1
	AW551984 expressed sequence AW551984	3.55	3.12E-28	771
	Zcchc12	zinc finger, CCHC domain containing 12	1.50	3.03E-27	3 567	1
	Cpne2	copine II	1.57	5.24E-26	3 808	1
	Stard5	START domain containing 5	2.75	8.83E-25	1 907
	Hap1	huntingtin-associated protein 1	1.76	9.75E-24	4 311	1
	Gda	guanine deaminase	1.28	1.92E-23	11 691	4
	Tcerg1l	transcription elongation regulator 1-like	2.71	3.05E-23	1 456
	Ntn1	netrin 1	2.56	1.79E-22	682
	Lin7a	lin-7 homolog A (C. elegans)	1.17	6.84E-22	8 413	4
	Kcnip1	Kv channel-interacting protein 1	2.45	3.36E-21	2 047
	Prkg1	protein kinase, cGMP-dependent, type I	2.01	1.07E-20	2 162
	Enah	enabled homolog (Drosophila)	1.09	5.00E-19	11 165	5
	Fat1	FAT atypical cadherin 1	1.58	4.40E-18	1 224
	Nnat	neuronatin	2.50	4.40E-18	15 066	1
	Dcx	doublecortin	1.25	6.54E-18	1 485
	Trhr	thyrotropin releasing hormone receptor	2.95	1.94E-17	553
	Soga1	suppressor of glucose, autophagy associated 1	1.57	4.74E-17	2 715
	Pea15a	phosphoprotein enriched in astrocytes 15A	1.40	2.37E-16	9 931	3
	Pde1a	phosphodiesterase 1A, calmodulin-dependent	1.87	2.40E-16	13 780	3
	Gm5607	predicted gene 5607	1.96	2.66E-15	1 730
	Tunar	Tcl1 upstream neural differentiation assoc. RNA	1.68	4.38E-15	2 170
	Sox1	SRY (sex determining region Y)-box 1	2.04	1.31E-14	4 256
	Zbtb7c	zinc finger and BTB domain containing 7C	2.84	1.98E-14	875
	Fam126a	family with sequence similarity 126, member A	1.64	2.11E-14	1 065
	Gabrg1	GABA-A receptor, subunit gamma 1	2.18	2.30E-14	411

Table 14 : Top 30 most significant genes with mRNA enriched in DS as compared to NAc in D1 neurons

 14

	Gene	Gene Description	log2 FC	padj	Nac mean	DS mean
	Cpne9	copine family member IX	-2.67	1.99E-47	136
	Nrgn	neurogranin	-2.12	4.96E-44	8 481	39
	Sgpp2	sphingosine-1-phosphate phosphatase 2	-2.65	2.64E-34	182	1
	Gcnt2	glucosaminyl transferase 2, I-branching enzyme	-1.76	3.62E-33	1 420	5
	Lpcat4	lysophosphatidylcholine acyltransferase 4	-1.58	9.87E-33	2 848	8
	Tpm2	tropomyosin 2, beta	-2.42	2.35E-32	245	1
	Pld5	phospholipase D family, member 5	-1.87	6.24E-32	463	1
	Synpo2	synaptopodin 2	-2.55	9.87E-30	171	1
	Gpr155	G protein-coupled receptor 155	-2.12	6.39E-28	1 554	7
	Gpx6	glutathione peroxidase 6	-2.32	2.90E-27	79
	Gpm6b	glycoprotein m6b	-1.32	5.61E-27	5 775	14
	Grm1	glutamate receptor, metabotropic 1	-1.58	3.37E-26	1 064	3
	Dach1	dachshund 1 (Drosophila)	-1.69	2.22E-24	893	3
	Zbtb18	zinc finger and BTB domain containing 18	-1.19	8.37E-24	1 919	4
	Ace	ACE (peptidyl-dipeptidase A) 1	-2.99	1.05E-22	71
	Acvr1c	activin A receptor, type IC	-1.58	7.68E-22	1 056	3
	Cd59a	CD59a antigen	-2.52	1.48E-21	106
	Entpd1	Ectonucleos. triphosphate diphosphohydrolase 1	-2.64	2.26E-21	97
	B3gnt2	UDP-GlcNAc:betaGal beta-1,3-N-ac. gluc.am.transf. 2	-1.62	2.39E-21	1 351	4
	Shb	SH2 domain-containing transforming protein B	-1.60	2.02E-20	345	1
	Pvalb	parvalbumin	-2.33	6.55E-20	122
	Scn4b	sodium channel, type IV, beta	-1.56	9.55E-20	7 121	22
	Ano3	anoctamin 3	-1.30	1.80E-19	9 865	25
	Ctsb	cathepsin B	-1.16	3.08E-19	5 315	12
	C030013G03Rik RIKEN cDNA C030013G03 gene	-2.82	5.95E-19	45
	Kcnh4	K+ voltage-gated chan., subfam H, eag-related, 4	-1.40	1.10E-18	361
	AI593442	expressed sequence AI593442	-1.42	1.85E-18	13 119	36
	Coch	cochlin	-2.74	3.06E-18	313	3
	Cnr1	cannabinoid receptor 1	-2.27	3.42E-18	2 099	12
	Cyp2s1	cyt P450, family 2, subfamily s, polypeptide 1	-2.34	3.47E-18	132

Table 15 : Top 30 most significant genes with mRNA enriched in NAc as compared to DS in D1 neurons

 15

	Gene	Gene Description	log2 FC	padj	Nac mean	DS mean
	AW551984	expressed sequence AW551984	3.86	7.30E-78	872
	Peg10	paternally expressed 10	3.04	2.71E-63	15 150	1
	Dgkk	diacylglycerol kinase kappa	2.77	3.73E-46	978
	Crym	crystallin, mu	2.00	1.71E-35	4 644	1
	Nnat	neuronatin	2.84	9.87E-33	15 712	1
	Stard5	START domain containing 5	3.32	3.13E-30	1 375
	Ahi1	Abelson helper integration site 1	1.51	1.01E-27	14 971	5
	Dlk1	delta-like 1 homolog (Drosophila)	3.21	1.72E-27	5 479
	Tmem255a transmembrane protein 255A	2.10	5.95E-27	1 272
	Fam196b	family with sequence similarity 196, member B	2.33	2.21E-26	708
	Prkg1	protein kinase, cGMP-dependent, type I	1.87	4.50E-25	2 226
	Zbtb7c	zinc finger and BTB domain containing 7C	3.52	3.28E-22	797
	Pde1c	phosphodiesterase 1C	1.46	8.76E-22	4 374	1
	Pea15a	phosphoprotein enriched in astrocytes 15A	1.60	3.23E-20	11 053	3
	Baiap3	BAI1-associated protein 3	2.88	2.63E-19	649
	Gabrg1	GABA-A receptor, subunit gamma 1	2.35	3.90E-19	422
	Ngb	neuroglobin	2.53	1.61E-18	248
	Cpne2	copine II	1.58	1.76E-18	4 460	1
	Zcchc12	zinc finger, CCHC domain containing 12	1.43	2.17E-18	3 695	1
	Hap1	huntingtin-associated protein 1	1.87	2.23E-18	4 908	1
	Tcerg1l	transcription elongation regulator 1-like	2.70	3.06E-18	1 608
	Scml4	sex comb on midleg-like 4 (Drosophila)	2.81	4.90E-18	169
	Ntn1	netrin 1	2.57	1.73E-17	761
	Zic3	zinc finger protein of the cerebellum 3	3.15	1.94E-17	130
	Plxnc1	plexin C1	1.53	3.36E-17	1 571
	Wnt7a	wingless-type MMTV integration site family, 7A	2.06	2.35E-16	418
	Carhsp1	calcium regulated heat stable protein 1	1.08	2.95E-16	7 923	3
	Gpr101	G protein-coupled receptor 101	1.99	1.16E-14	1 226
	Kctd12b	K+ channel tetramerisation domain containing 12b	2.97	2.05E-14	112
	Stra6	stimulated by retinoic acid gene 6	3.02	2.17E-14	395

Table 16 : IUPHAR data base-selected genes with mRNA enriched in DS as compared NAc in D1 neurons*

 16 Translated mRNAs were isolated from the dorsal striatum of Drd1::BAC-TRAP and Drd2::BAC-TRAP mice. Only identified in the IUPHAR nomenclature, with adjusted p value <0.05 and expression levels > 30 reads are included. Fold-change NAc/DS. Abbrev.: FC, fold-change, GPCR, G protein-coupled receptor, IC, ion channel, IUPHAR, International Union of Basic and Clinical Pharmacology, LGIC, ligand-gated ion channel, MGI, mouse genome informatics database symbol, NHR, nuclear hormone receptor, padj, adjusted p value, VGIC, voltage-gated ion channel. http://www.guidetopharmacology.org/download.jsp

	Type	Family name	MGI symbol	Mean	log2 FC	padj
		IL-17 receptor family	Il17rc		-1,39	1,66E-04
			Itga5		-1,38	5,03E-05
		Integrins	Itga8 Itga9		-1,60 -1,85	5,31E-04 1,59E-13
			Epor		-0,61	4,82E-03
		Type I receptor serine/threonine kinases	Acvr1c Acvrl1	2	-1,58 -2,60	7,68E-22 1,73E-11
	Catalytic	Type IX RTKs: MuSK	Musk		-1,20	3,38E-05
	receptors	Type V RTKs: FGF receptor family	Fgfr2		-0,96	2,65E-03
		Type VII RTKs: Neurotrophin receptor/Trk family Ntrk2	5	-0,57	2,86E-03
		Type VII RTKs: Neurotrophin receptor/Trk family Ntrk3	2	-0,57	3,88E-03
			Epha3		-1,47	1,09E-04
		Type XIII RTKs: Ephrin receptor family	Epha4 Ephb1	4 1	-0,77 -0,88	2,98E-07 8,72E-05
			Ephb6		-1,20	2,72E-08
		Type XVIII RTKs: LMR family	Lmtk2	4	-0,71	3,79E-03

*

Table 17 : IUPHAR data base-selected genes with mRNA enriched in NAc as compared DS in D1 neurons*

 17 Translated mRNAs were isolated from the dorsal striatum of Drd1::BAC-TRAP and Drd2::BAC-TRAP mice. Only identified in the IUPHAR nomenclature, with adjusted p value <0.05 and expression levels > 30 reads are included. Fold-change NAc/DS. Abbrev.: FC, fold-change, GPCR, G protein-coupled receptor, IC, ion channel, IUPHAR, International Union of Basic and Clinical Pharmacology, LGIC, ligand-gated ion channel, MGI, mouse genome informatics database symbol, NHR, nuclear hormone receptor, padj, adjusted p value, VGIC, voltage-gated ion channel. http://www.guidetopharmacology.org/download.jsp

	Type	Family name	MGI symbol Mean	log2 FC	padj
		GDNF receptor family	Gfra1		1.15	5.58E-04
	Catalytic	NOD-like receptor family	Nlrp10		1.38	4.16E-03
	receptors	Receptor tyrosine phosphatases (RTP)	Ptprg	1	0.91	3.20E-03
		Type XIX RTKs: Leukocyte tyrosine kinase (LTK)	Alk		1.59	1.97E-04

*

Table 18 : Top 30 most significant genes with mRNA enriched in DS as compared to NAc in D2 neurons

 18

	Gene	Gene Description	log2 FC	padj	NAc mean DS mean
	Clspn	claspin	-3.30 2.30E-20	82	1
	Synpo2	synaptopodin 2	-3.21 8.59E-20	141	1
	Reln	reelin	-2.29 1.78E-19	494	3
	Wnt8b	wingless-type MMTV integration site family, member 8B	-3.74 6.40E-16	3
	Coch	cochlin	-2.76 4.97E-15	271	3
	Lrrc10b	leucine rich repeat containing 10B	-2.25 3.03E-14	4 682	18
	Entpd1	ectonucleoside triphosphate diphosphohydrolase 1	-2.32 6.47E-13	129
	Cd72	CD72 antigen	-3.44 1.83E-12	0
	Hipk4	homeodomain interacting protein kinase 4	-2.24 2.18E-12	365	2
	Gabrd	GABA-A receptor, subunit delta	-1.87 6.86E-12	1 093	2
	Cldn1	claudin 1	-2.86 1.96E-11	16
	Arhgdib	Rho, GDP dissociation inhibitor (GDI) beta	-1.75 6.32E-11	1 141	4
	Ace	angiotensin I converting enzyme 1	-2.96 6.55E-11	20
	Prima1	proline rich membrane anchor 1	-2.35 1.49E-09	99
	Cnr1	cannabinoid receptor 1 (brain)	-2.43 1.63E-09	1 514	12
	Rasd2	RASD family, member 2	-1.41 2.46E-09	14 474	39
	Lpcat4	lysophosphatidylcholine acyltransferase 4	-2.11 3.59E-09	1 430	8
	Sgpp2	sphingosine-1-phosphate phosphotase 2	-2.36 1.40E-08	152	1
	Mmp17	matrix metallopeptidase 17	-1.48 1.55E-08	2 650	6
	Hs6st3	heparan sulfate 6-O-sulfotransferase 3	-2.50 4.50E-08	77
	Hpca	hippocalcin	-1.53 6.58E-08	118 750	341
	Meis1	Meis homeobox 1	-2.24 8.79E-08	176	1
	Actn2	actinin alpha 2	-1.76 3.17E-07	388	1
	Acvr1c	activin A receptor, type IC	-1.73 3.20E-07	605	2
	Sema7a	sema7A	-1.92 3.38E-07	681	3
	Cacna1h	Ca2+ chan., voltage-dependent, T type, alpha 1H subunit	-1.63 6.89E-07	430	1
	Deptor	DEP domain containing MTOR-interacting protein	-1.43 6.89E-07	1 535	5
	Slc41a1	solute carrier family 41, member 1	-1.59 8.11E-07	1 901	7
	Ldlrad4	LDL receptor class A domain containing 4	-1.86 1.25E-06	364	1
	Etl4	enhancer trap locus 4	-1.33 1.26E-06	3 315	10

Table 19 : Top 30 most significant genes with mRNA enriched in NAc as compared to DS in D2 neurons

 19

	Gene	Gene Description	log2 FC	padj	NAc mean DS mean
	Cartpt	CART prepropeptide	4.26	1.04E-31	3 110
	Fgf10	fibroblast growth factor 10	4.64	2.25E-29	870
	Dlk1	delta-like 1 homolog (Drosophila)	4.02	6.85E-21	1 616
	Dgkk	diacylglycerol kinase kappa	3.31	8.03E-20	1 421
	Stard5	StAR-related lipid transfer (START) domain containing 5	2.50	6.44E-14	3 020
	AW551984 expressed sequence AW551984	3.40	2.18E-12	528
	Kcnip1	Kv channel-interacting protein 1	2.78	1.96E-11	2 472
	Pcdh19	protocadherin 19	1.93	8.18E-11	1 607
	Plcxd3	phosphatidylinositol-specific PLC, X domain containing 3	2.19	1.13E-10	911
	Sox1	SRY (sex determining region Y)-box 1	2.50	1.97E-10	3 611
	Inadl	InaD-like (Drosophila)	2.16	6.97E-10	1 246
	Marcks	myristoylated alanine rich protein kinase C substrate	1.88	6.76E-09	10 253	5
	Fam196b	family with sequence similarity 196, member B	2.33	3.66E-08	669
	Adam12	a disintegrin and metallopeptidase domain 12	2.38	4.50E-08	604
	Crym	crystallin, mu	1.83	9.44E-08	4 907
	Phyh	phytanoyl-CoA hydroxylase	1.79	2.08E-07	1 906
	Phactr2	phosphatase and actin regulator 2	2.02	5.20E-07	806
	Dpp10	dipeptidylpeptidase 10	1.37	5.36E-07	3 611	1
	Ralyl	RALY RNA binding protein-like	1.92	7.17E-07	9 947	2
	Peg10	paternally expressed 10	1.50	9.13E-07	5 734	2
	Trhr	thyrotropin releasing hormone receptor	2.57	9.54E-07	728
	Enah	enabled homolog (Drosophila)	1.20	1.37E-06	10 830	5
	Rgs17	regulator of G-protein signaling 17	1.23	1.72E-06	15 453	8
	P2ry1	purinergic receptor P2Y, G-protein coupled 1	1.93	1.87E-06	8 025	2
	Tcerg1l	transcription elongation regulator 1-like	2.17	1.89E-06	1 084
	Slc8a1	solute carrier family 8 (Na+/Ca2+ exchanger), member 1	1.59	2.34E-06	4 062	1
	Fhl1	four and a half LIM domains 1	1.41	2.49E-06	3 285	1
	Prkg1	protein kinase, cGMP-dependent, type I	1.85	2.54E-06	1 960
	Gda	guanine deaminase	1.33	6.37E-06	11 248	5
	Npas3	neuronal PAS domain protein 3	2.26	7.29E-06	523

Table 20 : IUPHAR data base-selected genes with mRNA enriched in NAc as compared DS in D2neurons*

 20

	Type	Family name	MGI symbol	Mean	log2 FC	padj
		IL-17 receptor family	Il17ra		-1.44	2.60E-02
		Integrins	Itga9	1	-1.65	1.74E-06
		Receptor tyrosine phosphatases (RTP)	Ptprm	2	-0.76	1.92E-02
		Type I receptor serine/threonine kinases	Acvr1c	2	-1.73	3.20E-07
	Catalytic	Type I receptor serine/threonine kinases	Acvrl1		-1.39	3.15E-02
	receptors	Type VII RTKs: Neurotrophin receptor/Trk family Ntrk3	2	-0.73	3.85E-02
			Epha4	7	-0.86	8.43E-03
		Type XIII RTKs: Ephrin receptor family	Epha6	1	-1.11	1.61E-02
			Ephb6		-1.53	5.86E-03
		Type XVIII RTKs: LMR family	Lmtk2	4	-0.91	1.65E-03

* Translated mRNAs were isolated from the dorsal striatum of Drd1::BAC-TRAP and Drd2::BAC-TRAP mice. Only identified in the IUPHAR nomenclature, with adjusted p value <0.05 and expression levels > 30 reads are included. Fold-change NAc/DS.

Table 21 : IUPHAR data base-selected genes with mRNA enriched in NAc as compared DS in D2neurons*

 21 Translated mRNAs were isolated from the dorsal striatum of Drd1::BAC-TRAP and Drd2::BAC-TRAP mice. Only identified in the IUPHAR nomenclature, with adjusted p value <0.05 and expression levels > 30 reads are included. Fold-change NAc/DS. Abbrev.: FC, fold-change, GPCR, G protein-coupled receptor, IC, ion channel, IUPHAR, International Union of Basic and Clinical Pharmacology, LGIC, ligand-gated ion channel, MGI, mouse genome informatics database symbol, NHR, nuclear hormone receptor, padj, adjusted p value, VGIC, voltage-gated ion channel. http://www.guidetopharmacology.org/download.jsp

	Type	Family name	MGI symbol	Mean	log2 FC	padj
	Catalytic Receptor	Interferon receptor family TNF receptor family Type XIII RTKs: Ephrin receptor family	Ifngr2 Tnfrsf21 Epha5	1 2	1.02 1.05 1.04	4.41E-02 3.48E-04 8.43E-03
		1.1.1.42 Isocitrate dehydrogenases	Idh1		1.12	2.33E-02
		1.17.4.1 Ribonucleoside-diphos. reduct.	Rrm2		1.37	2.78E-02
		Adenylyl cyclases	Adcy7		1.26	4.09E-02
		Alkaline ceramidases	Acer2	1	0.94	4.51E-02
		Amino acid hydroxylases	Th		1.44	2.45E-02
		C14: Caspase	Casp3		2.00	4.47E-04
		CAMK2 family	Camk2d		1.37	1.02E-02
		Catecholamine turnover	Th		1.44	2.45E-02
		Eta subfamily	Prkch	3	0.75	3.15E-02
		Interleukin-1 receptor-assoc. kinase (IRAK) Irak1	1	0.88	1.89E-02
	Enzyme	Lanosterol biosynthesis pathway	Hmgcs1 Idi1	3	1.16 1.05	1.33E-03 1.26E-02
		M12: Astacin/Adamalysin	Adam12		2.38	4.50E-08
		Nucleoside synthesis and metabolism	Rrm2		1.37	2.78E-02
		PAKA subfamily	Pak3	7	0.71	3.93E-02
		Phosphatidylinositol kinases	Pik3r1	7	0.78	9.32E-03
		Phosphodiesterases, 3',5'-cyclic nucleotide	Pde1a Pde3b	3	1.75 1.45	8.62E-06 7.78E-03
		Protein kinase G (PKG)	Prkg1		1.85	2.54E-06
		S9: Prolyl oligopeptidase	Dpp4		1.15	4.23E-02
		Soluble guanylyl cyclase	Gucy1a3	35	0.67	4.94E-02
		5-Hydroxytryptamine receptors	Htr1a		1.44	2.50E-02
		Calcitonin receptors	Calcr		1.24	2.39E-02
		Chemokine receptors	Ackr3		1.52	1.53E-02
	GPCR	Class Frizzled GPCRs P2Y receptors	Fzd5 P2ry1	4	1.97 1.93	8.82E-05 1.87E-06
		Prostanoid receptors	Ptgdr		1.31	1.98E-02
		Thyrotropin-releasing hormone receptors	Trhr		2.57	9.54E-07
		Vasopressin and oxytocin receptors	Oxtr		1.70	3.88E-03
			Gabra5	1	0.97	3.47E-02
	LGIC	GABA-A receptors	Gabrg1 Gabrq		1.61 1.79	7.36E-03 1.83E-03
		Glycine receptors	Glra2		1.62	3.27E-03
	NHR	3A. Estrogen receptors	Esr1		1.56	6.55E-03
	Other proteins	Fatty acid-binding proteins Fatty acid-binding proteins RZ family	Fabp5 Rbp1 Rgs17	3 10	0.70 1.38 1.23	4.92E-02 3.57E-02 1.72E-06
		Mitochondrial nucleotide transporter	Slc25a24		1.85	9.14E-04
		SLC16 monocarboxylate transporters	Slc16a2		1.01	1.42E-02
	Transporters	SLC18 vesicular amine transporters SLC30 zinc transporter family	Slc18b1 Slc30a3		1.00 1.58	3.90E-02 1.12E-02
		SLC44 choline transporter-like family	Slc44a5		1.44	1.73E-02
		SLC8 family of sodium/calcium exchangers Slc8a1	2	1.59	2.34E-06
	VGIC	Cyclic nucleotide-regulated channels	Hcn4		1.44	2.69E-02

*

Table 4 : Genes differentially expressed in D1-SPNs-NAc. Comparison cCOC vs cSAL.

 4 Genes are ranked by FC. In blue the genes up-regulated and in orange the genes down-regulated.

		NAc-D2-SPNs: mHP vs yHP
				padj_Y
	Gene	GeneDescription	FC	asRef
		ATP-binding cassette, sub-family A (ABC1), member
	Abca1	1	2.41	0.002
	Ednrb	endothelin receptor type B	2.38	0.01
		phosphatidylinositol glycan anchor biosynthesis, class
	Pigf	F	2.20	0.01
	Megf10	multiple EGF-like-domains 10	2.16	0.01
	Acer3	alkaline ceramidase 3	2.13	0.01
		methylenetetrahydrofolate dehydrogenase (NADP+
	Mthfd2l	dependent) 2-like	2.13	0.02
	Zfp459	zinc finger protein 459	2.09	0.03
	Pde3b	phosphodiesterase 3B, cGMP-inhibited	2.04	0.03
	Zfp873	zinc finger protein 873	2.03	0.01
	Trim59	tripartite motif-containing 59	2.02	0.01
	Gm10033	predicted gene 10033	2.02	0.02
	Cbln4	cerebellin 4 precursor protein	1.96	0.01
	A230072C
	01Rik	RIKEN cDNA A230072C01 gene	1.96	0.00
	Zfp758	zinc finger protein 758	1.94	0.01
	Rasgrp3	RAS, guanyl releasing protein 3	1.93	0.03
	Cenpw	centromere protein W	1.92	0.05
	Snhg6	small nucleolar RNA host gene 6	1.90	0.03
	Rian	RNA imprinted and accumulated in nucleus	1.90	0.00
	BC029722	cDNA sequence BC029722	1.89	0.04
	Tspan6	tetraspanin 6	1.89	0.03
	Gpr165	G protein-coupled receptor 165	1.88	0.02
	Gm16532	NA	1.88	0.04
	March1	membrane-associated ring finger (C3HC4) 1	1.88	0.00
	AU041133	expressed sequence AU041133	1.87	0.03
	Ddx59	DEAD (Asp-Glu-Ala-Asp) box polypeptide 59	1.87	0.03
	Uvssa	UV stimulated scaffold protein A	1.86	0.05
	Plp1	proteolipid protein (myelin) 1	1.83	0.01
	Tmem229a	transmembrane protein 229A	1.83	0.03
		cysteine rich transmembrane BMP regulator 1 (chordin
	Crim1	like)	1.83	0.01
	Zfp932	zinc finger protein 932	1.82	0.02
	Rdx	Radixin	1.82	0.01
	Kantr	Kdm5c adjacent non-coding transcript	1.81	0.02
	Vps13a	vacuolar protein sorting 13A (yeast)	1.80	0.00
	Epyc	Epiphycan	1.80	0.04
	Zfp72	zinc finger protein 72	1.79	0.03
	Tmem161b	transmembrane protein 161B	1.79	0.03
	Itgbl1	integrin, beta-like 1	1.77	0.04
	Mbip	MAP3K12 binding inhibitory protein 1	1.77	0.04
	Lipo1	lipase, member O1	1.76	0.03
		protein tyrosine phosphatase, receptor type Z,
	Ptprz1	polypeptide 1	1.76	0.05

Table 5 : Genes differentially expressed in D2-SPNs-NAc. Comparison cCOC vs cSAL.

 5 Genes are ranked by FC. In blue the genes up-regulated and in orange the genes down-regulated.

	A total of 71 genes is differentially regulated in this comparison.

  . 9595; 39 ng/mL). For the study of phosphorylation, we added to all the solutions from homogenisation onwards a cocktail of five phosphatase inhibitors (Merck Millipore): imidazole, 2 mM; NaF, 1 mM; Na 2 MoO 4 , 1.15 mM; Na 3 VO 4 ,1m M; and sodium tartrate, 4 mM. The primary isotype controls were: mouseIgG (Abcam; ref. ab37355), mouse IgG1 (Abcam; ref. ab91353) and rabbit IgG

	ng/mL), CNPase (Abcam; ref.
	ab6319; 625 ng/mL), DARPP-32 rabbit IgG (Cell Signaling,
	Danvers MA, USA; ref. 2306; 156 ng/mL), me 3 H3K 9 rabbit IgG
	(Abcam, ref. ab8898; 625 ng/mL), methyl CpG binding domain pro-
	tein 2 (MBD2) rabbit IgG (Sigma-Aldrich, Saint-Quentin Fallavier,
	France; ref. M7318; 313 ng/mL), methyl CpG-binding protein 2
	(MeCP2) rabbit IgG (Sigma-Aldrich, ref. HPA001341; 625 ng/mL),
	NeuN mouse IgG1 (Merck-Millipore; ref. MAB377; 625 ng/mL),
	phospho-extracellular signal-regulated kinase (pERK) mouse IgG
	(Cell Signaling; ref. 5726; 313 ng/mL), phospho-mitogen-and
	stress-activated protein kinase 1 (pMSK1) rabbit IgG (Cell Signal-
	ing; ref

-Aims of the thesis -Aims of the thisis

-Materials & Methods -Materials & Methods 6

© 2014 Federation of European Neuroscience Societies and John Wiley & Sons Ltd European Journal of Neuroscience, 39, 1234-1244

© 2014 Federation of European Neuroscience Societies and John Wiley & Sons Ltd European Journal of Neuroscience, 39, 1234-1244 1244 L. Marion-Poll et al.

Acknowledgements

We thank Yohann Bertelle and Annie Rousseau for animal care, Mythili Savariradjane for assistance with the microscope, Patricia Gaspar for NeuN antibody and Jean-Christophe Poncer for PVCre::RCE mice, all from the Institut du Fer a Moulin. We are grateful to Paul Greengard (The Rockefeller University, New York) for initially providing the drd1a::EGFP and drd2:: EGFP mice. The Cell Imaging and Flow Cytometry facility of the IFR83 (Paris, France) is supported by the Conseil R egional Ile-de-France. This work was supported by an ERC grant to J.A.G., E.M. was supported by the Marie Curie early training network N-Plast. The authors declare no competing interest.

 ------------------------------------------DISCUSSION -------------------------------------

-Chapter 3 -

Long-lasting changes induced by cocaine

We compared the effects of the six different treatments (mHP, mST, yHP, yST, cocaine, saline) on the transcriptional profiles in PFC, NAc, and DS, taking into account the specific types of neuron (i.e. D1 or D2). Importantly, all the analyses that we reported until now were performed exclusively on the genes that showed an Adj p-value <0.05; here -in order to have the possibility to make all the comparisonswe did perform our first analysis taking into consideration all the genes that showed a nominal p-value <0.05.

Common genes were found in all comparison (see table below). For each population of neurons in each regions we compared the effect of the cocaine with: the effect of the non contingent presentation of the food (yHP-cCOC), the effect of the training for the ST food (mST-cCOC), and the effect of the training for the HP food (mHP-cCOC). In D1 NAc-SPNs, the yHP-cCOC comparison resulted in 85 common genes, 50 regulated in opposite fashion, 35 regulated in the same direction; the mHP-cCOC revealed 85 common genes. Interestingly only 15 are regulated in the same fashion. Lastly, the comparison mST-cCOC revealed 77 common genes, 37 regulated in the same direction and 41 in opposite direction. Contrary to the other comparisons of the PFC, when comparing the cocaine exposure with the operant training for the ST food we find a striking anti-correlation of the common genes: among the 96 genes commonly regulated only 4 are regulated in the same direction.

Conversely, in the comparison mHP-cCOC we can observe mainly a co-regulation of the gene expression, among the 87 common genes only 26 are regulated in opposite fashion.

In the D1-SPNs of the DS we found 25 genes in common in comparison yHP-cCOC, only 3 inversely regulated. In the mHP-cCOC 52 genes are in common, among them only 9 are regulated in the same direction. Lastly, for the comparison to mST-cCOC, 79 genes were common, with only 4 genes regulated in the same fashion. Lastly, in comparison with the PFC, we found 73 genes in common in yHP-cCOC, 23 co-regulated and 20 oppositely regulated.

Concerning the D2-DS-SPNs, we found 109 common genes in yHP-cCOC, 59 regulate in the opposite direction, 50 commonly regulated, 73 common genes in mHP-cCOC 36 co-regulated 37 anti-regulated, and only 5 genes common in comparison to mST-cCOC, 2 commonly regulated, 3 regulated in opposite fashion.

The comparison that revealed the most striking results was that between the effects of HP food and those of cocaine in the D2 neurons of the NAc. The analysis -Experimental results -7 -Discussion --Annexes -