Kristina Valentinova

Manuel Mameli
email: manuel.mameli@inserm.fr

mGluR-LTD at Excitatory and Inhibitory Synapses in the Lateral Habenula Tunes Neuronal Output

Excitatory and inhibitory transmission onto lateral habenula (LHb) neurons is instrumental for the expression of positive and negative motivational states. However, insights into the molecular mechanisms modulating synaptic transmission and the repercussions for neuronal activity within the LHb remain elusive. Here, we report that, in mice, activation of group I metabotropic glutamate receptors triggers long-term depression at excitatory (eLTD) and inhibitory (iLTD) synapses in the LHb. mGluR-eLTD and iLTD rely on mGluR1 and PKC signaling. However, mGluR-dependent adaptations of excitatory and inhibitory synaptic transmission differ in their expression mechanisms. mGluR-eLTD occurs via an endocannabinoid receptor-dependent decrease in glutamate release. Conversely, mGluR-iLTD occurs postsynaptically through PKC-dependent reduction of b2-containing GABA A -R function. Finally, mGluRdependent plasticity of excitation or inhibition decides the direction of neuronal firing, providing a synaptic mechanism to bidirectionally control LHb output. We propose mGluR-LTD as a cellular substrate that underlies LHb-dependent encoding of opposing motivational states.

Abstract

The capacity of the brain to anticipate and seek future rewards or alternatively escape aversive events allows individuals to adapt to their environment. A considerable research effort has focused on unraveling the cellular and synaptic mechanisms within the meso-cortico-limbic system underlying motivational processing both in physiological conditions and in pathologies such as addiction and depression. However, only recently we begin to understand the circuit substrates capable to control midbrain monoaminergic nuclei and their contribution to motivated behaviors.

The Lateral Habenula (LHb) has emerged in the last decade, as a major player encoding stimuli with motivational value and in controlling monoaminergic systems. The wiring of this epithalamic structure subserves discrete features of motivated behaviors, including preference and avoidance. Recent advances have also demonstrated that aberrant modifications in LHb function trigger negative emotional states in disorders including depression and addiction, highlighting the LHb as an important brain target for therapeutic intervention for these pathological states.

In my thesis work I first sought to investigate how modulation of synaptic transmission in the LHb controls neuronal activity, especially focusing on the role of metabotropic glutamate receptors. In a second study, I expanded my work examining how drug experience changes synaptic transmission in a precise habenular circuit that we discovered to be crucial for depressive states during cocaine withdrawal.

In an initial data set, we found that, in the LHb, metabotropic glutamate receptor 1 activation drives a PKC-dependent long term depression of excitatory (eLTD) and inhibitory (iLTD) synaptic transmission. Despite the common induction, eLTD and iLTD diverged in their expression mechanism. While eLTD required endocannabinoiddependent reduction of glutamate release, iLTD expressed postsynaptically through a decrease of 2-containing GABA A receptors function. Further, eLTD and iLTD bidirectionally controlled LHb neuronal output.

In a second study, we showed that chronic cocaine exposure leads to a persistent and projection-specific increase of excitatory synaptic transmission onto LHb neurons. This form of synaptic potentiation required membrane insertion of GluA1-containing AMPA receptors and a reduction in potassium channels function ultimately leading to increased LHb neuronal excitability both in vitro and in vivo. These cocaine-driven adaptations within the LHb were instrumental for depressive-like states emerging after drug withdrawal.

Altogether this work demonstrates how synaptic plasticity in the LHb affects neuronal output and thereby contributes to behaviors associated with the pathology of motivation.

Résumé

La survie des individus dépend de leur capacité d'anticiper la survenue d'une récompense ou d'un danger leur permettant ainsi de s'adapter à leur environnement. De considérables efforts ont été réalisés pour identifier les mécanismes cellulaires et synaptiques ayant lieu au niveau du circuit de la récompense afin d'avoir une meilleure compréhension des processus sous tendant des états motivationnels physiologiques et pathologiques tels que l'addiction et la dépression. Pour autant, ce n'est que récemment qu'on commence à comprendre les circuits capables de contrôler les systèmes monoaminergiques mésencéphaliques et leurs contributions aux comportements motivés.

Dans les dernières décennies l'habénula latérale (LHb) a émergé comme un acteur majeur capable d'encoder des stimuli de valeur motivationnelle et de contrôler les systèmes monoaminergiques. La connectivité de cette structure épithalamique joue un rôle clé dans différents aspects des comportements motivationnels, comme l'approche et la fuite. Des avancées récentes ont aussi démontré que des altérations de la fonction de la LHb entrainent des états émotionnels négatifs caractéristiques de la dépression et l'addiction. Ces observations suggèrent que la LHb pourrait s'avérer une cible importante pour le traitement de ces pathologies.

Au cours de mon travail de thèse, j'ai d'abord cherché à comprendre comment moduler la transmission synaptique au niveau de la LHb pouvait contrôler son activité. Pour répondre à cette question, je me suis focalisée sur le rôle des récepteurs métabotropiques au glutamate (mGluRs). Dans une seconde étude, j'ai examiné les mécanismes par lesquels les drogues d'abus modifient la transmission synaptique des neurones de la LHb. Ces modifications se produisent spécifiquement dans les neurones LHb se projetant vers le noyau tegmental rostral (RMT) et sont nécessaires pour l'émergence des états dépressifs.

Dans un premier temps, nous avons démontré qu'au niveau de la LHb les mGluRs de type I sont capables d'induire une dépression à long terme de la transmission synaptique excitatrice (eLTD) et inhibitrice (iLTD). Ces deux formes de plasticité dépendent de la signalisation PKC, mais requièrent des mécanismes d'expression différents. Tandis que eLTD réduit la probabilité de libération du glutamate via l'activation de récepteurs présynaptiques aux endocannabinoides (CB1), iLTD s'exprime par la réduction de la fonction des récepteurs GABA A postsynaptiques contenant la sous-unité 2. De plus, eLTD and iLTD exercent un contrôle bidirectionnel sur la décharge des neurones de la LHb.

Dans un second temps, nous avons mis en évidence qu'une exposition chronique à la cocaïne produit une augmentation persistante de la transmission excitatrice au niveau des neurones de la LHb ciblant le RMTg. Cette forme de potentialisation synaptique nécessite l'insertion membranaire de récepteurs contenant la sous-unité GluA1, ainsi que la réduction de conductances potassiques entrainant une hyperexcitabilité neuronale in vitro et in vivo dans la LHb. Ces modifications sont nécessaires pour l'établissement d'états dépressifs émergeant lors de la période de sevrage à la cocaïne.

En conclusion, ce travail a contribué à la compréhension de mécanismes de plasticité synaptique ayant lieu au niveau de la LHb et leurs répercussions pour son activité contrôlant ainsi des comportements motivationnels.

I would like to thank in first place the amazing lovable people in the lab. Starting with my advisor and dear friend Manuel. I thank you for your time, attention and help during all these years, for the countless days and nights you spent to guide me through my experience as a young scientist, for the discussions and fights, for the sharing and advices. I thank you for the inspiration and love for science that you are building in me with your example every single day. I thank you for making my experience in the lab an amazing adventure, overcoming difficulties and frustration and learning how to enjoy the good moments, for making me laugh when I am desperate and for showing me that there is a way out even in the most difficult moments. Thank you for being always available for me and the others in the lab. Thank you for supporting my downs and helping me get over them. Thank you for creating a positive and motivated environment where trust, mutual respect and enthusiasm are the driving forces. Thank you for challenging me and believing in me. And I thank you for all the great fun we had in the lab and outside, for all the celebrations and…… although I can continue thanking you for many other things most importantly I want to thank you for proving me that when you have positive attitude, enthusiasm and persistence everything is possible! I want to thank also Salvatore, Frank and Anna... you guys are not only colleagues and friends for me, but my bigger brothers and sister. I have not enough words to express how much I enjoyed having you around and how grateful I am for all the amazing time we spent together. I thank you so much for all the fun and enjoyable moments we shared, for all the laughing, for all the dinners, celebrations and birrettas, for all the foosball games, for the Italian and Dutch lessons, for the adventures in Sardinia, Sizel Montpellier and Copenhagen. I thank you also for being so understanding, caring and attentive, for listening and giving me your precious advices when I most need them. I thank you for being so creative and funny, for all the jokes you make, for being so positive and pleasant, I always enjoy your company. I thank you for sharing with me your knowledge and experience, for all the discussions we had, for your constructive feedback, for sharing your ideas, for being so much passionate and implicated in our work, you keep me motivated and make my days happier.

Finally, I want to thank all of you Manuel, Salvatore, Anna and Frank for keeping this unique family ambiance alive every day.

With all my love and respect a big THANK YOU!!!!! I also want to thank many people from the IFM institute with who I shared countless nice moments: Tamar, Quentin, Martin, Sana, Marie, Ferran, Jessica, Emily, Enrica, Assunta, Nicolas, Benoit, Mariano, Alfredo, Sebastian for all the enjoyable moments we spent together, sharing work space, but also knowledge and ideas, for creating friendly and convivial ambiance in the institute and for all the parties and celebrations. I thank Jean-Christophe, Sabine, Nicolas and Corentin for helpful discussions and advices. I want to thank also Jean-Antoine and Fiona for making the effort to keep this institute a dynamic and friendly place.

I want to thank my friend Silvana, who has been side by side with me throughout all these years (8!!!) I spent in Paris. I want to thank you for sharing all these precious moments and being around when I most needed you. You are very very special friend.

Table of figures

Introduction

The central nervous system (CNS) is designed to promote at best the survival of the species. Indeed the brain is able to encode the valence of environmental stimuli, which allow the individual to pursue rewards essential for survival or to avoid potential threats.

Single neurons within complex brain circuits represent such environmental information through changes in their spiking activity. During my PhD training I developed a strong interest in understanding how neurons encode and process valenced information at different space and time scales -from the single cell to the integrated circuit and from the millisecond duration of an action potential to the long-lasting synaptic remodeling.

My particular interest is to understand how reward and aversion-related experience modifies the synaptic properties of neurons and how experience-induced synaptic plasticity controls neuronal activity to drive motivational states crucial for survival.

In the course of evolution, highly organized circuits have emerged to promote survival through reward-seeking and threat-avoiding behaviors. The meso-cortico-limbic circuit has gained a central role in the processing of rewarding and aversive environmental information and in orchestrating adaptive behavioral responses. It also refers as a 'reward' circuit since a large body of evidence collected over the years has revealed its critical role in appetitive and reinforcing behaviors [START_REF] Berridge | What is the role of dopamine in reward: Hedonic impact, reward learning, or incentive salience?[END_REF][START_REF] Robbins | Neurobehavioural mechanisms of reward and motivation[END_REF]Schultz, 2007a;[START_REF] Wise | Brain dopamine and reward[END_REF]. It comprises a meso-limbic part represented by the ventral tegmental area (VTA) dopamine neurons projections to the nucleus accumbens (NAc) of the ventral striatum and a meso-cortical part represented by the VTA projection to the prefrontal cortex (PFC). However this system is highly interconnected and is tightly controlled by a much wider network of brain structures, including the lateral hypothalamus (LH), the amygdala and the lateral habenula (LHb) among others (Fig 1), which altogether operate to encode valenced stimuli and to transform them into behavioral output [START_REF] Ikemoto | Dopamine reward circuitry: two projection systems from the ventral midbrain to the nucleus accumbens-olfactory tubercle complex[END_REF][START_REF] Ikemoto | Basal ganglia circuit loops, dopamine and motivation: A review and enquiry[END_REF][START_REF] Ledoux | Emotion circuits in the brain[END_REF]Matsumoto and Hikosaka, 2007;[START_REF] Nieh | Optogenetic dissection of neural circuits underlying emotional valence and motivated behaviors[END_REF][START_REF] Stuber | Lateral hypothalamic circuits for feeding and reward[END_REF][START_REF] Wise | Forebrain substrates of reward and motivation[END_REF].

A key feature of the meso-cortico-limbic circuit is the attribution of motivational value to otherwise neutral stimuli. Indeed, a stimulus can acquire positive or negative valence through innate or learned mechanisms. Stimuli assigned with a positive value (rewards) are generally beneficial for survival and elicit approach behaviors. On the contrary, lifethreatening or harmful stimuli are ascribed with a negative value (aversion) and the corresponding behavioral reaction is to avoid or escape them. When animals are repetitively exposed to valenced stimuli in a particular environmental context (i.e. in presence of visual or auditory cues), cue-stimulus associations are formed allowing to predict the timing of occurrence and magnitude of the stimulus, a process necessary to orchestrate an appropriate behavioral response. Indeed, the ability to predict the outcome of a stimulus is at the base of conditioned behaviors, such as conditioned place preference and avoidance, paradigms widely used to assess motivational states [START_REF] Hollerman | Dopamine neurons report an error in the temporal prediction of reward during learning[END_REF][START_REF] Schultz | Predictive reward signal of dopamine neurons[END_REF][START_REF] Schultz | A neural substrate of prediction and reward[END_REF][START_REF] Tzschentke | Measuring reward with the conditioned place preference (CPP) paradigm: update of the last decade[END_REF]. In physiological conditions behaviors leading to obtaining a reward or escaping a danger can be reinforced, positively, when an animal actively works to obtain a reward or negatively, when the animal works to stop an aversive stimulus [START_REF] Valenstein | Interaction of positive and negative reinforcing neural systems[END_REF][START_REF] Ferster | Withdrawal of positive reinforcement as punishment[END_REF]). These behaviors are compromised in pathologies associated with deficits in reward and aversion processing such as depression or addiction [START_REF] Lüscher | The Emergence of a Circuit Model for Addiction[END_REF][START_REF] Russo | The brain reward circuitry in mood disorders[END_REF].

Over the last decades a great deal of attention has focused on the synaptic and cellular substrates of reward encoding, learning and behaviors within the meso-cortico-limbic system. However, it remains less known how this system processes aversion and which particular structures provide it with aversion informative signals. The lateral habenula is now emerging as a key structure capable to provide aversive information to the mesocortico-limbic system likewise contributing to avoidance behaviors.

In this thesis I will first briefly summarize key functional features of the reward system to provide a general context of positive and negative motivational processing. Next, I will introduce how the lateral habenula interacts with this system in the context of aversion.

Finally I will show how modulation of synaptic transmission in the lateral habenula can tune neuronal output potentially driving opposing motivational states and how drugexperience changes lateral habenula function to promote negative emotional states.

Figure 1 Simplified schematic of the meso-cortico-limbic and associated circuits in the rodent brain

The meso-cortico-limbic system comprises dopaminergic VTA-to-NAc and VTA-to-PFC projections, which release dopamine upon rewarding or aversive stimuli [START_REF] Abercrombie | Differential effect of stress on in vivo dopamine release in striatum, nucleus accumbens, and medial frontal cortex[END_REF][START_REF] Hernandez | Food reward and cocaine increase extracellular dopamine in the nucleus accumbens as measured by microdialysis[END_REF][START_REF] Kalivas | Selective activation of dopamine transmission in the shell of the nucleus accumbens by stress[END_REF]. The VTA also contains GABAergic interneurons which inhibit dopamine neurons. Apart from the input to the NAc and PFC, dopamine neurons also project to the amygdala (Amy), which in turn sends glutamatergic input to the NAc. VTA dopamine neurons receive GABAergic inhibitory inputs from local interneurons or from other structures including the rostromedial tegmental nucleus (RMTg) and the NAc. Excitatory glutamatergic afferents to the VTA arise from the LHb. LH and PFC. The VTA receives also both GABAergic and glutamatergic connections from the BNST (see Fig2).The LHb also projects to local GABA neurons in the VTA and RMTg, likewise exerting inhibitory control over dopamine neurons. The LH sends efferents to the LHb and VTA among others (not shown). The NAc receives glutamatergic innervation from the medial prefrontal cortex (mPFC) and amygdala (Amy) and projects to the LH and VTA. These various glutamatergic and GABAergic inputs to the VTA control aspects of reward and aversionrelated behaviors (adapted from [START_REF] Russo | The brain reward circuitry in mood disorders[END_REF]. The circuits of motivation -is it all about dopamine?

In an attempt to understand the molecular bases of motivation, early studies in the 1970s and 1980s have proposed that the neuromodulator dopamine has a central role in controlling reward-related behaviors [START_REF] Gerber | Pimozide attenuates lever pressing for water reinforcement in rats[END_REF][START_REF] Ungerstedt | Adipsia and aphagia after 6-hydroxydopamine induced degeneration of the nigro-striatal dopamine system[END_REF][START_REF] Wise | Neuroleptic-induced "anhedonia" in rats: pimozide blocks reward quality of food[END_REF]. Dopamine is a monoamine produced mainly by neurons within the VTA and substantia nigra pars compacta (SNc) which innervate the cerebral cortex and limbic forebrain regions [START_REF] Björklund | Dopamine neuron systems in the brain: an update[END_REF]. Initial studies using microdialysis measurements have suggested that dopamine release within the NAc (meso-limbic projection) is associated to self-stimulation reward [START_REF] Fiorino | Electrical stimulation of reward sites in the ventral tegmental area increases dopamine transmission in the nucleus accumbens of the rat[END_REF], while stress specifically activates the meso-cortical projection [START_REF] Thierry | Selective activation of mesocortical DA system by stress[END_REF]. Indeed, the function of dopamine in reward and aversive behaviors largely depend on the circuit connectivity of individual dopamine neurons [START_REF] Bromberg-Martin | Dopamine in Motivational Control: Rewarding, Aversive, and Alerting[END_REF][START_REF] Lammel | Projection-Specific Modulation of Dopamine Neuron Synapses by Aversive and Rewarding Stimuli[END_REF]. Furthermore, depending on their specific identity, dopamine neurons undergo different experience-driven synaptic modifications which are instrumental for behavioral adaptations underlying opposing motivational states [START_REF] Pignatelli | Role of Dopamine Neurons in Reward and Aversion: A Synaptic Plasticity Perspective[END_REF][START_REF] Volman | New insights into the specificity and plasticity of reward and aversion encoding in the mesolimbic system[END_REF].

It is now largely established that dopaminergic neurons undergo phasic changes of activity in response to unexpected salient stimuli. Seminal studies in behaving monkeys using single-unit recordings have demonstrated that dopamine neurons firing increases in phasic and burst-like manner when an unexpected reward is presented [START_REF] Schultz | A neural substrate of prediction and reward[END_REF]. Conversely, noxious foot pinch or foot shocks predominantly inhibit dopamine neurons burst activity [START_REF] Ungless | Uniform inhibition of dopamine neurons in the ventral tegmental area by aversive stimuli[END_REF], although some neurons also show phasic excitation [START_REF] Brischoux | Phasic excitation of dopamine neurons in ventral VTA by noxious stimuli[END_REF]. After several conditioning sessions where animals are trained to associate a cue with the delivery of a reward, the firing of dopamine neurons and the subsequent release of dopamine in the NAc no longer occur at the time of reward delivery, but shift to the cue that predicts it [START_REF] Schultz | A neural substrate of prediction and reward[END_REF][START_REF] Stuber | Reward-predictive cues enhance excitatory synaptic strength onto midbrain dopamine neurons[END_REF]. Moreover, the magnitude and direction of dopamine responses correlate with the predicted probability of the reward. Indeed, if an expected reward fails to occur or is smaller than expected, the activity of dopamine neurons is phasically inhibited, while if the reward is larger than expected dopamine neurons will fire at the time of reward delivery [START_REF] Fiorillo | Discrete coding of reward probability and uncertainty by dopamine neurons[END_REF][START_REF] Schultz | Predictive reward signal of dopamine neurons[END_REF][START_REF] Schultz | A neural substrate of prediction and reward[END_REF][START_REF] Tobler | Coding of predicted reward omission by dopamine neurons in a conditioned inhibition paradigm[END_REF].

These experiments led to the idea that dopamine neurons signal a reward prediction error, defined as the difference between the outcome of an expected and actual reward [START_REF] Schultz | Predictive reward signal of dopamine neurons[END_REF][START_REF] Keiflin | Review Dopamine Prediction Errors in Reward Learning and Addiction : From Theory to Neural Circuitry[END_REF]. Likewise, dopamine neurons serve as 'sensors' for any deviation from the expectancies, representing a teaching signal for future cuereward associations and reward-seeking behaviors. The short bursts of dopamine neurons trigger phasic release events in the NAc, where motivational signals are considered to be translated into a motor output leading to reward seeking behaviors [START_REF] Day | Associative learning mediates dynamic shifts in dopamine signaling in the nucleus accumbens[END_REF][START_REF] Mcclure | A computational substrate for incentive salience[END_REF][START_REF] Mogenson | From motivation to action: functional interface between the limbic system and the motor system[END_REF].

More recently, a causal relationship between dopamine neurons activity and conditioned learning have been provided by temporally precise and pattern-specific optogenetic modulation of VTA dopamine neurons activity. Indeed, physiologically relevant phasic activation of dopamine neurons expressing the light-activated cation channel rhodopsin 2 (ChR2) induces conditioned place preference behaviors while their inhibition via the hyperpolarizing and light-activated chloride ion pump Halorhodopsin (NpHR) produces conditioned place avoidance [START_REF] Tan | GABA neurons of the VTA drive conditioned place aversion[END_REF][START_REF] Tsai | Phasic firing in dopaminergic neurons is sufficient for behavioral conditioning[END_REF]. Moreover, these learning mechanisms and reward-oriented behaviors involve transient synaptic potentiation of excitatory transmission onto dopamine neurons [START_REF] Stuber | Reward-predictive cues enhance excitatory synaptic strength onto midbrain dopamine neurons[END_REF].

Similar synaptic plasticity occurs following single drug or stressful experience [START_REF] Saal | Drugs of abuse and stress trigger a common synaptic adaptation in dopamine neurons[END_REF][START_REF] Ungless | Single cocaine exposure in vivo induces long-term potentiation in dopamine neurons[END_REF]. However, in the case of prolonged drug administration synaptic changes in VTA dopamine neurons and the NAc become persistent contributing to cue-induced reinstatement of drug-seeking behaviors after drug withdrawal [START_REF] Chen | Cocaine but not natural reward self-administration nor passive cocaine infusion produces persistent LTP in the VTA[END_REF][START_REF] Mameli | Cocaine-evoked synaptic plasticity: persistence in the VTA triggers adaptations in the NAc[END_REF].

Taken together these data demonstrate an important role of dopamine neurons particularly in reward but also in aversion encoding and suggest that synaptic and circuit modifications within the meso-cortico-limbic system are instrumental for motivated behaviors. However, the dopamine system is far more complex in that it receives multiple inputs both from local GABAergic interneurons and from more distal structures, many of which also participate in motivational encoding (Fig1). Moreover, dopamine neurons present heterogeneity in terms of their physiological properties, input-output connectivity and their responses to valenced stimuli [START_REF] Lammel | Reward and aversion in a heterogeneous midbrain dopamine system[END_REF][START_REF] Lammel | Projection-Specific Modulation of Dopamine Neuron Synapses by Aversive and Rewarding Stimuli[END_REF][START_REF] Volman | New insights into the specificity and plasticity of reward and aversion encoding in the mesolimbic system[END_REF]. These aspects need to be taken into account when considering dopamine neurons function in reward or aversion.

Although motivational states can be driven by rewarding and aversive conditions, during my thesis I focused mainly on the cellular substrates devoted to aversion processing.

Neurobiological substrates of aversion

Aversion is a common term to designate the behavioral reaction of avoidance or escape in response to negative, unpleasant, painful, stressful or fearful events or stimuli. Acute and chronic exposure to such aversive stimuli produces short or long-lasting synaptic, structural and circuit modifications that can often lead to neuropsychiatric disorders including depression, anxiety, post-traumatic stress disorder and addiction [START_REF] Lüscher | Drug-evoked synaptic plasticity in addiction: from molecular changes to circuit remodeling[END_REF][START_REF] Nestler | The mesolimbic dopamine reward circuit in depression[END_REF][START_REF] Russo | The brain reward circuitry in mood disorders[END_REF]. Many studies have implicated the VTA dopamine system in such modifications [START_REF] Berton | Essential role of BDNF in the mesolimbic dopamine pathway in social defeat stress[END_REF][START_REF] Cao | Mesolimbic dopamine neurons in the brain reward circuit mediate susceptibility to social defeat and antidepressant action[END_REF][START_REF] Chaudhury | Rapid regulation of depression-related behaviours by control of midbrain dopamine neurons[END_REF][START_REF] Krishnan | Molecular adaptations underlying susceptibility and resistance to social defeat in brain reward regions[END_REF][START_REF] Lammel | Projection-Specific Modulation of Dopamine Neuron Synapses by Aversive and Rewarding Stimuli[END_REF].

However, it is now known that other structures directly or indirectly innervating the VTA, and often reciprocally connected, also contribute to different aspects of aversion (Fig1).

The picture is even more complex given the large heterogeneity of different cell types forming local microcircuits within the VTA and challenging our understanding of the contribution of distinct pathways to aversion encoding and avoidance behaviors. With the advances of new technologies such as the generation of specific mouse lines, viralbased circuit-and cell type-specific mapping as well as optogenetics it is now possible to interrogate the implication of distinct neuronal circuits and neuronal subtypes for different aspects of aversion. As a matter of example, specific cell type projections from the ventral bed nucleus of the stria terminalis (BNST) to GABAergic interneurons in the medial VTA have been involved in distinct motivational states. Indeed, glutamatergic BNST neurons, activating predominantly GABA neurons in the VTA, are excited by aversive conditions such as series of foot shocks or foot shock-associated cues. Moreover, ChR2-driven optogenetic activation of this pathway produces real time avoidance and elevated anxiety states(Fig2A) [START_REF] Jennings | Distinct extended amygdala circuits for divergent motivational states[END_REF]. This is consistent with evidence that activation of GABAergic VTA neurons inhibits dopamine neurons activity leading to conditioned place avoidance behaviors [START_REF] Tan | GABA neurons of the VTA drive conditioned place aversion[END_REF]. In contrast, GABA neurons of the BNST predominantly inhibit GABA neurons of the VTA and are preferentially silenced by foot shock exposure and foot shock-cues. Optogenetic activation of this pathway leads to real time preference and reward seeking behaviors (Fig2A) [START_REF] Jennings | Distinct extended amygdala circuits for divergent motivational states[END_REF].

Consistently, VTA GABA neurons inhibition drives preference, reinforces behavior and reduces anxiety states associated with previous aversive experience [START_REF] Jennings | Distinct extended amygdala circuits for divergent motivational states[END_REF]. Similarly to this, activation of dopamine neurons of the VTA also promotes reward-related behaviors [START_REF] Adamantidis | Optogenetic interrogation of dopaminergic modulation of the multiple phases of reward-seeking behavior[END_REF][START_REF] Tsai | Phasic firing in dopaminergic neurons is sufficient for behavioral conditioning[END_REF], suggesting that GABA neurons in the VTA may serve to break locally the activity of dopamine neurons and therefore control the expression of motivational states (Fig2B). These data raise the question whether other inputs onto dopamine neurons or onto GABA neurons of the midbrain can control different aspects of aversive behaviors. Indeed, a major challenge in the field is to identify the precise input-output and functional organization of the circuits of aversion. project to GABA neurons in the VTA. Their activation drives real time preference and reward-seeking (Jennings et al., 2013). (B). Direct optogenetic activation of VTA dopamine neurons [START_REF] Tsai | Phasic firing in dopaminergic neurons is sufficient for behavioral conditioning[END_REF][START_REF] Adamantidis | Optogenetic interrogation of dopaminergic modulation of the multiple phases of reward-seeking behavior[END_REF] or inhibition of GABA neurons [START_REF] Jennings | Distinct extended amygdala circuits for divergent motivational states[END_REF] promote reward, while activation of GABA neurons [START_REF] Tan | GABA neurons of the VTA drive conditioned place aversion[END_REF][START_REF] Van Zessen | Activation of VTA GABA neurons disrupts reward consumption[END_REF] and inhibition of dopamine neurons [START_REF] Tan | GABA neurons of the VTA drive conditioned place aversion[END_REF] is aversive.

A B

The lateral habenula: a control station of aversion

The lateral habenula (LHb) has gained considerable attention in the last decade because of its control on midbrain monoaminergic systems as well as for its crucial implication in aversion encoding and mood disorders [START_REF] Hikosaka | The habenula: from stress evasion to value-based decision-making[END_REF]. My PhD work has focused on unraveling some of the circuit and synaptic functions of the LHb in the context of aversion and drug experience.

Phylogeny and anatomy of the LHb

The LHb is part of the habenular complex, which is located at the posterior-dorsal end of the epithalamus close to the midline and at the border of the third ventricle (Fig3). It comprises a lateral (LHb) and medial division (MHb) which are anatomically, morphologically and functionally distinct [START_REF] Aizawa | Phylogeny and ontogeny of the habenular structure[END_REF][START_REF] Andres | Subnuclear organization of the rat habenular complexes[END_REF][START_REF] Bianco | The habenular nuclei: a conserved asymmetric relay station in the vertebrate brain[END_REF][START_REF] Kim | Dendritic morphology, local circuitry, and intrinsic electrophysiology of neurons in the rat medial and lateral habenular nuclei of the epithalamus[END_REF][START_REF] Sutherland | The dorsal diencephalic conduction system: a review of the anatomy and functions of the habenular complex[END_REF]. The habenula is phylogenetically conserved among vertebrates [START_REF] Aizawa | Phylogeny and ontogeny of the habenular structure[END_REF][START_REF] Bianco | The habenular nuclei: a conserved asymmetric relay station in the vertebrate brain[END_REF] 2009). In birds, reptiles and mammals the MHb and the LHb are homologous to the dorsal and ventral habenulae respectively in fish and amphibians [START_REF] Aizawa | Phylogeny and ontogeny of the habenular structure[END_REF][START_REF] Amo | Identification of the zebrafish ventral habenula as a homolog of the mammalian lateral habenula[END_REF]. The LHb and MHb relative proportion can vary across species. In mammals the size of the LHb is typically larger than the size of the MHb. Furthermore, a comparative analysis indicates that the proportional contribution of the LHb to the total surface of the habenular complex is considerably larger in humans compared to rats (Fig4A), supporting an increasing level of anatomical and functional specialization of the LHb throughout evolution [START_REF] Díaz | Morphologic and immunohistochemical organization of the human habenular complex[END_REF]. The LHb can be divided into lateral and medial portions that receive inputs in a segregated manner (Fig5B). Each of these main divisions of the LHb can be further subdivided into smaller subnuclei based on distinct and topographic cell morphology and cytoarchitecture. Indeed, a total of ten subnuclei, five within the medial and five within the lateral LHb, have been described in rats based on morphological criteria and differential immunoreactivity for cellular markers [START_REF] Andres | Subnuclear organization of the rat habenular complexes[END_REF][START_REF] Geisler | Morphologic and cytochemical criteria for the identification and delineation of individual subnuclei within the lateral habenular complex of the rat[END_REF]. The medial LHb comprises an anteriour subnucleus (LHbMA), located at the most rostral portion of the LHb, a superior (LHbMS), parvocellular (LHbMPc), central (LHbMC) and marginal subnuclei (LHbMMg). The lateral LHb contains a parvocellular (LHbLPc), magnocellular (LHbLMc), oval (LHbLO), marginal (LHbLMg) and basal subnuclei (LHbLB) (Fig4B). This subnuclear organization of the LHb seems to be well preserved in rodents since it is also present in the mouse [START_REF] Wagner | Correlating habenular subnuclei in rat and mouse by using topographic, morphological, and cytochemical criteria[END_REF]. In the human LHb five subnuclei have also been described

although some discrepancies exist at the level of their relative position, size, morphology and cell organization compared to rodents [START_REF] Díaz | Morphologic and immunohistochemical organization of the human habenular complex[END_REF]. Despite this detailed subnuclear organization and evidence indicating certain level of input-output-specific connectivity within some of the nuclei (Fig5B and Fig6B), LHb neurons seem to be relatively homogeneous in their basic cell properties [START_REF] Weiss | Morphological and electrophysiological characteristics of neurons within identified subnuclei of the lateral habenula in rat brain slices[END_REF]. A genetic profiling of LHb neurons would be required in order to identify potential discrimination criteria for different LHb subnuclei and would provide a tool to assess cell type-specific functions of LHb subpopulations in distinct aspects of motivated behaviors (Lecca et al., 2014;[START_REF] Proulx | Reward processing by the lateral habenula in normal and depressive behaviors[END_REF]. such as the GABAB receptor, the Kir3.2 potassium channel and neurofilament. (adapted from [START_REF] Geisler | Morphologic and cytochemical criteria for the identification and delineation of individual subnuclei within the lateral habenular complex of the rat[END_REF].

Inputs to the LHb

The habenular complex is positioned at the highway of a major information stream in the brain, connecting the limbic forebrain and basal ganglia regions with midbrain neuromodulatory systems. It receives most of its inputs through a fiber bundle called the stria medullaris and sends its projections to monoaminergic centers through the fasciculus retroflexus (Fig3B), which altogether form the diencephalic conduction system [START_REF] Sutherland | The dorsal diencephalic conduction system: a review of the anatomy and functions of the habenular complex[END_REF]. Although the MHb and LHb receive their inputs and send their outputs using the same fiber tracts, they are differently innervated and target different brain nuclei. A potential connectivity between the MHb and LHb has been debated, but has never been anatomically or functionally proven.

The MHb receives inputs from septal and diagonal band of Broca (DBB) areas and sends in turn its axons to the interpeduncular nucleus of the midbrain (Herkenham andNauta, 1979, 1977). In contrast, the LHb receives projections from the output structure of the basal ganglia -the entopeduncular nucleus (EPN; homologue of the globus pallidus interna in primates and humans) and from limbic forebrain regions including the lateral hypothalamus (LH), lateral preoptic area (LPO), ventral pallidum (VP), BNST, DBB, lateral septum (LS) as well as feedback projections from the laterodorsal tegmentum (LDT), the VTA and the dorsal and median raphe nuclei (DRN and MRN) (Fig5A) [START_REF] Herkenham | Afferent connections of the habenular nuclei in the rat. A horseradish peroxidase study, with a note on the fiber-of-passage problem[END_REF][START_REF] Nagy | Evidence for a GABA-containing projection from the entopeduncular nucleus to the lateral habenula in the rat[END_REF][START_REF] Parent | The origin of forebrain afferents to the habenula in rat, cat and monkey[END_REF][START_REF] Kowski | Differential Projections from Subfields in the Lateral Preoptic Area to the Lateral Habenular Complex of the[END_REF][START_REF] Li | Synaptic potentiation onto habenula neurons in the learned helplessness model of depression[END_REF][START_REF] Tripathi | Axonal branching patterns of ventral pallidal neurons in the rat[END_REF][START_REF] Lammel | Input-specific control of reward and aversion in the ventral tegmental area[END_REF]Swanson 1982;[START_REF] Aghajanian | Habenular and other midbrain raphe afferents demonstrated by a modified retrograde tracing technique[END_REF]. A projection from the PFC has also been described [START_REF] Kim | Topography of descending projections from anterior insular and medial prefrontal regions to the lateral habenula of the epithalamus in the rat[END_REF][START_REF] Li | Synaptic potentiation onto habenula neurons in the learned helplessness model of depression[END_REF][START_REF] Warden | A prefrontal cortex-brainstem neuronal projection that controls response to behavioural challenge[END_REF]. Some of the inputs to the LHb represent a topographical organization potentially important for LHb functions (Fig5B). lateral LHb (LHbLO), while this subregion is devoid of LH and LPO inputs [START_REF] Kowski | Differential Projections from Subfields in the Lateral Preoptic Area to the Lateral Habenular Complex of the[END_REF][START_REF] Poller | A glutamatergic projection from the lateral hypothalamus targets VTA-projecting neurons in the lateral habenula of the rat[END_REF]Shabel et al., 2012). LH axons are more concentrated in the medial aspect of the LHb (with the exception of the LHbMPc) [START_REF] Poller | A glutamatergic projection from the lateral hypothalamus targets VTA-projecting neurons in the lateral habenula of the rat[END_REF]. Other input terminals, such as the VTA ones are widely distributed throughout the LHb [START_REF] Hnasko | Ventral tegmental area glutamate neurons: electrophysiological properties and projections[END_REF]Root et al., 2014b). sm: stria medullaris; fr: fasiculust retroflexus, cc: corpus callosum; D3V: dorsal 3th ventricle and 4V: 4 th ventricle

Output connectivity of the LHb

LHb neurons are almost exclusively glutamatergic and long-range projecting [START_REF] Kim | Dendritic morphology, local circuitry, and intrinsic electrophysiology of neurons in the rat medial and lateral habenular nuclei of the epithalamus[END_REF][START_REF] Li | Synaptic potentiation onto habenula neurons in the learned helplessness model of depression[END_REF][START_REF] Weiss | Morphological and electrophysiological characteristics of neurons within identified subnuclei of the lateral habenula in rat brain slices[END_REF]. Although some studies have suggested the existence of local GABAergic neurons in the medial LHb, functional evidence about a potential LHb microcircuit is still lacking [START_REF] Li | Synaptic potentiation onto habenula neurons in the learned helplessness model of depression[END_REF][START_REF] Zhang | Thirst Is Associated with Suppression of Habenula Output and Active Stress Coping: Is there a Role for a Non-canonical Vasopressin-Glutamate Pathway? Front[END_REF]. Anatomical and physiological studies indicate that LHb neurons send their axons mainly to GABAergic neurons in the midbrain. Indeed, tracing experiments show that neurons located mainly within the lateral LHb project to the GABAergic rostro-medial tegmental nucleus (RMTg; [START_REF] Balcita-Pedicino | The inhibitory influence of the lateral habenula on midbrain dopamine cells: ultrastructural evidence for indirect mediation via the rostromedial mesopontine tegmental nucleus[END_REF][START_REF] Gonçalves | Differential projections from the lateral habenula to the rostromedial tegmental nucleus and ventral tegmental area in the rat[END_REF]Jhou et al., 2009b;Meye et al., 2016;[START_REF] Sego | Lateral habenula and the rostromedial tegmental nucleus innervate neurochemically distinct subdivisions of the dorsal raphe nucleus in the rat[END_REF], also called tail VTA [START_REF] Kaufling | Afferents to the GABAergic tail of the ventral tegmental area in the rat[END_REF][START_REF] Perrotti | DeltaFosB accumulates in a GABAergic cell population in the posterior tail of the ventral tegmental area after psychostimulant treatment[END_REF]. In contrast, LHb neurons originating from the medial aspect send their axons preferentially to monoaminergic nuclei [START_REF] Gonçalves | Differential projections from the lateral habenula to the rostromedial tegmental nucleus and ventral tegmental area in the rat[END_REF][START_REF] Sego | Lateral habenula and the rostromedial tegmental nucleus innervate neurochemically distinct subdivisions of the dorsal raphe nucleus in the rat[END_REF], where they form functional synapses with local GABAergic neurons [START_REF] Lammel | Input-specific control of reward and aversion in the ventral tegmental area[END_REF][START_REF] Weissbourd | Article Presynaptic Partners of Dorsal Raphe Serotonergic and GABAergic Neurons[END_REF] as well as with dopamine neurons of the VTA [START_REF] Balcita-Pedicino | The inhibitory influence of the lateral habenula on midbrain dopamine cells: ultrastructural evidence for indirect mediation via the rostromedial mesopontine tegmental nucleus[END_REF][START_REF] Lammel | Input-specific control of reward and aversion in the ventral tegmental area[END_REF] or serotonin neurons in the caudal dorsal raphe [START_REF] Dorocic | Article A Whole-Brain Atlas of Inputs to Serotonergic Neurons of the Dorsal and Median Raphe Nuclei[END_REF]Sego et al., 2014) (Fig 6B). Further, it has been shown that individual neurons project either to VTA or to raphe nuclei without collateralizing, suggesting that single LHb neurons have distinct output targets [START_REF] Bernard | Individual neurons in the rat lateral habenular complex project mostly to the dopaminergic ventral tegmental area or to the serotonergic raphe nuclei[END_REF][START_REF] Gonçalves | Differential projections from the lateral habenula to the rostromedial tegmental nucleus and ventral tegmental area in the rat[END_REF][START_REF] Li | Synaptic potentiation onto habenula neurons in the learned helplessness model of depression[END_REF]Maroteaux and Mameli, 2012). In addition, the LHb has been recently shown to send axons to GABA neurons within the LDT and to orexin-and melanin concentrating hormone (MCH)-expressing neurons in the LH [START_REF] González | Awake dynamics and brain-wide direct inputs of hypothalamic MCH and orexin networks[END_REF][START_REF] Lammel | Input-specific control of reward and aversion in the ventral tegmental area[END_REF][START_REF] Yang | Laterodorsal tegmentum interneuron subtypes oppositely regulate olfactory cue-induced innate fear[END_REF]. While the former has also been functionally proven, it remains to be confirmed whether LHb neurons establish functional synaptic connections in the LH (Fig6A).

Given that LHb neurons are of glutamatergic phenotype and that they project to a vast majority of GABA neurons in the midbrain it is plausible that they may disynaptically inhibit dopamine or serotonin neurons potentially providing aversive signals to the midbrain.

Function of the LHb in reward and aversion encoding

Seminal studies in rhesus monkeys have suggested a role of the LHb in encoding aversive stimuli and in controlling dopaminergic neurons activity, crucially contributing to our understanding of motivational processing. When monkeys were exposed to unexpected aversive air puff, a cue predicting it (after conditioning sessions) or an omission of expected reward, the majority of LHb neurons increased phasically their firing activity. On the contrary, when an unexpected rewarding stimulus or rewardpredictive cues were presented to the animals or alternatively an expected punishment was omitted, LHb neurons firing decreased or remained unchanged respectively (Fig7D) (Matsumoto andHikosaka, 2009a, 2007). Moreover, there was a linear relationship between the objective value of the stimulus and the conditioned stimulus (cue) response for negative outcomes, but not for positive ones (Fig7A). This has led to the idea that but also to dopamine and serotonin neurons in the VTA and dorsal raphe. The LHb also projects to MCH and Orexinexpressing neurons in the LH. (B). LHb neurons targeting the RMTg are mainly located in the lateral LHb [START_REF] Kowski | Differential Projections from Subfields in the Lateral Preoptic Area to the Lateral Habenular Complex of the[END_REF][START_REF] Poller | A glutamatergic projection from the lateral hypothalamus targets VTA-projecting neurons in the lateral habenula of the rat[END_REF]Shabel et al., 2012), while those targeting the VTA and dorsal raphe are located in the medial LHb [START_REF] Poller | A glutamatergic projection from the lateral hypothalamus targets VTA-projecting neurons in the lateral habenula of the rat[END_REF]. sm: stria medullaris; fr: fasiculust retroflexus, cc: corpus callosum; 3V and 4V: 3th and

4 th ventricle
LHb neuronal activity discriminates the valence of stimuli and preferentially represents negative-valued events with respect to dopamine neurons, the activation of which preferentially represents reward (Matsumoto and Hikosaka, 2009b;[START_REF] Mirenowicz | Preferential activation of midbrain dopamine neurons by appetitive rather than aversive stimuli[END_REF]. It is important to note that when a reward was fully predictable (100% reward predictive cue) LHb neurons did not respond to the unconditioned stimulus (reward delivery), whereas if the reward was not fully predictable (50% reward occurrence in previous sessions) LHb neurons were inhibited (Fig7B). The magnitude of the inhibitory response increased with the reward unpredictability. Similarly, in trials where an aversive stimulus was 100% predictable, LHb neurons responded with excitation to the unconditioned stimulus (air puff) and the response was reduced compared to trials where the aversive stimulus was not fully predictable (Fig7C). These data suggest that when an aversive or rewarding stimulus is predictable the response of LHb neurons during the stimulus presentation (excitation or inhibition) is decreased in magnitude or absent compared to when it occurs in an unpredictable manner (Matsumoto and Hikosaka, 2009a) (Fig7D). Altogether this evidence strongly indicates a role of LHb neurons in negative-reward prediction error. This behavior of LHb neurons appears opposite to the behavior of dopamine neurons in response to rewarding and aversive stimuli and their predictive cues (Fig7E). As discussed in the previous chapters, unpredicted rewarding stimuli or reward-predictive cues lead to rapid and brief bursts of activity in dopamine neurons. When the reward occurrence becomes predictable dopamine neurons no longer fire, whereas an omission of expected rewards inhibits them [START_REF] Schultz | A neural substrate of prediction and reward[END_REF]. In contrast, aversive stimuli such as noxious foot pinch or foot shock mainly decrease dopamine neurons firing rates and bursting activity [START_REF] Ungless | Uniform inhibition of dopamine neurons in the ventral tegmental area by aversive stimuli[END_REF], although few neurons respond with phasic excitation [START_REF] Brischoux | Phasic excitation of dopamine neurons in ventral VTA by noxious stimuli[END_REF]. This opposite processing of reward and aversion in the LHb and VTA led to the idea that LHb neurons provide a negative-reward predictive signal to dopamine neurons [START_REF] Bromberg-Martin | Dopamine in Motivational Control: Rewarding, Aversive, and Alerting[END_REF][START_REF] Keiflin | Review Dopamine Prediction Errors in Reward Learning and Addiction : From Theory to Neural Circuitry[END_REF]Schultz, 2007b). Importantly, the activity of LHb neurons is not only opposed, but it also precedes the activity of SNc and VTA dopamine neurons in non-rewarded trials, indicating that LHb neurons exert inhibitory drive onto dopamine neurons (Matsumoto and Hikosaka, 2007). This is in line with an inhibitory effect of LHb electrical stimulation onto dopamine neurons activity [START_REF] Christoph | Stimulation of the lateral habenula inhibits dopamine-containing neurons in the substantia nigra and ventral tegmental area of the rat[END_REF][START_REF] Ji | Lateral habenula stimulation inhibits rat midbrain dopamine neurons through a GABA(A) receptor-mediated mechanism[END_REF]. Moreover, blocking excitatory transmission within the LHb leads to transient increase of dopamine release in the NAc, dorsal striatum and PFC, suggesting that LHb activity exerts a tonic inhibition on dopaminergic transmission [START_REF] Lecca | Rescue of GABA B and GIRK function in the lateral habenula by protein phosphatase 2A inhibition ameliorates depression-like phenotypes in mice[END_REF]. However, in a recent study LHb lesions prevented a decrease in dopamine neurons activity only when a reward was omitted but not when an aversive stimulus was delivered, suggesting that LHb neurons are not the only source of negative reward prediction signal to dopamine neurons and that instead the LHb codes rather for disappointment [START_REF] Tian | Habenula Lesions Reveal that Multiple Mechanisms Underlie Dopamine Prediction Errors Article Habenula Lesions Reveal that Multiple Mechanisms Underlie Dopamine Prediction Errors[END_REF]. A limitation, nonetheless, of this study is the use of electrolytic lesion of the LHb, where circuit adaptations might have occurred as a compensatory mechanism. In this regard, the use of optogenetic or chemogenetic silencing of the LHb activity could be more informative to determine its contribution to negative reward prediction error when controlling midbrain structures.

In humans, functional magnetic resonance imaging (fMRI) showed increased LHb activity when healthy volunteers received negative feedback after failing to perform a task or when they were exposed to aversive stimuli [START_REF] Hennigan | Distinct Midbrain and Habenula Pathways Are Involved in Processing Aversive Events in Humans[END_REF][START_REF] Ullsperger | Error monitoring using external feedback: specific roles of the habenular complex, the reward system, and the cingulate motor area revealed by functional magnetic resonance imaging[END_REF]. Furthermore, similarly to monkeys the LHb of humans responded preferentially to cues signaling negative outcomes [START_REF] Lawson | The habenula encodes negative motivational value associated with primary punishment in humans[END_REF].

Altogether data in human and non-human primates suggest an important role of the LHb in predicting negative outcomes necessary for motivated behaviors. (A). Linear relationship between averaged conditioned stimulus response (cue) and the objective value for negative outcomes and non-linear relationship for rewarding outcomes. (B). Relationship between the averaged LHb unconditioned stimulus (reward) response and the probability for reward or reward omission (positive prediction error). (C). Relationship between averaged LHb unconditioned stimulus (air puff) response and the probability for airpuff or airpuff omission (negative prediction error). (From Matsumoto and Hikosaka, 2009a). (D). LHb responses to unpredictable (non-learned) rewarding or aversive stimuli. After learning, LHb neurons responses shift to the cues predicting 100% reward or 100% punishment. However, LHb neurons respond also to the unconditioned stimulus when it is aversive but with smaller magnitude. When a cue predicts omission of reward LHb neurons increase firing and when a cue predicts omission of air puff LHb neurons decrease firing. (E). Dopamine neurons show opposite responses to unpredictable rewards and to cues predicting rewards (increased firing) compared to LHb neurons.

When a cue predicts reward dopamine neurons fire, but if the reward is omitted dopamine neurons are phasically inhibited.

Role of LHb output for aversion processing

Following these studies an important question arose: given the excitatory nature of LHb neurons, how does their activity drive inhibition onto dopamine neurons? LHb neurons projections to GABA neurons of the midbrain might mediate LHb inhibitory control onto dopamine neurons. Indeed, an important link between the LHb and dopamine neurons function in reward/aversion encoding is the GABAergic RMTg. Anatomical and ultrastructural studies indicate that the LHb sends a major glutamatergic efferent to the RMTg. RMTg neurons in turn project to dopamine neurons within the VTA and SNc, although some synaptic contacts with non-dopaminergic cells have also been reported [START_REF] Balcita-Pedicino | The inhibitory influence of the lateral habenula on midbrain dopamine cells: ultrastructural evidence for indirect mediation via the rostromedial mesopontine tegmental nucleus[END_REF][START_REF] Brinschwitz | Glutamatergic axons from the lateral habenula mainly terminate on GABAergic neurons of the ventral midbrain[END_REF]Jhou et al., 2009b;[START_REF] Kaufling | Afferents to the GABAergic tail of the ventral tegmental area in the rat[END_REF]. In line with this, retrogradely labeled RMTg neurons projecting to the VTA were found in close apposition to anterogradely labelled LHb axon terminals [START_REF] Gonçalves | Differential projections from the lateral habenula to the rostromedial tegmental nucleus and ventral tegmental area in the rat[END_REF]. Importantly, RMTg neurons encode rewarding or aversive stimuli similarly to the LHb and opposite to dopamine neurons, by phasically decreasing (reward) or increasing (aversion) their firing (Fig8) (Jhou et al., 2009a;Matsumoto and Hikosaka, 2007).

Functional data in anesthetized rats and in behaving monkeys indicate that RMTg neurons receiving LHb input increase phasically their firing in response to aversive stimuli and inhibit dopamine neurons, further supporting a role of RMTg in relaying LHb signals to dopamine neurons [START_REF] Hong | Negative reward signals from the lateral habenula to dopamine neurons are mediated by rostromedial tegmental nucleus in primates[END_REF][START_REF] Lecca | Inhibitory inputs from rostromedial tegmental neurons regulate spontaneous activity of midbrain dopamine cells and their responses to drugs of abuse[END_REF][START_REF] Matsui | Opioid-sensitive GABA inputs from rostromedial tegmental nucleus synapse onto midbrain dopamine neurons[END_REF]. In line with a role of LHb output to midbrain GABA neurons in driving negative teaching signals, light-activation of ChR2-expressing LHb terminals in the RMTg of mice produced real-time and conditioned-place avoidance behaviors (Stamatakis and Stuber, 2012). Moreover stimulation of this pathway produced negative reinforcement since animals nose-poked to terminate stimulation, while it disrupted positive reinforcement as animals nose-poked less to obtain a reward, suggesting that LHb-to-RMTg pathway activity provides a punishing signal (Fig8) (Stamatakis and Stuber, 2012).

Although LHb neurons strongly project to the RMTg, they also send, direct projections to GABA and dopamine neurons in the VTA. Moreover there is a topographic organization of this connectivity since the VTA projecting neurons are found mainly in the medial division of the LHb and send their axons to the ventral medio-posterior aspects of the VTA [START_REF] Gonçalves | Differential projections from the lateral habenula to the rostromedial tegmental nucleus and ventral tegmental area in the rat[END_REF][START_REF] Omelchenko | Lateral habenula projections to dopamine and GABA neurons in the rat ventral tegmental area[END_REF]Swanson 1982;[START_REF] Phillipson | Dopamine neurones of the ventral tegmentum project to both medial and lateral habenula. Some implications for habenular function[END_REF][START_REF] Skagerberg | Origin, course and termination of the mesohabenular dopamine pathway in the rat[END_REF]. Importantly, light activation of ChR2-expressing LHb terminals in the VTA of mice evoked responses in half of non-dopaminergic cells (putative GABA neurons), whereas only a very small percentage of dopamine neurons responded to the stimulation (Stamatakis and Stuber, 2012). Altogether these data indicate that the LHb preferentially connects to GABA neurons in the midbrain, likewise exerting an inhibitory control on dopamine neurons [START_REF] Ji | Lateral habenula stimulation inhibits rat midbrain dopamine neurons through a GABA(A) receptor-mediated mechanism[END_REF]Matsumoto and Hikosaka, 2007). This is in line with the results obtained in monkeys showing that excitation of LHb neurons in response to aversive events is followed by inhibition of dopamine neurons (Matsumoto and Hikosaka, 2007). In agreement with this, light activation of ChR2-expressing GABA neurons in the VTA inhibits dopamine neurons firing, reduces reward-related behaviors (sucrose licking) and induces conditioned place avoidance (Fig2B), similarly to activation of the LHb-RMTg pathway (Fig8) (Stamatakis and Stuber, 2012;[START_REF] Tan | GABA neurons of the VTA drive conditioned place aversion[END_REF][START_REF] Van Zessen | Activation of VTA GABA neurons disrupts reward consumption[END_REF]. Moreover, inhibition of dopamine neurons expressing NpHR led to avoidance behaviors, further supporting that midbrain GABA-mediated inhibition of dopamine neurons is instrumental for the expression of aversive behaviors (Fig2B) [START_REF] Tan | GABA neurons of the VTA drive conditioned place aversion[END_REF].

The LHb also targets dopamine neurons in the ventromedial and posterior portion of the VTA, which preferentially send axons to the medial PFC and undergo aversive experience-dependent plasticity [START_REF] Lammel | Input-specific control of reward and aversion in the ventral tegmental area[END_REF][START_REF] Lammel | Projection-Specific Modulation of Dopamine Neuron Synapses by Aversive and Rewarding Stimuli[END_REF]. Furthermore, optogenetic activation of retrogradely labeled VTA-projecting LHb neurons produces conditioned place avoidance (Fig8) [START_REF] Lammel | Input-specific control of reward and aversion in the ventral tegmental area[END_REF].

In humans, together with an increased LHb activity a higher activation of the VTA, SNc and PFC regions were also detected after aversive stimuli and this was associated with increased functional connectivity between the habenula and VTA as well as VTA and PFC [START_REF] Hennigan | Distinct Midbrain and Habenula Pathways Are Involved in Processing Aversive Events in Humans[END_REF], suggesting that both in rodents and in humans the habenulo-meso-cortico-limbic circuit is involved in aversion processing.

Collectively, all these anatomical and functional data strongly suggest that LHb output conveys aversive signals via different anatomical connections and ultimately underlies aspects of aversive behaviors. The stimulation of each of these distinct LHb pathways to the midbrain is sufficient, but it remains still unclear whether it is necessary for aversive behaviors. Other important questions arising following these studies are whether these pathways are simultaneously active or occur at different instances and which particular aspects of aversive behaviors do they encode.

+

Role of inputs to the LHb for aversion processing

Data collected so far suggest that the LHb lacks local GABAergic control [START_REF] Li | Synaptic potentiation onto habenula neurons in the learned helplessness model of depression[END_REF]. Instead its activity is largely shaped by multiple long-range synaptic inputs (Root et al., 2014b;Shabel et al., 2014Shabel et al., , 2012;;[START_REF] Stamatakis | Lateral Hypothalamic Area Glutamatergic Neurons and Their Projections to the Lateral Habenula Regulate Feeding and Reward[END_REF]. Indeed, many forebrain and midbrain nuclei send glutamatergic and GABAergic terminals directly to the LHb contributing to its function in reward and aversion. Recent studies have proposed that some inputs to the LHb, including the EPN, VTA and potentially the LH are capable to co-release GABA and glutamate from the same synaptic terminal and to form functional postsynaptic connections with LHb neurons (Root et al., 2014b;Shabel et al., 2014;[START_REF] Stamatakis | Lateral Hypothalamic Area Glutamatergic Neurons and Their Projections to the Lateral Habenula Regulate Feeding and Reward[END_REF]. Activating ChR2 specifically in glutamatergic neurons in a

Vglut2-Cre-dependent manner (Vglut2: vesicular glutamate transporter 2) in any of these three inputs led to both AMPA receptor-and GABA A receptor-mediated postsynaptic currents. Similarly, a Vgat-Cre-or GAD67-Cre-dependent activation of ChR2 (Vgat:

vesicular GABA transporter and GAD67: glutamate decarboxylase, the enzyme synthetizing GABA from glutamate) in GABAergic neurons of the VTA or EPN respectively triggered both excitatory and inhibitory postsynaptic currents (EPSCs and IPSCs respectively) in the LHb. A fine ultrastructural analysis in mouse and rat has shown that the majority of VTA-to-LHb neurons co-release glutamate and GABA from the same synaptic terminal forming functional asymmetrical and symmetrical synapses (Root et al., 2014b). Activation of the EPN and LH inputs drives LHb neurons to fire, while the predominant effect of VTA-to-LHb pathway stimulation in vivo was inhibitory (Fig9) (Root et al., 2014b;Shabel et al., 2014;[START_REF] Stamatakis | Lateral Hypothalamic Area Glutamatergic Neurons and Their Projections to the Lateral Habenula Regulate Feeding and Reward[END_REF]. Interestingly, VTA-to-LHb optostimulation resulted in inhibition followed by rebound excitation in some LHb neurons, raising the possibility that the co-release of glutamate and GABA from the same synapse may have a role to temporally control LHb neuronal firing (Root et al., 2014b). This hypothesis is supported by previous evidence showing that phasic electrical stimulation of the midbrain decreases the firing rate of a subset of LHb neurons, whereas a tetanic stimulation tends to increase it [START_REF] Shen | Stimulation of midbrain dopaminergic structures modifies firing rates of rat lateral habenula neurons[END_REF].

Importantly, optogenetic activation of specific inputs to the LHb can drive distinct motivational states. Indeed, stimulation of the EPN-to-LHb synapses, which has an overall excitatory effect on LHb firing, drives real-time place avoidance behavior (Fig9), consistent with the idea that an increased LHb activity is required for the expression of negative states (Shabel et al., 2012). EPN inputs impinge onto neurons of the lateral division of the LHb, which project mainly to GABA neurons of the midbrain, further supporting the role of this pathway in aversion encoding and avoidance behaviors (Fig5B and Fig6B) [START_REF] Gonçalves | Differential projections from the lateral habenula to the rostromedial tegmental nucleus and ventral tegmental area in the rat[END_REF]Lecca et al., 2014;[START_REF] Proulx | Reward processing by the lateral habenula in normal and depressive behaviors[END_REF]Shabel et al., 2012, Meye 2016). Interestingly, although the EPN participates mainly in the control of body movements [START_REF] Delong | Activity of pallidal neurons during movement[END_REF], some EPN neurons encode negativereward prediction error similarly to the LHb, by phasic excitation upon unexpected aversive stimuli or reward omissions and phasic inhibition by unexpected rewards. The response of these negative-reward encoding neurons in the EPN precedes that of LHb neurons, suggesting that they may excite LHb neurons during aversive stimuli or reward omission (Fig9) [START_REF] Hong | The globus pallidus sends reward-related signals to the lateral habenula[END_REF].

Optostimulation of the VTA-to-LHb pathway also produced avoidance of the lightassociated chamber after conditioning, suggesting that this input may be involved in avoidance learning (Fig9) (Root et al., 2014a). Nevertheless, this effect seems puzzling considering that the VTA-to-LHb input inhibits the majority of LHb neurons, which would presumably lead to disinhibition of downstream dopamine neurons and would drive rewarding states. A possible explanation to this issue may reside in that in this study a continuous light activation has been employed when the animal was in the lightassociated chamber, which may result in progressive increase of LHb neuronal activity and therefore to increased inhibitory drive onto downstream dopamine targets [START_REF] Shen | Stimulation of midbrain dopaminergic structures modifies firing rates of rat lateral habenula neurons[END_REF]. Whether the balance of excitation and inhibition at co-releasing synapses is frequency or time-dependent remains however an open question. Alternatively, VTA inputs may target LHb neurons projecting directly onto dopamine neurons, therefore inhibiting them and contributing to avoidance behaviors.

Another important question arising is whether VTA neurons projecting to the LHb release dopamine and what the functional consequences would be on LHb neuronal activity. Indeed, early anatomical and tracing studies have described afferent projection from the ventromedial portion of the VTA to the medial aspect of the LHb (Swanson 1982;[START_REF] Phillipson | Dopamine neurones of the ventral tegmentum project to both medial and lateral habenula. Some implications for habenular function[END_REF][START_REF] Skagerberg | Origin, course and termination of the mesohabenular dopamine pathway in the rat[END_REF], where thyrosin hydroxylase positive fibers (TH, the rate limiting enzyme for dopamine synthesis) are found [START_REF] Aizawa | Molecular characterization of the subnuclei in rat habenula[END_REF][START_REF] Geisler | Morphologic and cytochemical criteria for the identification and delineation of individual subnuclei within the lateral habenular complex of the rat[END_REF][START_REF] Gruber | Dopaminergic projections from the VTA substantially contribute to the mesohabenular pathway in the rat[END_REF]. Some more recent studies instead report that VTA fibers are widely distributed across the LHb and are predominantly TH negative and Vglut2 positive [START_REF] Hnasko | Ventral tegmental area glutamate neurons: electrophysiological properties and projections[END_REF]Root et al., 2014b). Despite this evidence, characterization of the expression profile of LHb-projecting VTA neurons showed that a non-negligible population expresses TH in addition to glutamate and GABA markers (Root et al., 2014b), suggesting that these neurons are potentially capable to release dopamine. Cre-dependent ChR2 expression in the VTA of TH-Cre mice led to detection of ChR2 + terminals in the LHb, but a weak expression of TH.

Optostimulation of these VTA TH+/ChR2+ fibers in the LHb failed to release detectable RMTg neurons, whereas the same stimulation increased VTA activity. Moreover, stimulation of this pathway led to conditioned place preference as well as to positive reinforcement (Fig9) (Stamatakis et al., 2013). This is in discrepancy with the aversive effect of VTA-to-LHb stimulation reported by Root et al., 2014a. This contradiction may arise from the different genetic approaches employed and the potential targeting of different LHb neuronal subpopulations which may in turn project to distinct downstream targets. Notably, it has also been shown recently that the TH-Cre line can present some ectopic expression in non-TH-expressing neurons, raising the issue of choosing appropriate Cre-driver lines to study complex circuits [START_REF] Lammel | Diversity of Transgenic Mouse Models for Selective Targeting of Midbrain Dopamine Neurons Matters Arising Diversity of Transgenic Mouse Models for Selective Targeting of Midbrain Dopamine Neurons[END_REF][START_REF] Stuber | Considerations when using cre-driver rodent lines for studying ventral tegmental area circuitry[END_REF].

Other inputs to the LHb have also been implicated in different aversive behaviors.

Anatomical studies indicate that predominantly glutamatergic LH inputs target mainly VTA-projecting LHb neurons in the medial division of the LHb (Fig5 and Fig6) [START_REF] Gonçalves | Differential projections from the lateral habenula to the rostromedial tegmental nucleus and ventral tegmental area in the rat[END_REF][START_REF] Poller | A glutamatergic projection from the lateral hypothalamus targets VTA-projecting neurons in the lateral habenula of the rat[END_REF]. Excitatory, Vglut2-expressing LH neurons projecting to the LHb (LH Vglut2 -to-LHb) bidirectionally modulate motivational states.

Indeed, optical stimulation of LH Vglut2 -to-LHb pathway increases LHb neurons activity and induces real-time place avoidance, whereas NpHR-driven inhibition of this pathway leads to real-time preference (Fig9) [START_REF] Stamatakis | Lateral Hypothalamic Area Glutamatergic Neurons and Their Projections to the Lateral Habenula Regulate Feeding and Reward[END_REF]. This is in line with the necessity of increased LHb output for the expression of aversive behaviors and a decreased LHb output for reward-related behaviors [START_REF] Lammel | Input-specific control of reward and aversion in the ventral tegmental area[END_REF]Stamatakis and Stuber, 2012).

Another glutamatergic projection described to date arises from the mPFC [START_REF] Kim | Topography of descending projections from anterior insular and medial prefrontal regions to the lateral habenula of the epithalamus in the rat[END_REF][START_REF] Li | Synaptic potentiation onto habenula neurons in the learned helplessness model of depression[END_REF][START_REF] Warden | A prefrontal cortex-brainstem neuronal projection that controls response to behavioural challenge[END_REF]. Indeed, a study in rats shows that activating glutamatergic mPFC inputs expressing ChR2 in the LHb leads to behavioral despair as shown by decreased mobility in the forced-swim test (FST), a paradigm widely use to screen for depressive-like behaviors (Fig9) [START_REF] Warden | A prefrontal cortex-brainstem neuronal projection that controls response to behavioural challenge[END_REF]. However, the authors did not further investigate the contribution of this input for LHb neurons activity, leaving it open for further investigation.

Altogether, these studies demonstrate that distinct excitatory or inhibitory pathways can drive LHb neuronal activity ultimately contributing to the encoding and expression of motivated behaviors. Importantly, excitatory inputs arising from the EPN, LH, VTA and mPFC are capable to drive avoidance behaviors, whereas inhibitory inputs such as the GABAergic VTA-to-LHb component can inhibit neuronal activity and produce preference behaviors (Fig9). A caveat however of these studies is the use of non-specific stimulation patterns to activate exogenously-expressed opsins, which may overcome other physiological mechanisms relevant for these behaviors. It is also interesting to address whether different frequency patterns lead to different glutamate/GABA release ratios from the co-releasing synapses. Another limitation of the so far described approaches is that in the majority of the cases, it has been shown sufficiency of a pathway in inducing specific behaviors, but not its necessity. Nevertheless these studies demonstrate the importance of investigating specific circuits within the LHb to the level of single cell-types for different aspects of motivated behaviors. Furthermore, it is of major interest to closely investigate whether and how synaptic plasticity occurs in the LHb following rewarding or aversive experience and what is its contribution on LHb neuronal output both in physiological and pathological conditions. This is particularly important in light of the role of LHb neurons activity in aversive behaviors (Lecca et al., 2014;[START_REF] Proulx | Reward processing by the lateral habenula in normal and depressive behaviors[END_REF]. A thorough examination of specific plasticity mechanisms

Dysfunction of the LHb: implications in depression and addiction

LHb in depression

Substantial evidence links LHb hyperactivity to the pathophysiology of mood disorders including depression and anxiety both in humans and rodent models (Lecca et al., 2014;[START_REF] Li | Synaptic potentiation onto habenula neurons in the learned helplessness model of depression[END_REF][START_REF] Li | βCaMKII in lateral habenula mediates core symptoms of depression[END_REF][START_REF] Proulx | Reward processing by the lateral habenula in normal and depressive behaviors[END_REF][START_REF] Sartorius | Deep brain stimulation of the lateral habenula in treatment resistant major depression[END_REF]. In humans, positron emission tomography shows increased neural activity in the LHb and DRN with depletion of plasma levels of tryptophan (the precursor for serotonin), which also correlates with the degree of depressed mood [START_REF] Morris | Covariation of activity in habenula and dorsal raphé nuclei following tryptophan depletion[END_REF]. In line with this, the metabolic activity of the LHb of rodents correlates with the vulnerability to develop learned helpless behavior after uncontrollable, inescapable and unpredictable stress [START_REF] Mirrione | Increased metabolic activity in the septum and habenula during stress is linked to subsequent expression of learned helplessness behavior[END_REF]. Moreover, data from congenitally helpless rats susceptible to develop learned helplessness show a hypermetabolic activity in the habenular complex and a hypometabolic activity in the VTA-striatum pathway [START_REF] Shumake | Brain systems underlying susceptibility to helplessness and depression[END_REF]. The spontaneous firing activity within the LHb of such congenitally helpless rats is also increased [START_REF] Li | Synaptic potentiation onto habenula neurons in the learned helplessness model of depression[END_REF][START_REF] Li | βCaMKII in lateral habenula mediates core symptoms of depression[END_REF]. Consistently, lesioning the LHb of depressed rats reduces depressive-like behaviors and increases the levels of serotonin in the DRN [START_REF] Yang | Lateral habenula lesions improve the behavioral response in depressed rats via increasing the serotonin level in dorsal raphe nucleus[END_REF]. Furthermore, pharmacological inactivation of the LHb with the GABA A receptor agonist muscimol has antidepressant effects in a rat model of treatment-resistant depression [START_REF] Winter | Pharmacological inhibition of the lateral habenula improves depressive-like behavior in an animal model of treatment resistant depression[END_REF]. Collectively, these data strongly suggest that an increased activity of LHb neurons can be responsible for behavioral despair both in humans and rodents.

In line with these data, the LHb activity of humans diagnosed with major depressive disorder shows abnormal responses to cues that predict punishment. As conditioned cues became more shock predictive the LHb activity of patients affected by major depressive disorders (MDD) decreased, while it increased in healthy volunteers. This also suggests that aberrant negative-reward prediction errors in the LHb can lead to impaired coping strategies with aversive situations characteristic of depressed patients [START_REF] Lawson | Disrupted habenula function in major depression[END_REF].

Deep brain stimulation (DBS), which has been extensively used to suppress neuronal hyperexcitability [START_REF] Vitek | Mechanisms of deep brain stimulation: excitation or inhibition[END_REF], applied in the LHb of both humans and rodents had beneficial effects on depressed patients mood and animal performance in the forced swim test [START_REF] Kiening | A new translational target for deep brain stimulation to treat depression[END_REF][START_REF] Li | Synaptic potentiation onto habenula neurons in the learned helplessness model of depression[END_REF][START_REF] Lim | Electrical stimulation alleviates depressive-like behaviors of rats: investigation of brain targets and potential mechanisms[END_REF][START_REF] Sartorius | Deep brain stimulation of the lateral habenula in treatment resistant major depression[END_REF]. Rats subjected to chronic mild stress, a paradigm that induces depressivelike states, had a significant improvement of their behavior following DBS in the LHb [START_REF] Meng | Chronic deep brain stimulation of the lateral habenula nucleus in a rat model of depression[END_REF]. Similarly, DBS in the stria medullaris led to substantial improvement of depressed mood in a patient with treatment-resistant depression [START_REF] Sartorius | Remission of major depression under deep brain stimulation of the lateral habenula in a therapy-refractory patient[END_REF]. A potential way DBS exerts its beneficial effects in the LHb is by reducing excitatory drive [START_REF] Li | Synaptic potentiation onto habenula neurons in the learned helplessness model of depression[END_REF], however the exact mechanisms remain to be established.

LHb in addiction

Addiction is defined as chronic and relapsing condition characterized by compulsive drug use despite the negative consequences. It is considered to be a disease of learning and memory, where addicted individuals overlearn drug-associated cues and contexts and narrow their behavior to seeking and using the drug, while ignoring their health and social status [START_REF] Hyman | Addiction: a disease of learning and memory[END_REF]. One of the major problems of addiction is that individuals present a high risk of relapse even after long drug-free periods, which is often triggered by stress and negative emotional states [START_REF] Self | Relapse to drug-seeking: neural and molecular mechanisms[END_REF][START_REF] Sinha | Chronic stress, drug use, and vulnerability to addiction[END_REF]. One theory of addiction posits that drug-seeking and drug-relapse result from opponent processes represented by both the positive and negative reinforcing properties of the abused drugs. Indeed, the positive reinforcement after psychostimulant intake is produced by an initial euphoric 'high', whereas the negative reinforcement results from a following 'down' phase characterized by increased anxiety and drug craving. The desire to alleviate these aversive effects would further motivate the addicted subject to continue using the drug (Koob and Le Moal, 2008;[START_REF] Koob | Negative reinforcement in drug addiction : the darkness within[END_REF]. Addictive psychostimulants have the common feature to target the DAT, likewise increasing dopamine levels in the NAc and other target regions of the dopamine system therefore acting as reinforcers. The reinforcing properties of abused drugs characterize the initial stages of addiction [START_REF] Di Chiara | Drugs abused by humans preferentially increase synaptic dopamine concentrations in the mesolimbic system of freely moving rats[END_REF][START_REF] Vaughan | Mechanisms of dopamine transporter regulation in normal and disease states[END_REF]. However, after persistent drug-taking, withdrawal and negative affective symptoms arise, involving a circuit reorganization and recruitment of anti-reward and stress systems [START_REF] Ettenberg | The runway model of drug self-administration[END_REF][START_REF] George | Negative reinforcement via motivational withdrawal is the driving force behind the transition to addiction[END_REF][START_REF] Knackstedt | Evidence for opponent-process actions of intravenous cocaine and cocaethylene[END_REF][START_REF] Koob | Negative reinforcement in drug addiction : the darkness within[END_REF].

The LHb has been proposed to participate in the pathophysiology of drug addiction because of its role in encoding rewarding and aversive stimuli as well as of its functional connection to the dopamine system. Indeed, the LHb is innervated by dopaminergic fibers, expresses dopamine receptors and the dopamine transporter, making it a potential target for drug-driven synaptic and behavioral adaptations [START_REF] Freed | Dopamine transporter immunoreactivity in rat brain[END_REF][START_REF] Good | Dopamine D4 receptor excitation of lateral habenula neurons via multiple cellular mechanisms[END_REF][START_REF] Jhou | Cocaine drives aversive conditioning via delayed activation of dopamine-responsive habenular and midbrain pathways[END_REF]Kowski et al., 2009;Root et al., 2014b;Zuo et al., 2013). The LHb has been implicated in different aspects of drug-related behaviors. LHb metabolic activity negatively correlates with locomotor activity after cocaine administration (Fig10) [START_REF] Porrino | Selective alterations in cerebral metabolism within the mesocorticolimbic dopaminergic system produced by acute cocaine administration in rats[END_REF], while its pharmacological inactivation or lesion increases locomotor responses to psychostimulants [START_REF] Gifuni | Lesions of the lateral habenula dissociate the reward-enhancing and locomotor-stimulant effects of amphetamine[END_REF][START_REF] Gill | Inactivation of the lateral habenula reduces anxiogenic behavior and cocaine seeking under conditions of heightened stress[END_REF], suggesting that LHb activity is decreased during the rewarding effects of drugs (i.e. locomotor sensitization). This is consistent with the idea that LHb activity needs to be decreased to relieve the inhibitory tone onto dopamine neurons allowing them to encode reward-related signals. In contrast, an increased expression of c-fos (c-fos is an immediate early gene, a marker for neuronal activity) in LHb neurons projecting to the VTA occurs in the withdrawal phase after extinction of cocaine-seeking behavior or in response to conditioned stimuli predicting absence of cocaine reward [START_REF] Mahler | Fos activation of selective afferents to ventral tegmental area during cue-induced reinstatement of cocaine seeking in rats[END_REF]. In line with this, rats that had unilateral lesions of the LHb failed to extinguish cocaine-seeking behavior, consistent with reduced inhibitory drive onto dopamine neurons. Interestingly, low-frequency stimulation of the LHb (10Hz) led to increased cocaine self-administration, while a mixed pattern of stimulation alternating low and high-frequencies (10 and 100Hz respectively) reduced this behavior [START_REF] Friedman | Electrical stimulation of the lateral habenula produces enduring inhibitory effect on cocaine seeking behavior[END_REF].

The LHb has also been associated to the susceptibility of drug relapse. Studies in mice have shown that some animals present higher vulnerability to cocaine relapse and that this correlates with increased c-fos activity in the LHb [START_REF] Brown | Identification of brain nuclei implicated in cocaine-primed reinstatement of conditioned place preference: a behaviour dissociable from sensitization[END_REF][START_REF] James | Propensity to "relapse" following exposure to cocaine cues is associated with the recruitment of specific thalamic and epithalamic nuclei[END_REF]. While the LHb seems to be a target for psychostimulants, relapse from opiates has also been linked to increased LHb activity, suggesting that the LHb is a common target for different classes of drugs [START_REF] Madsen | Investigation of the neuroanatomical substrates of reward seeking following protracted abstinence in mice[END_REF][START_REF] Zhang | Increased c-Fos expression in the medial part of the lateral habenula during cue-evoked heroin-seeking in rats[END_REF]. In contrast, LHb pharmacological inactivation does not affect cue-induced reinstatement of cocaine seeking, but reduces stress-induced reinstatement, in line with a role of the LHb in encoding stressful events [START_REF] Gill | Inactivation of the lateral habenula reduces anxiogenic behavior and cocaine seeking under conditions of heightened stress[END_REF]. Recently, a study in our laboratory has demonstrated that the input from the EPN to the LHb contributes to withdrawal symptoms and relapse behaviors after chronic cocaine treatment (Meye et al., 2016).

Accumulating evidence suggests that the LHb plays a role in the aversive component of drug-taking associated to the withdrawal phase after acute drug experience. Indeed, a single cocaine injection in rats produces real-time place preference immediately after the injection and this behavior occurs along with reduction of LHb firing rate. Around 15 minutes later, when drug rewarding effects start to wear off, the animals exhibit a mild place avoidance which is paralleled with a rebound excitation of LHb neurons (Fig10). 30 minutes post-injection, when the effects of the drug should be almost completely dissipated no preference or avoidance occurs and LHb neurons activity returns to baseline levels [START_REF] Ettenberg | Evidence for opponentprocess actions of intravenous cocaine[END_REF][START_REF] Jhou | Cocaine drives aversive conditioning via delayed activation of dopamine-responsive habenular and midbrain pathways[END_REF]. This supports a scenario where negative symptoms emerge during the down phase of acute cocaine exposure when the rewarding effects of the drug start to wear off. Importantly, acute cocaine administration also increases c-fos levels in the LHb and RMTg (Fig10) [START_REF] Jhou | Cocaine drives aversive conditioning via delayed activation of dopamine-responsive habenular and midbrain pathways[END_REF](Jhou et al., , 2009b;;[START_REF] Zahm | Fos after single and repeated self-administration of cocaine and saline in the rat: emphasis on the Basal forebrain and recalibration of expression[END_REF]. Indeed, a considerable proportion of c-fos positive LHb neurons are RMTg-projecting. In contrast, the majority of VTA-projecting LHb neurons do not express c-fos. In line with this, LHb and fasciculus retroflexus lesions reduce in half the number of c-fos expressing neurons in the RMTg after cocaine, suggesting that the cocaine-activated LHb neurons also activate downstream RMTg neurons (Jhou et al., 2013(Jhou et al., , 2009b)). In the light of these data it has been proposed that LHb-to-RMTg neurons might encode an early rewarding phase (reflected by an inhibition of LHb neurons activity) and a later aversive phase (reflected by the rebound excitation after the acute cocaine effects have faded away). The runway operant paradigm has been used to assess such conflicting rewarding and aversive conditioning. In this paradigm animals have to traverse a runway to self-administer cocaine. Progressively they show longer latencies to reach the goal compartment as well as increased frequency of approach and retreat, reflecting opponent processes and hesitating-like behavior after cocaine administration [START_REF] Ettenberg | Qualitative and quantitative differences in the operant runway behavior of rats working for cocaine and heroin reinforcement[END_REF]Geist & Ettenberg 1997). Rats showed substantial decrease in this progressive hesitation-like behavior when lesioning the RMTg or the fasciculus retroflexus. Similarly, optogenetically silencing RMTg neurons 15-25 minutes after cocaine injection also reduced hesitation behavior in the runway task, further supporting that a delayed activity of the LHb-to-RMTg pathway may be involved in the aversive effects of cocaine [START_REF] Jhou | Cocaine drives aversive conditioning via delayed activation of dopamine-responsive habenular and midbrain pathways[END_REF]. This is also in line with data showing aversive conditioning after LHb-to-RMTg pathway stimulation (Stamatakis and Stuber, 2012). Altogether these data support a role of the LHb-to-RMTg pathway activity in aversive behaviors related to the acute and potentially long-lasting effects of drug withdrawal.

Taken together all these data support that aspects of addiction and depression share similar neurobiological substrates leading to increased activity in the LHb. The exact molecular, synaptic and circuit mechanisms leading to such modifications are underway investigation and new insights are emerging regarding potential targets within the LHb to reverse pathological neuroadaptations. During my thesis I addressed some of the molecular mechanisms leading to increased LHb output to the midbrain after cocaine withdrawal.

LHb b LHb

early phase 0-15min late phase 15-30min -LHb metabolic activity [START_REF] Porrino | Selective alterations in cerebral metabolism within the mesocorticolimbic dopaminergic system produced by acute cocaine administration in rats[END_REF] -LHb firing activity [START_REF] Jhou | Cocaine drives aversive conditioning via delayed activation of dopamine-responsive habenular and midbrain pathways[END_REF] -place preference [START_REF] Jhou | Cocaine drives aversive conditioning via delayed activation of dopamine-responsive habenular and midbrain pathways[END_REF] -c-fos in LHb-RMTg [START_REF] Jhou | Cocaine drives aversive conditioning via delayed activation of dopamine-responsive habenular and midbrain pathways[END_REF] -LHb firing activity [START_REF] Jhou | Cocaine drives aversive conditioning via delayed activation of dopamine-responsive habenular and midbrain pathways[END_REF] -place avoidance [START_REF] Jhou | Cocaine drives aversive conditioning via delayed activation of dopamine-responsive habenular and midbrain pathways[END_REF] acute psychostimulant exposure

Properties of lateral habenula neurons

Cell morphology and electrophysiology

The LHb is characterized by loosely dispersed cells with spherical, fusiform or polymorphic shape and rather small size. They have few very long dendrites stemming from the soma that can extend distally in the dorso-ventral and horizontal axis covering a large surface of the LHb. The dendritic arborization is not very branched as primary dendrites can give rise to few secondary ones [START_REF] Weiss | Morphological and electrophysiological characteristics of neurons within identified subnuclei of the lateral habenula in rat brain slices[END_REF]. The majority of LHb cells have spines along their dendrites (Fig12B) [START_REF] Kim | Dendritic morphology, local circuitry, and intrinsic electrophysiology of neurons in the rat medial and lateral habenular nuclei of the epithalamus[END_REF][START_REF] Li | Synaptic potentiation onto habenula neurons in the learned helplessness model of depression[END_REF]Maroteaux and Mameli, 2012) although aspiny neurons have also been described [START_REF] Weiss | Morphological and electrophysiological characteristics of neurons within identified subnuclei of the lateral habenula in rat brain slices[END_REF]. LHb neurons are almost exclusively glutamatergic and longrange projecting. The axons branch directly from the soma or from primary dendrites and run ventro-laterally to reach the root of the fasiculus retroflexus. Axonal reconstructions show the presence of varicosities along the axons, suggesting that they may form en passant synaptic connections and despite the lack of local collaterals they may interact in within the LHb [START_REF] Kim | Dendritic morphology, local circuitry, and intrinsic electrophysiology of neurons in the rat medial and lateral habenular nuclei of the epithalamus[END_REF]. A recent study has proposed that single neurons with dense dendritic arbors can have their axonal terminals within the LHb, presumably forming a local microcircuit. These cells were mainly located within the medial division of the LHb and expressed GABAergic markers [START_REF] Li | Synaptic potentiation onto habenula neurons in the learned helplessness model of depression[END_REF][START_REF] Zhang | Thirst Is Associated with Suppression of Habenula Output and Active Stress Coping: Is there a Role for a Non-canonical Vasopressin-Glutamate Pathway? Front[END_REF][START_REF] Zhu | A thalamic input to the nucleus accumbens mediates opiate dependence[END_REF]. However, whether they can release GABA and inhibit locally other LHb neurons remains still unknown. Despite the different morphologies of LHb neurons, no clear correlation between their electrophysiological properties and a given morphological type has been observed.

LHb neurons present three major activity patterns: silent, tonically active and bursting.

Most of LHb neurons have spontaneous tonic activity and a regular spiking pattern, although irregularly firing neurons also exist. The resting membrane potential of these neurons is around -55mV and they have a rather high input resistance ~ 450Min mice (Meye et al., 2015) and ~ 1.16 Gin rats [START_REF] Weiss | Morphological and electrophysiological characteristics of neurons within identified subnuclei of the lateral habenula in rat brain slices[END_REF], which contributes to the spontaneous activity of these neurons. The mean spontaneous firing frequency is ~ 4-5Hz in vitro [START_REF] Weiss | Morphological and electrophysiological characteristics of neurons within identified subnuclei of the lateral habenula in rat brain slices[END_REF]) and ~10-12Hz in vivo [START_REF] Jhou | Cocaine drives aversive conditioning via delayed activation of dopamine-responsive habenular and midbrain pathways[END_REF]Kowski et al., 2009). Virtually all LHb neurons have the capacity to generate a rebound burst discharge of action potentials in response to membrane hyperpolarization, which requires low-threshold Ca 2+ channels activation [START_REF] Chang | Ionic mechanism of long-lasting discharges of action potentials triggered by membrane hyperpolarization in the medial lateral habenula[END_REF][START_REF] Huguenard | Transient Ca2+ currents in neurons isolated from rat lateral habenula[END_REF][START_REF] Wilcox | Electrophysiological properties of neurons in the lateral habenula nucleus: an in vitro study[END_REF]. LHb neurons also express hyperpolarization activated cyclic nucleotide-gated cation (HCN) channels, responsible for the generation of pacemaker activity and potentially underlying the tonic inhibitory effect that LHb neurons exert on dopamine neurons [START_REF] Lecca | Rescue of GABA B and GIRK function in the lateral habenula by protein phosphatase 2A inhibition ameliorates depression-like phenotypes in mice[END_REF][START_REF] Poller | Lateral habenular neurons projecting to reward-processing monoaminergic nuclei express hyperpolarization-activated cyclic nucleotid-gated cation channels[END_REF]. Importantly, LHb neurons discharge properties strongly depend on the voltage. Cells that are more depolarized at rest show tonic regular firing and can shift their spiking pattern to burst firing if membrane hyperpolarization occurs. Inversely, more hyperpolarized cells present bursting activity that can turn into regular firing if the membrane is depolarized [START_REF] Chang | Ionic mechanism of long-lasting discharges of action potentials triggered by membrane hyperpolarization in the medial lateral habenula[END_REF][START_REF] Weiss | Morphological and electrophysiological characteristics of neurons within identified subnuclei of the lateral habenula in rat brain slices[END_REF]. In line with these observations, brief high frequency stimulation of the stria medullaris, the fiber bundle conveying the majority of inputs to the LHb, induces brief hyperpolarization followed by rebound excitation and this phenomenon was mainly observed in the medial portion of the LHb [START_REF] Chang | Ionic mechanism of long-lasting discharges of action potentials triggered by membrane hyperpolarization in the medial lateral habenula[END_REF]. The membrane characteristics of LHb neurons including membrane resting potential, action potential duration and after-hyperpolarization potential (AHP) amplitude are similar [START_REF] Weiss | Morphological and electrophysiological characteristics of neurons within identified subnuclei of the lateral habenula in rat brain slices[END_REF].

Taken together, these data suggests that LHb neurons cannot be distinguished on the bases of their morphology or electrophysiological properties alone. Their functional differences may rather arise from the integration of distinct inputs on their long dendritic arborization or alternatively from distinct expression of synaptic and cellular proteins important for their activity. Indeed, LHb neurons receive their inputs in a segregated manner, but also express differentially neuronal markers involved in synaptic transmission and neuronal activity [START_REF] Geisler | Morphologic and cytochemical criteria for the identification and delineation of individual subnuclei within the lateral habenular complex of the rat[END_REF][START_REF] Zhang | Thirst Is Associated with Suppression of Habenula Output and Active Stress Coping: Is there a Role for a Non-canonical Vasopressin-Glutamate Pathway? Front[END_REF]. A genetic profiling as well as the generation of transgenic Cre-driver mouse lines can be of help to identify and target specific cell types within the LHb. It remains also to be established how synaptic inputs are organized along LHb dendrites as well as what type of dendritic integration these neurons perform to orchestrate their neuronal output.

Fast excitatory transmission via ionotropic glutamate receptors

Apart from voltage-gated ion channels, synaptic inputs onto the LHb also largely contribute to control neuronal activity. As described previously, the LHb receives several glutamatergic afferent inputs from forebrain and basal ganglia regions, whose activation can generate action potential firing (Root et al., 2014b;Shabel et al., 2012;[START_REF] Stamatakis | Lateral Hypothalamic Area Glutamatergic Neurons and Their Projections to the Lateral Habenula Regulate Feeding and Reward[END_REF]. Glutamate release in the LHb leads to activation of both AMPA-type and NMDA-type of receptors.

AMPA receptors are cation channels responsible for the majority of the fast excitatory transmission in the brain. They are composed of a combination of the four subunits GluA1 to GluA4 forming homo-or hetrotetramere [START_REF] Henley | Synaptic AMPA receptor composition in development, plasticity and disease[END_REF][START_REF] Malinow | AMPA receptor trafficking and synaptic plasticity[END_REF][START_REF] Nicoll | Auxiliary subunits assist AMPA-type glutamate receptors[END_REF]. The C-terminal domain of each subunit harbors different interaction sites with kinases, phosphatases or other auxiliary proteins necessary for receptor trafficking and function during synaptic plasticity [START_REF] Swope | Regulation of ligand-gated ion channels by protein phosphorylation[END_REF][START_REF] Wang | Phosphorylation of AMPA receptors: mechanisms and synaptic plasticity[END_REF]. Likewise, the C-terminal domain of the GluA1 subunit contains PKA and CaMKII phosphorylation sites controlling surface insertion of the receptor (Fig11A) [START_REF] Ehlers | Reinsertion or degradation of AMPA receptors determined by activitydependent endocytic sorting[END_REF][START_REF] Esteban | PKA phosphorylation of AMPA receptor subunits controls synaptic trafficking underlying plasticity[END_REF][START_REF] Lee | Regulation of distinct AMPA receptor phosphorylation sites during bidirectional synaptic plasticity[END_REF] as well as its channel properties [START_REF] Banke | Control of GluR1 AMPA receptor function by cAMP-dependent protein kinase[END_REF][START_REF] Barria | Identification of the Ca2+/calmodulin-dependent protein kinase II regulatory phosphorylation site in the alpha-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate-type glutamate receptor[END_REF][START_REF] Derkach | Ca2+/calmodulin-kinase II enhances channel conductance of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate type glutamate receptors[END_REF]. GluA1containing and GluA2-lacking AMPA receptors have particular biophysical properties such as Ca 2+ permeability, high single channel conductance and inward rectification due to polyamine obstruction of the channel at positive potentials (Fig11B) [START_REF] Burnashev | Divalent ion permeability of AMPA receptor channels is dominated by the edited form of a single subunit[END_REF][START_REF] Donevan | Intracellular polyamines mediate inward rectification of Ca(2+)-permeable alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors[END_REF][START_REF] Liu | Ca2+-permeable AMPA receptors in synaptic plasticity and neuronal death[END_REF]. In contrast, GluA2containing receptors are impermeable to Ca 2+ , have smaller single channel conductance and are insensitive to polyamine block, therefore yielding linear current-voltage relationship (Fig11B). These different properties of GluA2-lacking and GluA2-containg AMPA receptors result from postnatal mRNA editing which leads to a replacement of a single amino acid, from glutamine to arginine, in the pore-lining domain of the GluA2 subunit [START_REF] Barbon | Glutamate receptor RNA editing in health and disease[END_REF][START_REF] Lomeli | Control of kinetic properties of AMPA receptor channels by nuclear RNA editing[END_REF][START_REF] Swanson | Single-channel properties of recombinant AMPA receptors depend on RNA editing, splice variation, and subunit composition[END_REF]. The arginine residue present in the GluA2-containing receptors does not allow the flow of big ions such as Ca 2+ and contributes to its lower single channel conductivity [START_REF] Burnashev | Divalent ion permeability of AMPA receptor channels is dominated by the edited form of a single subunit[END_REF]Mameli et al., 2007).

AMPA receptors undergo experience-dependent modifications including trafficking and interactions with other synaptic proteins [START_REF] Bellone | Cocaine triggered AMPA receptor redistribution is reversed in vivo by mGluR-dependent long-term depression[END_REF][START_REF] Clem | Pathway-specific trafficking of native AMPARs by in vivo experience[END_REF]Rumpel et al., 2005) leading to augmentation or reduction of the efficacy of excitatory transmission. These phenomena known as long-term depression (LTD) and long-term potentiation (LTP) comprise multiple expression mechanisms depending on the structure and developmental stage: from changes in surface expression levels of functional receptors, to changes of their biophysical properties or their interactions with scaffolding proteins. Such synaptic remodeling is thought to be the corollary of learning and memory [START_REF] Huganir | Perspective AMPARs and Synaptic Plasticity : The Last 25 Years[END_REF][START_REF] Malenka | LTP and LTD: an embarrassment of riches[END_REF][START_REF] Malinow | AMPA receptor trafficking and synaptic plasticity[END_REF]. The LHb expresses GluA2-lacking AMPA receptors as shown by inwardly rectifying current-voltage relationships (Fig12A,C) [START_REF] Li | Synaptic potentiation onto habenula neurons in the learned helplessness model of depression[END_REF]Maroteaux and Mameli, 2012).

These receptors are of particular interest in the context of synaptic plasticity. Notably, GluA2-lacking AMPA receptors are known to underlie anti-Hebbian type of plasticity, which requires paired afferent stimulation together with postsynaptic hyperpolarization for its induction as opposed to the Hebbian plasticity which instead relies on paired preand post-synaptic activity [START_REF] Lamsa | Anti-Hebbian long-term potentiation in the hippocampal feedback inhibitory circuit[END_REF][START_REF] Le Roux | Input-specific learning rules at excitatory synapses onto hippocampal parvalbumin-expressing interneurons[END_REF]. These receptors are also involved in experience-dependent plasticity including reward-and drug-driven experience. Indeed, GluA2-lacking AMPA receptors are recruited at glutamatergic synapses onto VTA dopamine neurons after a single cocaine injection and are responsible for the increased strength of these synapses [START_REF] Bellone | Cocaine triggered AMPA receptor redistribution is reversed in vivo by mGluR-dependent long-term depression[END_REF].

Moreover, they are permissive for further potentiation of the synapse since they allow the induction of an anti-Hebbian LTP in cocaine treated mice [START_REF] Mameli | Cocaine inverts rules for synaptic plasticity of glutamate transmission in the ventral tegmental area[END_REF].

Similar experience-driven changes have been also reported in the NAc, where GluA2lacking AMPA receptors accumulate following prolonged withdrawal from cocaine selfadministration (McCutcheon et al., 2011a) or in the amygdala during acquisition of fear memories [START_REF] Clem | Calcium-permeable AMPA receptor dynamics mediate fear memory erasure[END_REF]. This information is also relevant for LHb function.

Indeed in the LHb anti-Hebbian type of protocol (somatic hyperpolarization + 10Hz afferent stimulation) induces LTD of excitatory synaptic currents both in VTA-projecting and RMTg-projecting LHb neurons (Maroteaux and Mameli, 2012). Importantly, brief stimulation of the stria medullaris hyperpolarizes LHb neurons likewise providing gating conditions for anti-Hebbian plasticity [START_REF] Chang | Ionic mechanism of long-lasting discharges of action potentials triggered by membrane hyperpolarization in the medial lateral habenula[END_REF].

The NMDA receptors are also ion channels and are typically di-heterotetrameres ot triheterotetramears. There are generally three subfamilies of NMDA receptors comprising in total seven distinct subunits: the GluN1, four GluN2 (GluN2A, GluN2B, GluN2C and GluN2D) and two GluN3 (GluN3A and GluN3B). The relative composition of the receptor is regulated throughout development and brain regions and confers distinct biophysical properties and synaptic plasticity mechanisms to each receptor subtype [START_REF] Paoletti | NMDA receptor subunit diversity: impact on receptor properties, synaptic plasticity and disease[END_REF]. NMDA receptors are one of the best studied triggers of long-term synaptic plasticity, the underlying expression mechanisms of which vary across synapses, developmental stages and structures [START_REF] Malenka | LTP and LTD: an embarrassment of riches[END_REF]. NMDA receptors are expressed in the LHb (Fig12A). However, their relative contribution to excitatory transmission is relatively low (Fig12C) [START_REF] Li | Synaptic potentiation onto habenula neurons in the learned helplessness model of depression[END_REF]Maroteaux and Mameli, 2012).

The subunit composition and function of NMDA receptors in the LHb is poorly investigated and their potential role in synaptic plasticity remains to be addressed.

Fast inhibitory synaptic transmission via ionotropic GABA A receptors

The LHb is as well densely innervated by GABAergic axons [START_REF] Araki | Retrograde HRP tracing combined with a pharmacohistochemical method for GABA transaminase for the identification of presumptive GABAergic projections to the habenula[END_REF][START_REF] Smith | Distribution of GABA-immunoreactive neurons in the thalamus of the squirrel monkey (Saimiri sciureus)[END_REF]. These inputs originate from the EPN, the LH and the VTA as optogenetic stimulation of these afferents evokes GABA A receptor-mediated currents (Root et al., 2014b;Shabel et al., 2014;[START_REF] Stamatakis | Lateral Hypothalamic Area Glutamatergic Neurons and Their Projections to the Lateral Habenula Regulate Feeding and Reward[END_REF]. Other potential inhibitory projections may arise from LPO,VP, DBB and NAc, although their functional relevance remains to be tested [START_REF] Hong | Diverse sources of reward value signals in the basal ganglia nuclei transmitted to the lateral habenula in the monkey[END_REF]Meye et al., 2013). Electrophysiological evidence suggests that fast inhibitory transmission in the LHb is mediated by GABA A receptors.

GABA A receptors are homo or hetero-pentameric ligand-gated ion channels permeable to chloride (Cl -).They are composed of a combination of the 19 existing subunits (1-6;

1-3; 1-3; 1-3;), which determine their channel and pharmacological properties, scaffolding, subcellular localization and trafficking [START_REF] Birnir | The impact of sub-cellular location and intracellular neuronal proteins on properties of GABA(A) receptors[END_REF][START_REF] Luscher | GABAA receptor trafficking-mediated plasticity of inhibitory synapses[END_REF][START_REF] Olsen | GABA A receptors: subtypes provide diversity of function and pharmacology[END_REF]. GABA A receptors are phasically activated upon GABA release. However, when GABA spills out of the synaptic cleft it activates extrasynaptic receptors (GABA A and GABA B ) which may underlie tonic inhibition. Most of synaptically expressed GABA A receptors contain a combination of 1-3,  and  subunits, while extrasynaptic ones often contain 4-6 and a  subunit instead of  [START_REF] Farrant | Variations on an inhibitory theme: phasic and tonic activation of GABA(A) receptors[END_REF]. GABA A receptors are highly regulated by phosphorylation-dephosphorylation processes, which play a major role in receptors trafficking and function [START_REF] Comenencia-Ortiz | Phosphorylation of GABAA receptors influences receptor trafficking and neurosteroid actions[END_REF]Kittler and Moss, 2003). Indeed, the 1-3 and 2 subunits can be phosphorylated by cAMP-dependent protein kinase (PKA) and protein kinase C (PKC). Phosphorylation events can modify receptors conductance, but also can facilitate the interaction of specific subunits with membrane proteins controlling their insertion or removal from the synapse (Herring et al., 2005;[START_REF] Luscher | GABAA receptor trafficking-mediated plasticity of inhibitory synapses[END_REF]. This functional modulation of GABA A receptors by kinases, typically triggered by metabotropic receptors signaling, may represent important mechanisms of synaptic plasticity controlling the efficacy of inhibitory transmission.

In situ hybridization data suggests that LHb neurons express the 1-3, 1 and 1-3 subunits in the mouse, with the strongest expression of 1 [START_REF] Hörtnagl | Patterns of mRNA and protein expression for 12 GABAA receptor subunits in the mouse brain[END_REF].

Another study in the rat reports the highest expression of 1 and 2 and 2 subunits in the LHb, suggesting a synaptic rather than extrasynaptic localization (Fig12A) [START_REF] Pirker | GABA(A) receptors: immunocytochemical distribution of 13 subunits in the adult rat brain[END_REF]. Whether other subunits are also expressed in the LHb remains to be established.

LHb neurons activity is shaped by a balance of excitation and inhibition especially represented by the capacity of several inputs to co-release glutamate and GABA.

Inhibition has a particularly important role in maintaining this fine balance to ensure the correct functioning of LHb neurons. Indeed, a decrease in inhibitory control over excitation at EPN synapses drives negative states in depression and drug withdrawal (Meye et al., 2016;Shabel et al., 2014). Therefore an important question which still needs to be addressed is how inhibitory transmission is modulated and what plasticity mechanisms govern GABAergic function in the LHb.

Modulation of fast excitatory and inhibitory transmission: role of mGluRs

Metabotropic glutamate receptors (mGluRs) are widely expressed throughout the CNS.

They have been implicated in controlling excitatory and inhibitory transmission across synapses as well as neuronal excitability (Anwyl, 1999;[START_REF] Lüscher | Group 1 mGluR-dependent synaptic long-term depression: mechanisms and implications for circuitry and disease[END_REF]. The group I mGluRs are typically expressed postsynaptically and at the extra-or peri-synaptic membrane [START_REF] Lujan | Perisynaptic location of metabotropic glutamate receptors mGluR1 and mGluR5 on dendrites and dendritic spines in the rat hippocampus[END_REF]. These receptors have seven transmembrane domains and are coupled to the Gq proteins triggering the PLC signaling cascade. This signaling leads to PKC activation and Ca 2+ release from intracellular membrane compartments such as the endoplasmic reticulum (Fig13A) [START_REF] Kim | Metabotropic glutamate receptors: phosphorylation and receptor signaling[END_REF][START_REF] Lüscher | Group 1 mGluR-dependent synaptic long-term depression: mechanisms and implications for circuitry and disease[END_REF]. The group I mGluR comprises the mGluR1 and mGluR5 subtypes which share the same signaling and can be expressed together or separately at synapses. mGluRs control excitatory and inhibitory synaptic transmission and most commonly they act to reduce their efficacy via different pre-and postsynaptic mechanisms [START_REF] Bellone | Mechanisms of synaptic depression triggered by metabotropic glutamate receptors[END_REF][START_REF] Lüscher | Group 1 mGluR-dependent synaptic long-term depression: mechanisms and implications for circuitry and disease[END_REF].

A very well studied form of mGluR-dependent long term depression (mGluR-LTD) occurs through a reduction of presynaptic transmitter release. Indeed, mGluRs trigger postsynaptically the generation of retrograde messengers such as endocannabinoids which subsequently diffuse through the lipid membrane and bind to their receptor, the cannabinoid type 1 receptor (CB1). CB1 receptors are typically expressed at presynaptic boutons and their activation inhibits Ca 2+ channels thereby reducing the probability of both glutamate and GABA release throughout the brain (Fig13A) [START_REF] Chevaleyre | Endocannabinoid-mediated metaplasticity in the hippocampus[END_REF][START_REF] Kreitzer | Dopamine modulation of state-dependent endocannabinoid release and long-term depression in the striatum[END_REF][START_REF] Maejima | Presynaptic inhibition caused by retrograde signal from metabotropic glutamate to cannabinoid receptors[END_REF].

Postsynaptically, mGluRs strongly modulate synapses that express GluA2-lacking AMPA receptors. In many brain structures their activation induces rapid internalization of GluA2-lacking and insertion of GluA2-containing AMPA receptors ultimately leading to reduced efficacy of excitatory transmission (Fig13B) [START_REF] Bellone | Cocaine triggered AMPA receptor redistribution is reversed in vivo by mGluR-dependent long-term depression[END_REF][START_REF] Clem | Calcium-permeable AMPA receptor dynamics mediate fear memory erasure[END_REF]Kelly et al., 2009;McCutcheon et al., 2011b). In the VTA this plasticity requires the mTOR signaling for rapid de novo synthesis of GluA2 subunits and their insertion to the membrane via the protein interacting with C kinase 1 (PICK1). This exchange of subunits leads to reduced single channel conductance of AMPA receptors [START_REF] Bellone | Cocaine triggered AMPA receptor redistribution is reversed in vivo by mGluR-dependent long-term depression[END_REF]Mameli et al., 2007). In the NAc, similar mechanisms require PKC signaling, likely through its interaction with PICK1 (McCutcheon et al., 2011b). In the cerebellum, the switch of subunits requires both mGluR and GluA2lacking AMPA receptors activation leading to Ca 2+ elevation and protein synthesis (Kelly et al., 2009).

Collectively, these data illustrate the variety of mechanisms that can be employed at

Synaptic plasticity in the LHb: a cellular substrate for motivated states in disease

Increasing evidence points to a crucial role of aberrant synaptic and cellular plasticity within the LHb in the pathophysiology of disorders of motivation. Changes in the balance of excitation and inhibition can drive LHb hyperactivity. Excessive activity of LHb neurons has been linked to the emergence of negative emotional states in depression and following withdrawal from drugs of abuse [START_REF] Li | Synaptic potentiation onto habenula neurons in the learned helplessness model of depression[END_REF][START_REF] Li | βCaMKII in lateral habenula mediates core symptoms of depression[END_REF]Meye et al., 2016;[START_REF] Neumann | Increased excitability of lateral habenula neurons in adolescent rats following cocaine self-administration[END_REF]. This suggests that converging mechanisms can underlie the pathophysiology of these two highly comorbid disease states [START_REF] Russo | The brain reward circuitry in mood disorders[END_REF].

Cellular mechanisms in the LHb in depression

Recent studies have begun to elucidate the cellular underpinnings of LHb dysfunction during depression. In rodent models of learned helplessness the excitatory drive onto LHb-to-VTA neurons is substantially increased and correlates with the extent of depressive-like behaviors. These adaptations occur along with increased spontaneous activity of LHb-to-VTA neurons [START_REF] Li | Synaptic potentiation onto habenula neurons in the learned helplessness model of depression[END_REF]. In the same depressive model, the  form of the calcium/calmoduline-dependent kinase type II (CaMKII) has higher expression levels in the LHb and occurs together with increased GluA1 membrane expression leading to increased spontaneous firing of LHb neurons [START_REF] Li | βCaMKII in lateral habenula mediates core symptoms of depression[END_REF]. In parallel, the balance of excitation/inhibition at the EPN-to-LHb synapse is shifted towards more excitation in congenitally learned helpless rats (Shabel et al., 2014).

These studies suggest that different synaptic modifications potentially at different synapses or in different LHb output pathways converge to increase the overall activity of LHb neurons. Future studies are needed to address the exact induction mechanisms of these synaptic and cellular adaptations as well as their circuit relevance.

Recently, GABA B receptors plasticity in the LHb has also been implicated in depressivelike behaviors after acute aversive experience as well as in depression mouse models.

Animals submitted to unescapable and randomly delivered foot-shocks led to a reduction of GABA B receptors and their inwardly rectifying potassium channels (GIRKs) effectors function, whose activation normally leads to hyperpolarization and acts as a break for excessive excitability (Lecca et al., 2016;[START_REF] Lüscher | Emerging roles for G protein-gated inwardly rectifying potassium (GIRK) channels in health and disease[END_REF]. These changes led to increased spontaneous firing and excitability of LHb neurons as soon as one hour after the procedure and persisted up to two weeks. Electron microscopy data showed that both the GABA B receptor and the GIRK are internalized following acute foot shock exposure (Lecca et al., 2016). Importantly, the protein phosphatase 2A (PP2A), which has a role in regulating GABA B -GIRK complex surface expression [START_REF] Hearing | Repeated cocaine weakens GABA(B)-Girk signaling in layer 5/6 pyramidal neurons in the prelimbic cortex[END_REF][START_REF] Padgett | Methamphetamine-evoked depression of GABA(B) receptor signaling in GABA neurons of the VTA[END_REF][START_REF] Terunuma | Functional modulation of GABAB receptors by protein kinases and receptor trafficking[END_REF], had an increased activity in these animals and was required for the increased LHb excitability and depressive phenotype.

These results raise the interesting hypothesis that GABA B -GIRK complexes in the LHb serve as a general substrate to control excitability of these neurons and that their function is affected in pathologies characterized with depressive symptoms. It is interesting to understand whether this is a general phenomenon occurring after traumatic life events permissive for further pathological adaptations to take place or whether there are other vulnerability traits necessary for abnormal plasticity in the LHb in depression. or D2 receptor antagonists, although a combination of both does not further decrease firing or mini EPSCs frequency (Zuo et al., 2013). As already mentioned, some LHb neurons show bidirectional spontaneous firing responses to cocaine (initial inhibition followed by excitation) both in vivo and in vitro [START_REF] Jhou | Cocaine drives aversive conditioning via delayed activation of dopamine-responsive habenular and midbrain pathways[END_REF]. Another subset of neurons, which are phasically excited by tail pinch were also excited by cocaine and glucocorticoids, while those that were inhibited by tail pinch were also inhibited by cocaine and did not respond to glucocorticoids, suggesting that cocaine may share common pathways with stress-related factors in the LHb [START_REF] Zhang | The lateral habenula is a common target of cocaine and dexamethasone[END_REF].

Cellular mechanisms in the LHb in addiction

Altogether, these data suggest that LHb neurons respond in a non-homogeneous way to acute cocaine, raising the possibility that different subsets of LHb neurons, potentially dependent on their output targets, display different physiological responses to cocaine.

Chronic psychostimulant exposure produces long-lasting circuit and synaptic adaptations [START_REF] Kasanetz | Transition to addiction is associated with a persistent impairment in synaptic plasticity[END_REF][START_REF] Mameli | Cocaine-evoked synaptic plasticity: persistence in the VTA triggers adaptations in the NAc[END_REF]. Indeed, cocaine administration for two consecutive days leads to increased number of activated postsynaptic AMPA receptors specifically onto RMTg-projecting LHb neurons and 24 hours after the last injection. Moreover, the same protocol changes the rules for synaptic plasticity in these same RMTg-projecting neurons. Indeed, in saline treated mice pairing afferent stimulation (10Hz) with postsynaptic hyperpolarization induced long-term depression, while this protocol switched the direction of the plasticity to an LTP in cocaine treated ones (Maroteaux and Mameli, 2012). This suggests that cocaine induces long term adaptations of excitatory synaptic transmission in a target-specific fashion.

Synaptic long-lasting modifications of inhibitory synaptic transmission in the LHb after chronic psychostimulant intake have been recently reported. Five consecutive cocaine injections followed by two or fourteen days of withdrawal produced a reduction of GABA A /AMPA and GABA A /NMDA ratios at EPN-to-LHb synapses without significantly changing its glutamatergic component. This occurred along with decreased Vgat labeling specifically at EPN terminals, suggesting a decreased GABA filling into synaptic vesicles and the subsequent reduction of the amount of GABA released at this synapse.

These modifications led to reduced GABAergic modulation of evoked EPN-to-LHb neurons firing, shifting the balance of excitation/inhibition towards more excitation. This cocaine-evoked and synapse-specific plasticity was instrumental for withdrawal depressive-like symptoms and stress-induced relapse since rescuing the synaptic modifications by overexpressing Vgat specifically at EPN-to-LHb terminals ameliorated the behavioral phenotype (Meye et al., 2016).

Altogether these studies suggest that LHb synapses undergo long-lasting modifications after cocaine withdrawal which generally increases excitatory synaptic transmission and decreases inhibitory transmission, overall promoting an increase in LHb neuronal activity. This is supported also by evidence indicating that LHb neurons are hyperexcitable following withdrawal from cocaine self-administration [START_REF] Neumann | Increased excitability of lateral habenula neurons in adolescent rats following cocaine self-administration[END_REF].

In conclusion, LHb neurons are not only a direct target of psychostimulants but also depressive-like states. This work will be described in detail in the results section.

Context and objectives for the studies I. mGluR-LTD at excitatory and inhibitory synapses controls lateral habenula output

The so far presented literature illustrates that the lateral habenula has a key position to control downstream monoaminergic systems therefore participating in motivational processing. A large effort has been focused in the recent years to elucidate the functional connectivity of the LHb as it receives dense innervation from and projects to multiple structures implicated in reward and aversion processing. With the advances of optogenetics and circuit mapping tools it has been possible to probe the contribution of specific inputs and output targets of the LHb in motivated behaviors. This is how it became clear that excitatory inputs activation increases LHb neuronal output likewise contributing to aversive behaviors, whereas inhibitory inputs tend to decrease LHb firing therefore promoting preference behaviors (Root et al., 2014b;Stamatakis et al., 2013;[START_REF] Stamatakis | Lateral Hypothalamic Area Glutamatergic Neurons and Their Projections to the Lateral Habenula Regulate Feeding and Reward[END_REF][START_REF] Warden | A prefrontal cortex-brainstem neuronal projection that controls response to behavioural challenge[END_REF]. However, in physiological conditions LHb neuronal activity is shaped by the simultaneous activity of all inputs and depends on the synaptic and integration properties of each neuron. Indeed, many of the so far described studies lack information regarding the precise synaptic mechanisms engaged following specific input stimulation and whether long-term synaptic plasticity at these inputs is required for the establishment of the observed behaviors.

We aimed to understand how synaptic transmission in the LHb can be modulated under different activity conditions and how synaptic plasticity impacts neuronal output. We focused on the group I mGluR receptors because they are capable to induce long-term synaptic changes throughout the CNS as well as to modify intrinsic neuronal properties (Anwyl, 1999;[START_REF] Bellone | Mechanisms of synaptic depression triggered by metabotropic glutamate receptors[END_REF]. The group I mGluRs mostly reduce the efficacy of excitatory, but also inhibitory synaptic transmission and have been implicated in several pathological conditions also linked to LHb dysfunction, such as addiction [START_REF] Lüscher | Group 1 mGluR-dependent synaptic long-term depression: mechanisms and implications for circuitry and disease[END_REF]. Whether mGluRs are functionally expressed and whether they underlie long-term synaptic modifications in the LHb remains so far elusive. We hypothesized that if mGluRs are present in the LHb they may trigger long lasting decrease in the efficacy of excitatory and/or inhibitory neurotransmission. Given that LHb neurons express predominantly GluA2-lacking AMPA receptors, a potential mGluR modulation could occur postsynaptically via a switch of AMPA receptors subunits (from rectifying, larger conductance GluA2-lacking to non-rectifying, smaller conductance GluA2containing AMPA receptors) [START_REF] Bellone | Cocaine triggered AMPA receptor redistribution is reversed in vivo by mGluR-dependent long-term depression[END_REF]Kelly et al., 2009;McCutcheon et al., 2011b). Alternatively they could induce retrograde messengers capable to modify presynaptic release probability at excitatory and inhibitory synapses (Chevaleyre et al., 2006;Heifets and Castillo, 2009). Here we probed the mechanisms of mGluRdependent modulation of synaptic transmission and its impact on LHb neuronal activity.

Our results provide insights on so far unexplored cellular mechanisms, through which mGluRs control excitatory and inhibitory synaptic strength and highlight the functional repercussions of mGluR-plasticity for LHb neuronal output. In Brief

Valentinova and Mameli show that mGluR1s in the lateral habenula (LHb) triggers PKC-dependent depression of excitatory and inhibitory transmission, allowing for bidirectional tuning of neuronal output via distinct presynaptic and postsynaptic mechanisms.

INTRODUCTION

Excitatory and inhibitory projections onto the lateral habenula (LHb) control the direction of neuronal output, contributing to the encoding of rewarding and aversive stimuli (Shabel et al., 2012(Shabel et al., , 2014;;Stamatakis et al., 2013). Moreover, in rodent models of addiction and depression, glutamatergic and GABAergic synaptic plasticity modulates LHb neuronal firing, which is in turn instrumental for depression-like phenotypes (Lecca et al., 2016;Maroteaux and Mameli, 2012;Meye et al., 2015;Shabel et al., 2014). This highlights the behavioral relevance of synaptic adaptations in the LHb, heightening the need of understanding its underlying cellular processes.

Group I metabotropic glutamate receptor (mGluR) signaling and expression undergo modifications in disorders such as addiction and depression, disease states also characterized by aberrant LHb neuronal firing [START_REF] Bellone | mGluR-Dependent Synaptic Plasticity in Drug-Seeking[END_REF][START_REF] Hovelsø | Therapeutic potential of metabotropic glutamate receptor modulators[END_REF]Lecca et al., 2014). Group 1 mGluRs consist of mGluR1 and mGluR5 subtypes (L€ uscher and Huber, 2010). Their activation modulates the strength of excitatory and inhibitory synapses through G q /G 11 -mediated calcium mobilization and activation of downstream effectors, including protein kinase C (PKC) [START_REF] L€ Uscher | Group 1 mGluR-dependent synaptic long-term depression: mechanisms and implications for circuitry and disease[END_REF][START_REF] Page | Modulation of the neuronal dopamine transporter activity by the metabotropic glutamate receptor mGluR5 in rat striatal synaptosomes through phosphorylation mediated processes[END_REF]. Pre-and postsynaptic mechanisms underlie mGluR-dependent longterm plasticity, but its relevance for controlling neuronal activity remains poorly understood [START_REF] Galante | Group I metabotropic glutamate receptors inhibit GABA release at interneuron-Purkinje cell synapses through endocannabinoid production[END_REF][START_REF] Kammermeier | Homer proteins regulate coupling of group I metabotropic glutamate receptors to N-type calcium and M-type potassium channels[END_REF]Mameli et al., 2007).

We combine electrophysiology in LHb-containing acute slices with pharmacology and find that activation of mGluR1 receptors, but not of mGluR5, triggers long-term depression of excitatory and inhibitory synaptic transmission (mGluR-eLTD and mGluR-iLTD, respectively). mGluR-eLTD and -iLTD induction requires postsynaptic PKC signaling, but their maintenance relies on divergent expression mechanisms. mGluR-eLTD occurs via a presynaptic cannabinoid 1 receptor (CB1-R)-dependent decrease in glutamate release. In contrast, mGluR-iLTD is independent of presynaptic changes. Instead, mGluR-iLTD is postsynaptically expressed and requires PKC targeting onto GABA A -R b2-subunits and a reduction in GABA A -R single-channel conductance. The functional relevance of mGluR activation in the LHb is represented by opposing effects on neuronal output. Indeed, in the LHb, the mGluR-driven modulation of synaptic responses and output firing correlate positively. These data unravel the distinct molecular mechanisms underlying mGluR control of synaptic strength and the subsequent regulation of LHb neuronal activity.

RESULTS mGluRs Drive Long-Term Synaptic Depression in the LHb

To examine the presence of group I mGluRs, we microdissected the LHb of mice and employed RT-PCR, which revealed mGluR1 and mGluR5 expression (Figure 1A). Accordingly, bath application (3-5 min) of the mGluR1/5 agonist 3,5-dihydroxyphenylglycine (DHPG, 50 mM) led to a transient inward current (Figure 1B; [START_REF] Gee | Group I metabotropic glutamate receptors activate a calcium-sensitive transient receptor potential-like conductance in rat hippocampus[END_REF]. These data indicate the presence of functional postsynaptic group I mGluRs in LHb neurons.

To investigate whether mGluR activation modulates neurotransmission in the LHb, we tested the effect of DHPG application (5 min) on pharmacologically isolated AMPA receptor (AMPA-R)-mediated excitatory and GABA A -R-mediated inhibitory postsynaptic currents (excitatory postsynaptic currents [EPSCs] and inhibitory postsynaptic currents [IPSCs], respectively). DHPG produced long-term depression of EPSCs and IPSCs (Figures 1C and1D), termed eLTD and iLTD, respectively. mGluRs are activated by wide ranges of presynaptic activity (L€ uscher and Huber 2010; Chevaleyre et al., 2006). Accordingly, we found that low-frequency stimulation (LFS) of presynaptic fibers (1 Hz) led to eLTD (Figure 1E). Instead, at inhibitory synapses, high-frequency stimulation (HFS) of presynaptic afferents (100 Hz at 0 mV) triggered iLTD (Figure 1F). Thus, mGluR activation and a distinct pattern of presynaptic activity in the LHb efficiently reduce excitatory and inhibitory synaptic transmission.

mGluR-eLTD and -iLTD Require mGluR1 and PKC Signaling Group I mGluRs comprise mGluR1 and mGluR5 subtypes. To assess the induction requirement for mGluR-eLTD and -iLTD, we first exposed slices to either mGluR1 or mGluR5 antagonists (LY367385 or 3-2-methyl-4-thiazolyl-ethynyl-pyridine [MTEP], respectively). The mGluR1 antagonist LY367385 prevented DHPG eLTD/iLTD as well as LFS eLTD and HFS iLTD (Figures 2A and2B; Figures S1A andS1B). Although the LFS protocol also reduced IPSCs, LY367385 failed to block this form of plasticity, indicating a different mechanism of induction (Figure S1C). Importantly, DHPG eLTD and iLTD remained intact in presence of the mGluR5 antagonist MTEP (Figures 2C and2D). Downstream of mGluRs, the G q -coupled cascade leads to PKC activation, which targets a wide spectrum of synaptic proteins crucial for synaptic adaptations (L€ uscher and Huber, 2010). To test PKC implication for mGluR-eLTD and -iLTD, we dialyzed neurons through a patch pipette with a pseudosubstrate peptide inhibitor of PKC, PKC [19][20][21][22][23][24][25][26][27][28][29][30][31][32][33][34][35][36] [START_REF] Oliet | Two distinct forms of longterm depression coexist in CA1 hippocampal pyramidal cells[END_REF]. mGluR-eLTD and -iLTD were abolished in the presence of PKC [19][20][21][22][23][24][25][26][27][28][29][30][31][32][33][34][35][36] (Figures 2E and2F). If PKC underlies mGluR-eLTD and -iLTD, we reasoned that its activation would occlude mGluR-driven synaptic plasticity. To test this, we bath-applied the PKC activator phorbol-12-myristate-13-acetate (PMA). When PMA successfully decreased EPSCs and IPSCs (seven of ten and five of six cells, respectively; Figures 2G and2H), subsequent DHPG application failed to further reduce excitatory and inhibitory synaptic responses (Figures 2G and2H). These data indicate that mGluR activation decreases excitatory and inhibitory synaptic transmission via a common mechanism requiring mGluR1-driven PKC signaling.

Presynaptic Expression Mechanism of eLTD in the LHb

Excitatory synapses in the LHb contain GluA2-lacking AMPA-Rs, as indicated by inwardly rectifying EPSCs (Maroteaux and Mameli, 2012). In brain structures such as the ventral tegmental area, nucleus accumbens, and cerebellum, the presence of GluA2-lacking AMPA-Rs is a requirement for mGluRs to trigger postsynaptic LTD. This form of plasticity occurs via a switch from GluA2-lacking high-conductive to GluA2-containing lowconductive AMPA-Rs [START_REF] Bellone | mGluRs induce a long-term depression in the ventral tegmental area that involves a switch of the subunit composition of AMPA receptors[END_REF]Kelly et al., 2009;McCutcheon et al., 2011). To test whether this scenario also applies to the LHb, we evoked EPSCs at different holding potentials (-60, 0, and +40 mV) before and after mGluR-eLTD (Figure 3A). EPSCs at baseline were inwardly rectifying, yielding a rectification index of >1, indicative of GluA2-lacking AMPA-R expression. DHPG reduced EPSC amplitude at negative and positive potentials, leaving the rectification index unaltered (Figure 3A). Thus, mGluR-eLTD in the LHb does not require postsynaptic modifications of AMPA-R subunit composition.

Aside from postsynaptic modifications, mGluRs can also trigger presynaptic long-term adaptations. To examine whether a decrease in presynaptic glutamate release underlies mGluR-eLTD, we monitored the paired-pulse ratio (PPR) of EPSCs before and after DHPG and LFS. Along with the reduced EPSC amplitude, DHPG application as well as the LFS produced a long-lasting increase in the PPR, indicating reduced glutamate release (Figure 3B; Figures S2A-S2C). In line with the mGluR1 and PKC requirements for mGluR-eLTD, the PPR remained unaltered after DHPG in the presence of the mGluR1 antagonist and PKC inhibitor but not in the presence of the mGluR5 blocker (Figure 3B). Interestingly, PMA-driven reduction in EPSCs occurred along with an increased PPR, which remained unaffected after subsequent DHPG application (Figure 3B). The different pharmacological agents did not alter the baseline PPR, suggesting the absence of drug-induced modifications in the probability of glutamate release (Figure 3B; black columns for all conditions). To corroborate our findings on the presynaptic mechanism underlying mGluR-eLTD, we examined quantal release by recording miniature EPSCs (mEPSCs). In the presence of tetrodotoxin, DHPG application led to a decrease in mEPSC frequency without significant changes in mEPSC amplitude (Figure 3C). This supports a scenario for a presynaptic expression of mGluR-eLTD. mGluR activation can trigger the release of endocannabinoids from postsynaptic neurons in several brain structures, including the striatum, hippocampus, and ventral tegmental area. mGluR-driven endocannabinoid mobilization acts retrogradely on presynaptic CB1-Rs,

Figure 2. mGluR1 and PKC-Dependent Induction for eLTD and iLTD

(A) DHPG effect on EPSCs in the presence of the mGluR1 antagonist LY367385 (91.1 ± 5.6%, t 9 = 2.063, p > 0.05). (B) The same as (A) but for IPSCs (88.7 ± 6.7%, t 8 = 1.680, p > 0.05). (C) DHPG effect on EPSCs in the presence of the mGluR5 antagonist MTEP (74.5 ± 10.8%, t 8 = 2.377, *p < 0.05). (D) The same as (C) but for IPSCs (71 ± 8.7%, t 6 = 3.425, *p < 0.05). (E) DHPG effect on EPSCs during intracellular dialysis of PKC [19][20][21][22][23][24][25][26][27][28][29][30][31][32][33][34][35][36] negatively modulating neurotransmitter release (Heifets and Castillo, 2009). However, whether mGluRs trigger endocannabinoid signaling within the LHb is unknown. We first tested whether CB1-Rs are functionally expressed in the LHb. The CB1-R agonist WIN-55,212-2 reduced EPSC amplitude and increased the PPR (Figures 3D and3E). This intervention occluded DHPG eLTD, suggesting that mGluR-eLTD expresses through CB1-R activation (Figures 3D and3E). We pharmacologically confirmed that CB1-Rs are required for WIN-55,212-2-driven EPSC reduction because this was prevented by bath application of the CB1-R neutral antagonist NESS-0327 (Meye et al., 2013;Figures 3F and 3G). Consistent with the idea that CB1-Rs underlie the presynaptic expression of mGluR-eLTD, NESS-0327 also prevented mGluR-dependent plasticity and the concomitant increase in PPR (Figures 3H and3I). This suggests that mGluR-eLTD requires a PKC-dependent and CB1-Rs-mediated reduction in presynaptic glutamate release.

Postsynaptic Mechanisms for mGluR-iLTD in the LHb

Because mGluR-driven endocannabinoid mobilization can also modulate GABA transmission (Chevaleyre et al., 2006), we questioned whether mGluR-iLTD requires a reduction in GABA release. We first examined the PPR of IPSCs before and after DHPG or HFS. mGluR-iLTD and HFS-iLTD occurred without PPR modifications, independent of the pharmacological intervention, suggesting the absence of presynaptic adaptations at inhibitory synapses (Figure 4A; Figures S2B andS2D). In line with this finding, mIPSC frequency remained unchanged, whereas mIPSCs amplitude decreased after DHPG application (Figure 4B). Moreover, NESS-0327 did not prevent the mGluR-dependent reduction in GABAergic transmission (Figure 4C). Together, these findings support that mGluR-iLTD is independent of endocannabinoiddriven presynaptic modifications. These data suggest instead a postsynaptic expression mechanism for mGluR-iLTD in contrast to the presynaptically expressed mGluR-eLTD. (G) The same as (D) but in the presence of intracellular GABA A -b2 peptide (97.7 ± 10.4%, t 13 = 0.225, p > 0.05). (H) The same as (G) but for EPSCs (78.8 ± 6.3%, t 6 = 3.488, *p < 0.05). (I) The same as (G) but in the presence of intracellular GABA A -g2 peptide (67.7 ± 5.5%, t 8 = 6.141, ***p < 0.001). (J) The same as (I) but for EPSCs (77.7 ± 7.4%, t 7 = 3.014, *p < 0.05). Error bars represent SEM. n indicates number of recorded neurons.

Whether and how mGluR-PKC signaling modulates postsynaptic GABA A -R function remains unknown. PKC can directly target GABA A -Rs as well as auxiliary proteins modifying receptors' membrane expression and function (Kittler and Moss, 2003). For instance, PKC activation can increase GABA A -R internalization via a dynamin-dependent mechanism (Herring et al., 2005). To examine whether mGluR-iLTD in the LHb requires GABA A -R internalization, we dialyzed neurons with a membrane-impermeable dynamin inhibitor to prevent endocytosis. This intervention left mGluR-iLTD intact (Figure 4D), suggesting that GABA A -R internalization is not required. To corroborate the absence of changes in the number of postsynaptic GABA A -Rs during mGluR-iLTD, we employed peak-scaled non-stationary fluctuation analysis (NSFA) of mIPSCs (Maroteaux and Mameli, 2012;[START_REF] Nusser | Synapse-specific contribution of the variation of transmitter concentration to the decay of inhibitory postsynaptic currents[END_REF]. Based on the stochastic closing of ion channels, this statistical method allows us to estimate the number of receptors opened (N) by neurotransmitter release as well as their single-channel conductance (g). Plotting the decay variance as a function of the mean current amplitude for all recorded neurons yielded g GABA-A-R values comparable to previous studies (31.4 ± 1 pS; Figures 4E and4F; [START_REF] Nusser | Synapse-specific contribution of the variation of transmitter concentration to the decay of inhibitory postsynaptic currents[END_REF]. DHPG decreased estimated g GABA-A-Rs without altering estimated N GABA-A-Rs (Figures 4E and4F). Together, this supports the absence of mGluR-driven GABA A -R internalization. Conversely, a reduction in g GABA-A-R suggests a decrease in GABA A -R function, a modulation that may result from subunitspecific PKC-mediated phosphorylation (Kittler and Moss, 2003). Consistently, PKC-driven phosphorylation of specific serine residues on the GABA A -R b1-3 and g2 subunits reduces receptor function without altering the total receptor pool (Brandon et al., 2002a;Feng et al., 2001;Kittler and Moss, 2003). Given the reported expression of GABA A -R b2 and g2 subunits within the LHb [START_REF] Ho ¨rtnagl | Patterns of mRNA and protein expression for 12 GABAA receptor subunits in the mouse brain[END_REF], we predicted that the described mGluR-iLTD results from the direct PKC modulation of specific GABA A -R subunits. To test this, we dialyzed dominant-negative peptides corresponding to the PKC-targeted sequences of GABA A -R b2 or g2 subunits [START_REF] Brandon | GABAA receptor phosphorylation and functional modulation in cortical neurons by a protein kinase C-dependent pathway[END_REF]Feng et al., 2001). The presence of the b2 peptide (GABA A -b2) prevented mGluR-iLTD. In contrast, mGluR-eLTD and the concomitant PPR increase remained intact, ruling out nonspecific actions of GABA A -b2 dialysis (Figures 4G and4H; Figures S2C andS2D). Intracellular infusion of the g2 peptide (GABA A -g2) did not affect the expression of mGluR-iLTD or mGluR-eLTD (Figures 4I and4J; Figures S2C andS2D). These data suggest that mGluRs trigger a PKC-dependent reduction in GABA A -R conductance, likely occurring via phosphorylation of the b2 but not g2 receptor subunits.

mGluRs Decide the Direction of LHb Neuronal Output Opposed motivational states (i.e., reward and aversion) require bidirectional modification of LHb neuronal output, which can result in part from glutamatergic and GABAergic synaptic adaptations (Shabel et al., 2012;Stamatakis et al., 2013;Meye et al., 2015). What would be the functional repercussions of mGluR-eLTD and iLTD for LHb activity? To test the consequences of mGluR-LTD on LHb neuronal output, we recorded synaptically evoked postsynaptic potentials (PSPs) in current clamp mode. In the absence of synaptic blockers, PSPs result from a mixture of glutamatergic and GABAergic components and are therefore susceptible to mGluR-eLTD and -iLTD. We delivered trains of ten stimuli (20 Hz) and set the stimulation intensity so that $50% of evoked PSPs would produce action potentials (APs) (Figure 5A). Ten minutes after DHPG washout, a time point where mGluR-eLTD and -iLTD are fully expressed, AP numbers either increased or decreased (>20% change in APs) in $64% of neurons. Because of this dual modulation, DHPG did not, on average, modify the extent of evoked APs (Figure 5A). However, the bidirectional mGluR-driven change in neuronal activity may result from the expression of either mGluR-eLTD or -iLTD. Therefore, we examined whether the direction of LHb neuronal output after DHPG application correlates with mGluR-mediated modulation of PSPs. The area under individual PSPs (not including APs) was computed and averaged before and after DHPG to assess the PSPs potentiation or inhibition after mGluR activation. We predicted that the mGluR-mediated increase in PSPs would facilitate firing as a consequence of mGluR-iLTD. Conversely, predominant mGluReLTD would reduce the PSP area, decreasing neuronal output. In line with this scenario, the mGluR-driven change in PSP area positively correlated with the DHPG-driven modulation of AP number (Figures 5A and5B). To determine the causality between mGluR-eLTD/iLTD and the firing adaptations, we prevented the expression mechanisms underlying mGluR-LTD at excitatory and inhibitory synapses. Concomitantly blocking CB1-R and PKC action on GABA A -b2 receptors led to DHPG-driven modulation of LHb neuronal firing (>20% change in APs) in only 12.5% of recorded neurons. Under this condition, no correlation occurred between PSP area and firing after mGluR activation (Figures 5C and5D). In contrast, independently preventing either mGluR-eLTD or -iLTD expression mechanisms revealed a marked bidirectional DHPG-induced modulation of evoked firing (Figures S3A andS3B). Furthermore, input resistance and AP properties did not change or correlate with DHPG-mediated firing changes (Figures S3C-S3H).

If the occurrence of mGluR-dependent plasticity differs at excitatory or inhibitory synapses from specific inputs, this would partly explain the predominant influence of either mGluR-eLTD or -iLTD on neuronal output. LHb neurons receive axons from the entopeduncular nucleus (EPN, EPN LHb ) that co-release glutamate and GABA (Shabel et al., 2012). This allows us to examine whether mGluR-LTD occurs in a neurotransmission-specific fashion at a precise synaptic input. As a proof of concept, we virally expressed channelrhodopsin-2 (ChR2) in the EPN. This led to ChR2 + terminals within the lateral aspect of the LHb (Shabel et al., 2012;Meye et al., 2016;Figure S4A). DHPG bath application triggered a LTD of light-evoked EPN LHb IPSCs, whereas light-evoked EPN LHb EPSCs remained unaffected (Figures S4B andS4C). Together, these findings suggest that mGluRs in the LHb can control the direction of neuronal activity, likely via input-specific eLTD or iLTD.

DISCUSSION

Here we demonstrate that group I mGluRs decrease excitatory and inhibitory synaptic transmission in the LHb in a PKC-dependent manner. On one hand, mGluR1-driven PKC activation in LHb represents a common process at excitatory and inhibitory synapses. On the other hand, mGluR1 signaling diverges at the level of PKC, targeting distinct substrates but leading to decreased glutamatergic and GABAergic neurotransmis- sion. mGluR-eLTD and -iLTD modulate PSPs to decide the direction of LHb neuronal output. Our data support a scenario in which mGluRs modulate glutamatergic and GABAergic synapses in the LHb, contributing to adaptations in their computational properties potentially relevant for motivational states.

Notably, excitatory synapses in the LHb contain rectifying GluA2-lacking AMPA-Rs (Maroteaux and Mameli, 2012). A recent hypothesis posits that GluA2lacking AMPA-R expression represents a predictive factor for a postsynaptic mGluR1-LTD requiring a subunit composition switch [START_REF] Loweth | Using metabotropic glutamate receptors to modulate cocaine's synaptic and behavioral effects: mGluR1 finds a niche[END_REF]. However, mGluR1 activation in the LHb reduces excitatory synaptic transmission while leaving the GluA2-lacking AMPA-R current-voltage relationship unchanged. This unaffected AMPA-Rs rectification may result from LHb-specific interactions between receptors and scaffolding proteins or, alternatively, from unidentified AMPA-Rs subtypes that would need to be further investigated. In contrast, mGluR1s in the LHb act through postsynaptic PKC signaling to reduce glutamate release via CB1-R activation. Together with evidence indicating that LHb contains the endocannabinoid-synthesizing enzyme diacylglicerol lipase [START_REF] Sua ´rez | Distribution of diacylglycerol lipase alpha, an endocannabinoid synthesizing enzyme, in the rat forebrain[END_REF], our data support functional endocannabinoid signaling within the LHb. mGluR-driven endocannabinoid LTD is also observed at inhibitory synapses (Chevaleyre et al., 2006); however, this does not hold true in the LHb. Indeed, mGluR-iLTD is independent of presynaptic modifications and remains intact in the presence of CB1-R blockers. Although mGluR-iLTD does not require CB1-Rs, other G q protein-coupled receptors (GPCRs) may mobilize endocannabinoids to drive iLTD. Future studies need to address whether CB1-R activation modifies GABA transmission or whether other GPCRs mediate endocannabinoid-dependent iLTD in the LHb.

Although PKC mediates the presynaptic expression of mGluR-eLTD, it reduces GABA transmission through a postsynaptic mechanism. Importantly, postsynaptic PKC signaling controls the strength of inhibitory neurotransmission (Kittler and Moss, 2003). For instance, PKC can induce rapid internalization of GABA A -Rs through its actions on specific serine residues [START_REF] Chapell | Activation of protein kinase C induces gamma-aminobutyric acid type A receptor internalization in Xenopus oocytes[END_REF]Herring et al., 2005). However, mGluR-iLTD in the LHb is independent of dynamin-mediated endocytosis and does not involve a reduction in the number of activated receptors. Instead, mGluRs promote a reduction in GABA A -R singlechannel conductance. This modification in GABA A -R function may result from alterations in subunit composition, scaffolding proteins, and phosphorylation events (Kittler and Moss, 2003). Indeed, PKC reduces GABA A -R function, but not receptor expression, via phosphorylation of key residues on the GABA A -R b and g subunits [START_REF] Brandon | GABAA receptor phosphorylation and functional modulation in cortical neurons by a protein kinase C-dependent pathway[END_REF](Brandon et al., , 2002b;;Feng et al., 2001). We report that PKC action on GABA A -R b2-subunits, but not on g2 subunits, is crucial for mGluR-iLTD in the LHb. Interestingly, different subtypes of G q -PCRs other than group I mGluRs (i.e., muscarinic acetylcholine and serotonin receptors) also reduce GABA A -R function by PKC targeting of GABA A -R b1 and g2 subunits (Feng et al., 2001;Brandon et al., 2002a). This evidence therefore raises the possibility that different classes of G q -PCRs across the CNS may reduce synaptic inhibition via PKC phosphorylation of specific GABA A -R subunits (i.e., b2, g2, b1) (Brandon et al., 2002b;Feng et al., 2001;Kittler and Moss, 2003).

mGluR-eLTD and -iLTD are widespread across many synapses (Chevaleyre et al., 2006), but their functional repercussions on neuronal output remain elusive. mGluR1 can affect potassium and calcium conductances, crucial for neuronal activity (Anwyl, 1999). However, the reported absence of changes in input resistance and AP properties suggests that mGluR-driven modulation of neuronal activity likely arises from synaptic adaptations. The mGluR-dependent potentiation and inhibition of PSPs indeed predicts the direction of neuronal output after mGluR activation. Moreover, precluding mGluR-eLTD and -iLTD concomitantly or independently unravels the causality between synaptic plasticity and mGluR-dependent control of LHb neuronal firing. This result also suggests that mGluR-eLTD and -iLTD likely do not occur simultaneously at the same locus and with the same extent. Instead, one predominates over the other, to drive, in different neurons, opposite neuronal output changes. mGluR-eLTD and -iLTD may occur together with similar magnitude but on distinct postsynaptic sites or even distinct LHb neuronal populations. In both cases, mGluR plasticity would ultimately lead to a bidirectional modulation of LHb global activity. These scenarios may rely, to some degree, on circuit specificity. The observation that the EPN LHb GABAergic but not glutamatergic component is affected by mGluRs strongly suggests that, in the LHb, mGluR1 modulation may occur in a neurotransmission-and input-specific fashion. This is in in line with our data indicating that different patterns of activity trigger either mGluR-eLTD or -iLTD, and it is further supported by findings describing that input/outputspecific plasticity controls LHb output firing (Shabel et al., 2014;Meye et al., 2016). In conclusion, these findings identify how mGluR1 signaling in the LHb diverges at the level of PKC, leading to reduced presynaptic glutamate release and postsynaptic GABA A -R function. Based on our results, we speculate that mGluR-LTD in the LHb can decide the direction of neuronal activity, potentially influencing opposing motivational states.

EXPERIMENTAL PROCEDURES Animals

C57Bl/6J male mice ($30 days old) were used in accordance with the guidelines of the French Agriculture and Forestry Ministry for handling animals, and protocols were validated by the Darwin#5 ethical committee of the University Pierre et Marie Curie. Mice were anesthetized (i.p.) with ketamine (150 mg/kg)/ xylazine (100 mg/kg) (Sigma-Aldrich) prior to brain slice preparation or viral injections (Supplemental Experimental Procedures).

In Vitro Electrophysiology

Sagittal slices (250 mm) containing the LHb were prepared, and recordings were performed as described previously (Maroteaux and Mameli, 2012). For voltage clamp experiments, the internal solution contained 130 mM CsCl, 4 mM NaCl, 2 mM MgCl 2 , 1.1 mM EGTA, 5 mM HEPES, 2 mM Na 2 ATP, 0.6 mM Na 3 GTP, 5 mM Na + creatine phosphate, 2 mM QX-314, and 0.1 mM spermine ; (pH 7.3), osmolarity $300 mOsm. The holding potential was -50 mV. Synaptic currents were evoked through a glass pipette placed in the stria medullaris (60 ms at 0.1 Hz). The PPR was monitored (2 pulses, 20 Hz) and calculated as follows: EPSC 2 /EPSC 1 . mGluRs were activated by DHPG (50 mM) in the presence of 2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo[f]quinoxaline-2,3-dione (NBQX; 10 mM) and D-(2R)-amino-5-phosphonovaleric acid; (2R)-amino-5phosphonopentanoate (D-APV; 50-100 mM) or picrotoxin (100 mM). An LFS protocol (1 Hz,15 min) or an HFS protocol (100 Hz at 0 mV for 1 s, 5 times every 10 s) was used for synaptic activation of mGluRs. The rectification index of AMPA-EPSCs was calculated as follows: ((I EPSC(-60) /I EPSC(+40) )/1.5). Experiments assessing the postsynaptic effects of DHPG (voltage clamp) and output firing (current clamp) were performed with internal solution containing 140 mM KGluconate, 5 mM KCl, 10 mM HEPES, 0.2 mM EGTA, 2 mM MgCl 2 , 4 mM Na 2 ATP, 0.3 mM Na 3 GTP, and 10 mM creatine phosphate; (pH 7.3), osmolarity $300 mOsm. The input resistance was calculated via a 50-ms hyperpolarizing current (I = 20 pA) step (Ri = resting membrane potential [RMP]/I).

Non-stationary Fluctuation Analysis

A peak-scaled non-stationary fluctuation analysis was made from mIPSCs (Synaptosoft; Supplemental Experimental Procedures).

Drugs and Peptides

Drugs were obtained from Abcam, Tocris, Hello Bio, or Latoxan and dissolved in water. Tetrodotoxin (TTX) was dissolved in citric acid (1%); picrotoxin, NESS-0327, and PMA in DMSO;and LY367385 in NaOH 10%. For PMA experiments, only cells responding to drug application were included in the analysis. Peptides used in the study were custom-made (GeneScript) or obtained from Tocris (Supplemental Experimental Procedures) and, when indicated, included in the internal solution.

Analysis

Analysis was performed using IGOR-6 (Wavemetrics) and MiniAnalysis (Synaptosoft). Kolmogorov-Smirnov (KS) test, Student's t test, or ANOVA were used throughout the study. n in the figures indicates number of recorded (D). PPR of IPSCs after HFS (baseline 0.79±0.1 vs post-HFS 0.79±0.1, t 6 =0.1, p>0.05); PPR of IPSCs in presence of LY367385 (baseline 0.61±0.06 vs post-DHPG 0.65±0.06, t 8 =1.166, p>0.05); of MTEP (baseline 0.63±0.12 vs post-DHPG 0.8±0.1, t 6 =2.179, p>0.05); of PKC [19][20][21][22][23][24][25][26][27][28][29][30][31][32][33][34][35][36] in the recording pipette (baseline 0.83±0.08 vs post-DHPG 0.8±0.04, t 6 =0.318, p>0.05); after PMA and DHPG (baseline 0.56±0.2 vs PMA 0.56±0.16, t 4 =0.107, p>0.05; baseline-PMA vs PMA-post-DHPG 0.5±0.2, t 4 =0.794, p>0.05; PMA vs post-DHPG+PMA, t 4 =1.812, p>0.05); in presence of NESS-0327 (baseline 0.82±0.09 vs post-DHPG 0.8±0.06, t 10 =0.310, p>0.05); of dynamin inhibitor in the recording pipette (baseline 0.8±0.09 vs post-DHPG 0.83±0.2, t 8 =0.280, p>0.05); of the GABA-2 peptide in the recording pipette (baseline 0.79±0.12 vs post-DHPG 0.8±0.1, t 13 =0.192, p>0.05); of the GABA-2 peptide in the recording pipette (baseline 0.57±0.08 vs post-DHPG 0.67±0.09, t 8 =0.973, p>0.05).

One way ANOVA among all baseline PPR conditions: F (8, 80) =0.693, p>0.05. N in the figures indicates number of recorded neurons.

Supplemental Experimental Procedures

Stereotactic injections of viral constructs

Bilateral injections (~300 nl per hemisphere) in anaesthetized mice were performed using a glass pipette on a stereotactic frame (Kopf, France). Viral constructs allowing the expression of Channelrhodopsin-2 or Cheta (rAAV2/1-CAG-hChR2(H134R)-mCherry; rAAV9-hSyn-Cheta-EYFP; titers: 1*10 12 -1.3*10 13 gc/ml; University of Pennsylvania, USA) were injected in the entopeduncular nucleus (EPN) (-1.25 mm AP, 1.80 mm ML, -4.65 mm DV). Animals were allowed to recover for a minimum of 3 weeks before recordings were made. Only mice showing fluorescence within the lateral portion of the LHb were used for recordings.

In vitro electrophysiology

Sagittal slices (250 µm) containing the LHb were prepared as previously described (Maroteaux and Mameli, 2012). Slices were used for recordings in artificial cerebrospinal fluid (ACSF) equilibrated with 95% O 2 /5 % CO 2 and containing (in mM): NaCl 124; NaHCO 3 26.2; glucose 11; KCl 2.5; CaCl 2 2.5; MgCl 2 1.3; NaH 2 PO 4 1. The ACSF flow rate was 2 ml/min and the temperature was set at ~30°C. Currents were amplified, filtered at 2.5-5 kHz and digitized at 10 kHz. Access resistance was monitored by a 4 mV hyperpolarizing step, and experiments were discarded if access resistance increased >20%. For voltage-clamp experiments the internal solution contained (in mM): CsCl 130; NaCl 4; MgCl 2 2; EGTA 1.1; HEPES 5; Na 2 ATP 2; Na 3 GTP 0.6; Na + -creatine-phosphate 5, QX-314 2, and spermine 0.1, pH 7.3, osmolarity ~300 mOsm. The liquid junction potential was -3 mV and pipette resistance was 3-4 MΩ. Holding potential was -50 mV. Synaptic currents were evoked through a glass pipette placed in the stria medullaris (60 µs at 0.1 Hz). Paired pulse ratio (PPR) was monitored (2 pulses, 20 Hz) and calculated as follows: EPSC 2 /EPSC 1 . mGluRs were activated by R,S-3,5-dihydroxyphenylglycine (DHPG, 50 µM) in presence of NBQX (10 µM) and D-APV (50-100 µM) or Picrotoxin, 100 µM. mGluRs were synaptically activated using a low frequency stimulation protocol (LFS, 1 Hz, 15 min) or a high frequency stimulation protocol (HFS, 100 Hz at 0 mV for 1 sec; 5 times every 10 sec). Experiments assessing postsynaptic effects of DHPG (voltage-clamp) and output firing (current-clamp) were performed with internal solution containing (in mM): KGluconate 140; KCl 5; HEPES 10; EGTA 0.2; MgCl 2 2; Na 2 ATP 4; Na 3 GTP 0.3; Creatine Phosphate 10, pH 7.3, osmolarity ~300mOsm. QX-314 2mM was included when blocking action potentials. Liquid junction potential ( 12mV) was not corrected. AMPA-EPSCs current-voltage relationships were performed at -60, 0 and +40 mV. The rectification index was calculated as follows: ((I EPSC(- 60) /I EPSC(+40) )/1.5). Miniature EPSCs (mEPSCs) or IPSCs (mIPSCs) were recorded at -60mV in presence of Tetrodotoxin (TTX, 1 µM) and frequency and amplitudes were computed during baseline and 20 minutes after DHPG. Current clamp recordings were performed in ACSF or in presence of the CB1-Rs neutral antagonist NESS-0327 (0.5 µM) and the GABA A -2 peptide (50 µM) in the patch pipette. Cells were kept around their resting membrane potential (RMP -55 mV) throughout the recording. EPSPs and action potentials were evoked by 10 electrical pulses 50 ms apart through a glass pipette. Stimulation intensity was set to evoke ~50% of spikes out of the 10 delivered pulses. 10-20 min following DHPG the number of spikes were counted for a period of 3 minute (19 sweeps) and compared to the same period at baseline. The changes in firing following DHPG were normalized to the baseline number of action potentials. Only regular firing neurons were included in the analysis.

The area under individual EPSPs (Synaptosoft Inc, USA) during baseline and following DHPG was averaged and the normalized total area was correlated to the normalized firing after DHPG. The input resistance was calculated via a 50ms hyperpolarizing current (I=20pA) step (Ri=RMP/I). Action potential waveform analysis (Synaptosoft Inc, USA) of the amplitude, width and afterhyperpolarization amplitude for traces at baseline and after DHPG allowed to assess action potential properties changes.

Non-stationary fluctuation analysis

A peak-scaled non-stationary fluctuation analysis was made from mIPSCs (Synaptosoft Inc., USA). mIPSCs were selected by: fast rise time alignment; stable baseline holding current; absence of spurious fluctuations during the mIPSC decay.

Variance-amplitude relationship of mIPSC decay was plotted and fitted with the equation: 𝜎2 = 𝑖𝐼 -𝐼2/𝑁 + 𝜎𝑏2, (i: mean single-channel GABA current; I: mean current; N: the number of channels activated at the peak, N = mean amplitude/i; σ b 2 : baseline variance). i was estimated as the slope of the linear fit of the first portion of the parabola. The goodness of the fit was assessed with a least-square algorithm.

Unitary current was converted in conductance based on holding potential (-60mV).

Conductance and average IPSC amplitude, mean rise time, mean decay time, access resistance or background noise variance had no correlation (P > 0.4) (Maroteaux and Mameli, 2012).

Reverse Transcription-PCR

Total RNA from LHb (4 mice) was extracted using Trizol (Invitrogen) and transcribed into cDNA using SuperScript II Reverse Transcriptase (Invitrogen). PCR were conducted with Taq DNA polymerase (Invitrogen) using the following primers: mGluR1: forward primer 5'-CAAATCGCCTATTCTGCCAC-3', reverse primer 5'-CCATTCCACTCTCGCCGTAATTC-3'; mGluR5: forward primer 5'-GAGGAGGGGCTCGTCTGGGG-3', reverse primer 5'-ACAGTCGCTGCCACAGGTGC -3'

Drugs and Peptides

Drugs were obtained from Abcam (Cambridge, UK), Tocris (Bristol, UK), Hello Bio (Bristol, UK) or Latoxan (France) and dissolved in water. TTX was dissolved in citric acid (1%), Picrotoxin, NESS-0327, . For PMA experiments, only cells responding to drug application were included in the analysis. When indicated peptides were included in the internal solution: PKC inhibitor PKC [19][20][21][22][23][24][25][26][27][28][29][30][31][32][33][34][35][36] (20 µM), dynamin inhibitor (QVPSRPNRAP; 1.5 mM), GABA A R-2 subunit peptide (KSRLRRRASQLKITI; 50 µM), GABA A R-2 subunit peptide (SNRKPSKDKDKKKKNPAPT; 50 µM). Peptide sequences for 2 and 2 were custom-synthesized (GeneScript, USA). When recording in presence of peptides, baseline was taken ~10 min after entering in whole-cell to allow intracellular dialysis.

Analysis

Analysis was performed using IGOR-6 (Wavemetrics, USA) and MiniAnalysis (Synaptosoft Inc,USA). Cumulative plots for mPSCs were obtained by compiling all recorded neurons at baseline and after DHPG conditions. Data were binned at 2.5 pA and 150 ms to normalize the sample size across recordings and significance was assessed by the Kolmogorov-Smirnov test (KS). A linear fit was used for correlations and significance was tested with Pearson Coefficient (r). N in the figures indicates number of recorded neurons. All data are expressed as mean ± s.e.m. Significance was set at alpha=0.05 using paired t-test.

II. Cocaine-evoked negative symptoms require AMPA receptor trafficking in the lateral habenula

One of the theories of addiction posits that addiction is a process engaging initial hedonic effects after drug intake, but with repetitive use soon after the drug effects dissipate a multitude of aversive states arises motivating further use and ending up in an uncontrollable intake (Koob and Le Moal, 2008;[START_REF] Koob | Negative reinforcement in drug addiction : the darkness within[END_REF]. These aversive effects are characterized by increased anxiety and depressed mood. However, the neurobiological substrates of psychostimulant-induced aversive states are poorly understood.

As presented above, the lateral habenula has been proposed to participate in the pathophysiology of addictive disorders mainly because of its function in encoding aversive stimuli (Matsumoto and Hikosaka, 2009a), in establishing memories associated with negative events (Stamatakis and Stuber, 2012) and its ability to control the dopamine system [START_REF] Hong | Negative reward signals from the lateral habenula to dopamine neurons are mediated by rostromedial tegmental nucleus in primates[END_REF][START_REF] Ji | Lateral habenula stimulation inhibits rat midbrain dopamine neurons through a GABA(A) receptor-mediated mechanism[END_REF]Matsumoto and Hikosaka, 2007). Indeed, the LHb has an indirect inhibitory effect on dopamine neurons via its projections to GABA neurons of the midbrain (Jhou et al., 2009b;Meye et al., 2016). Moreover, single cocaine exposure directly modifies LHb neuronal function and induces aversive conditioning via LHb connection to the RMTg when drugs effects start to wear off [START_REF] Jhou | Cocaine drives aversive conditioning via delayed activation of dopamine-responsive habenular and midbrain pathways[END_REF]Zuo et al., 2013).

Repeated cocaine exposure induces synaptic adaptations in the LHb leading to increased excitatory transmission specifically in LHb neurons projecting to the RMTg (Maroteaux and Mameli, 2012). However, the persistence of these modifications, their precise molecular mechanisms and impact on neuronal activity remain so far unknown. Given that synaptic potentiation onto LHb neurons can drive hyperexcitable states and depressive-like behaviors [START_REF] Li | Synaptic potentiation onto habenula neurons in the learned helplessness model of depression[END_REF], we tested the hypothesis that cocaine-induced synaptic plasticity may trigger persistent increase in neuronal output specifically to GABA neurons of the midbrain therefore contributing to the establishment of depressive-like symptoms. Our results provide causal relationship between synaptic and cellular drug-induced adaptations in the LHb and the emergence of negative emotional states potentially leading to subsequent compulsive drug use. Withdrawal from addictive substances, including cocaine, produces negative symptoms such as a depressive-like phenotype that contributes to compulsive drug abuse 1,2 . The lateral habenula (LHb) inhibits monoaminergic systems via the GABAergic rostromedial tegmental nucleus (RMTg), encoding aversion-related stimuli 3,4 . Notably, functional modifications of the LHb are associated with neuropsychiatric disorders, including addiction and depression 5-7 . However, whether a drug-mediated depressive-like phenotype is a consequence of drug-evoked adaptations in the habenulo-mesencephalic pathway remains unknown.

We examined the effects of cocaine on glutamatergic synaptic transmission onto mouse retrogradely labeled RMTg-projecting LHb neurons (LHb →RMTg ; Fig. 1a and Supplementary Fig. 1a,b). Miniature excitatory postsynaptic current (mEPSC) amplitudes, but not frequencies, were larger in LHb →RMTg neurons 24 h after two consecutive cocaine injections (20 mg per kg of body weight, intraperitoneal; Fig. 1b,c) 6 . This cocaine-induced synaptic potentiation persisted 1 week after cocaine exposure, as well as 14 d after a chronic cocaine regimen (Supplementary Fig. 1c,d). This time course parallels the emergence of strong negative symptoms during drug-free periods 1 . Drug-evoked plasticity was detected only in LHb →RMTg , but not LHb neurons sending axons to the ventral tegmental area (LHb →VTA ; Supplementary Fig. 1e). This suggests that cocaine promotes a projection-specific and enduring postsynaptic strengthening of excitatory transmission onto LHb →RMTg neurons. However, whether cocaine alters LHb subpopulations other than RMTg/VTA-projecting neurons remains to be tested.

LHb synapses largely express GluA1-containing rectifying AMPARs 8 . Based on this subunit composition, we tested the necessity of GluA1 trafficking for cocaine-induced synaptic potentiation. We designed a viral vector encoding a dominant-negative form of the GluA1 C terminus (GluA1ct) to temporally prevent activity-dependent synaptic delivery of GluA1 (ref. 9) via doxycycline-driven recombination 10 . Doxycycline administration led to GluA1ct expression solely in the LHb (Fig. 1d and Supplementary Fig. 1f,g). We performed consecutive recordings from neighboring LHb →RMTg neurons infected or uninfected with GluA1ct (Fig. 1d and Online Methods). GluA1ct overexpression left basal transmission unaltered (Fig. 1e,f) 9 . After cocaine administration, mEPSC amplitude was increased in GluA1ct-uninfected LHb →RMTg neurons. Notably, in neighboring neurons expressing GluA1ct, the cocaineevoked potentiation was prevented (Fig. 1f). Accordingly, cocaine increased evoked AMPAR-EPSCs (-70 mV) and AMPAR-NMDAR ratios (+50 mV; Fig. 1g,h and Supplementary Fig. 1h,i), which was precluded in LHb →RMTg neurons expressing GluA1ct. We detected no substantial change in EPSC rectification index, suggesting that, despite the insertion of new AMPARs at the synapse, the overall AMPAR subunit composition remained similar after cocaine treatment (Supplementary Fig. 1j).

The phosphorylation of serine 845 (S845) and serine 831 (S831) on the GluA1 C terminus crucially controls AMPAR membrane delivery in various structures 11 . Immunoblots from microdissected LHbs revealed that phosphorylation of S845 was markedly increased following cocaine treatment. We observed no modifications of phospho-S831 or total GluA1 (Supplementary Fig. 2a,b). Accordingly, S845 phospho-mutant mice (S845A) showed no cocaine-induced mESPC amplitude enhancement, whereas this plasticity in S831 phosphomutant mice (S831A) was left intact (Supplementary Fig. 2c-f). Thus, cocaine drives S845 phosphorylation-dependent GluA1 delivery onto LHb →RMTg neurons.

We assessed the importance of cocaine exposure on the in vivo firing rate of LHb →RMTg neurons in anesthetized mice (Fig. 2a and Supplementary Fig. 2g). LHb →RMTg neuronal firing rate was higher in cocaine-than saline-treated mice (Figs. 1c and2b,c). Given that single-cell GluA1ct expression is difficult to assess in vivo, we evaluated the necessity of GluA1 trafficking for LHb →RMTg neuronal hyperexcitability in acute brain slices. Cocaine increased the excitability of LHb →RMTg neurons not expressing GluA1ct. However, excitability in GluA1ct-infected LHb →RMTg neurons was comparable to that of saline controls (Figs. 1f and2d,e). Similar to the synaptic adaptations, hyperexcitability was still present 14 d after chronic cocaine exposure (Fig. 2g,h and Supplementary Fig. 2h). Notably, blockade of AMPARs by NBQX in slices did not affect cocaine-evoked hyperexcitability, suggesting a causal, but indirect, effect of GluA1

Cocaine-evoked negative symptoms require AMPA receptor trafficking in the lateral habenula Frank J Meye 1-3,8 , Kristina Valentinova 1-3,8 , Salvatore Lecca 1-3,8 , Lucile Marion-Poll 1-3 , Matthieu J Maroteaux 1-3 , Stefano Musardo 4 , Imane Moutkine 1-3 , Fabrizio Gardoni 4 , Richard L Huganir 5 , François Georges 6,7 & Manuel Mameli 1-3 Addictive substances mediate positive and negative states promoting persistent drug use. However, substrates for aversive effects of drugs remain elusive. We found that, in mouse lateral habenula (LHb) neurons targeting the rostromedial tegmental nucleus, cocaine enhanced glutamatergic transmission, reduced K + currents and increased excitability. GluA1 trafficking in LHb was instrumental for these cocaine-evoked modifications and drug-driven aversive behaviors. Altogether, our results suggest that long-lasting adaptations in LHb shape negative symptoms after drug taking.

1 Institut du Fer à Moulin, Paris, France. 2 Inserm, UMR-S 839, Paris, France. 3 Université Pierre et Marie Curie, Paris, France. 4 DiSFeB, Dipartimento di Scienze Farmacologiche e Biomolecolari, Università degli Studi di Milano, Milano, Italy. 5 Solomon H. Snyder Department of Neuroscience, The Johns Hopkins University School of Medicine, Baltimore, Maryland, USA. 6 Centre National de la Recherche Scientifique, Interdisciplinary Institute for Neuroscience, UMR 5297, Bordeaux, France. 7 Université de Bordeaux, Bordeaux, France. 8 These authors contributed equally to this work. Correspondence should be addressed to M.M. (manuel.mameli@inserm.fr). 2i,j). Consistently, we found that input resistance was larger following cocaine treatment, an effect that was prevented by GluA1ct expression.

The cell input resistance was highly correlated with LHb →RMTg neuronal excitability (Fig. 2f,g). To understand the cocaine-driven GluA1dependent mechanisms causing LHb →RMTg neuron hyperexcitability, Recording we focused on K + conductance, an inhibitory driving force that is critical for neuronal excitability 12 . LHb →RMTg neurons from cocaine-exposed mice exhibited a marked reduction of K + channelmediated peak and sustained currents, which are mediated by multiple K + channel subtypes (Figs. 1f and2h,i) 12 . Similar to the modifications described above, the reduction in K + currents was prevented by the expression of GluA1ct in LHb →RMTg neurons. This suggests that cocaine triggers a GluA1-dependent synaptic potentiation, resulting in a functional reduction of K + conductance and LHb →RMTg neuron hyperexcitability. Future studies will need to identify the specific K + component underlying cocaine-evoked adaptations of intrinsic excitability.

Is GluA1 trafficking-dependent plasticity in LHb a requirement for cocaine-driven behavioral phenotypes emerging during withdrawal? We employed the forced swim test (FST) and the tail suspension test (TST) to assess drug-evoked depressive-like states 13,14 . Exposing control mice (AAV-Venus) to five cocaine injections followed by a 2-week drug-free period resulted in depressive-like behaviors, with reduced latency to first immobility in the FST and increased total immobility in the FST and TST (Fig. 3a and Supplementary Fig. 3a,c). Notably, GluA1ct expression in the LHb prevented the cocaine-induced depressive-like phenotype (Fig. 3a and Supplementary Fig. 3c). Precluding GluA1 trafficking in the LHb specifically abolished the drug-mediated aversive component, as cocaine-induced locomotor sensitization or place preference remained intact following GluA1ct overexpression (Fig. 3b,c and Supplementary Fig. 3b,d,e).

We found that cocaine-evoked GluA1 trafficking mediated the potentiation of AMPAR transmission, reduction of K + conductance and hyperexcitability in the LHb. This ultimately contributes to the emergence of negative depressive-like symptoms during drug withdrawal. The GluA1-driven reduction in K + conductance provides a previously unidentified mechanism, potentially representing a common substrate for disorders characterized by LHb dysfunction 13 . The cocaine-driven hyperactivity of LHb →RMTg neurons may provide an 'anti-reward' signal 1 that likely inhibits the dopamine (DA) system 15 . Indeed, cessation of cocaine intake diminishes striatal DA levels, and reduced DA neuron activity produces depressive-like behaviors 16,17 . Notably, deep-brain stimulation in the LHb rescues a depressive-like phenotype in the learned helplessness model by restoring the excitatory drive onto LHb 5 . Whether this intervention would also efficiently reverse the aversive component of cocaine remains an open question. In conclusion, our data provide mechanistic insights for cocaine-driven adaptations in the LHb. These findings support the idea that increased LHb output is instrumental for drug-evoked negative symptoms, potentially contributing to compulsive drug use.

MetHods

Methods and any associated references are available in the online version of the paper.

Note: Any Supplementary Information and Source Data files are available in the online version of the paper.

oNLINe MetHods

Surgery. C57Bl/6J wild-type mice, S845A knock-in mice and S831A knock-in mice (males of 4-9 weeks) were used in accordance with the guidelines of the French Agriculture and Forestry Ministry for handling animals. Animals were anesthetized with ketamine (90 mg per kg)/xylazine (15 mg per kg intraperitoneal) (Sigma-Aldrich) before bilateral injection with a herpes simplex virus (McGovern Institute) expressing enhanced GFP allowing for retrograde tracing, into the RMTg or VTA. Retrobeads (Lumafluor) were used for recordings performed 2 weeks after withdrawal 18 . The following coordinates were used (from bregma, in mm) 18 : RMTg: A-P, -2.9; M-L, ±0.5; D-V, -4.3; VTA: A-P, -2.5; M-L, ±0.8; D-V, -4.4. Recovery was allowed for 5-15 d. When indicated, an adeno-associated virus (AAV) coding for an 89 amino-acid sequence of the GluA1 carboxy-terminal tail was used (AAV-GluA1ct) 19 . The GluA1ct sequence is fused to mCherry and inserted under the control of a TET-ON system. AAV-GluA1ct-mCherry and AAV-Venus as a control were injected bilaterally in the LHb at a final volume of 300-500 nl. The following coordinates were used (from bregma, in mm): A-P, -1.7; M-L, ±0.45; D-V, -3.1. After 3 weeks, mice were subjected to doxycycline treatment that consisted of intragastric administrations of 5 mg per 100 µl twice per day for three consecutive days. The injection sites were carefully examined for all electrophysiology experiments and only animals with correct injections were used for recordings. Similarly, for behavioral studies only animals with correct injection sites were included in the analysis. Brain slices from mice injected with AAVs or HSV were directly examined under a fluorescent microscope.

electrophysiology. Animals injected with saline or cocaine (20 mg Neighboring neurons positive for the EGFP retrograde labeling and negative or positive for the mCherry GluA1ct expression were recorded sequentially, counterbalancing the order of patching. EPSCs were evoked through glass electrodes placed 200 µm from the recording site. In the case of the sequential recording of neurons, the stimulation electrode was left in place using the same intensity. AMPA:NMDA ratios of evoked-EPSC were obtained by AMPA-EPSC -70 mV/NMDA-EPSCs at +50 mV as previously described, using the late component of the EPSC at 30 ms after the onset 18 . Rectification indexes were computed by AMPA-EPSC -70 /AMPA-EPSC +50 . Current-clamp recordings were obtained using an internal solution containing (in mM): potassium gluconate 140; KCl 5; HEPES 10; EGTA 0.2; MgCl 2 2; Na 2 ATP 4; Na 3 GTP 0.3 mM; creatine phosphate 10. Current-clamp experiments were performed by a series of current steps (negative to positive) injected to induce action potentials (20-pA injection current per step, 500 ms). To isolate voltage-gated K + channel-mediated currents, 4-s incremental 10-mV voltage steps were given from -70 to +40 starting at -50 mV holding potential in ACSF containing 1 µM tetrodotoxin, 200 µM CdCl 2 , 20 µM NBQX, 100 µM AP5 and 100 µM picrotoxin.

In vivo recordings in anesthetized mice. Stereotaxic surgery for electrophysiology experiments was performed under isoflurane anesthesia as previously described 20 . Recording pipettes and stimulating electrodes were inserted into the LHb and RMTg, respectively. A glass micropipette (tip diameter = 2-3 µm, 4-6 MΩ) filled with a 2% pontamine sky blue solution (wt/vol) in 0.5 M sodium acetate was lowered into the LHb. LHb neurons were identified according to recently established electrophysiological features 21 , these include a broad triphasic extracellular spike (>3 ms), and a tonic regular, tonic irregular or bursting spontaneous activity. Through these electrodes, the extracellular potential was recorded with an Axoclamp2B amplifier in the bridge mode. The extracellular potential amplified tenfold by the Axoclamp2B amplifier was further amplified 100-fold and filtered (low-pass filter at 300 Hz and high-pass filter at 0.5 kHz) via a differential AC amplifier (model 1700, A-M Systems). Single neuron spikes were discriminated and digital pulses were collected on-line using a laboratory interface and software (CED 1401, SPIKE 2, Cambridge Electronic Design).

Electrical stimulation of the RMTg was used to test for antidromic activation of LHb neurons using high frequency following collision methods, as described previously 20 . A bipolar concentric stimulation electrode was inserted into the RMTg and driven impulses were considered antidromic if they met the following criteria: 1) constant latency of spike response, 2) driving by each paired stimulus pulses at frequencies of 100 Hz or greater, and 3) collision of driven spikes by spontaneous impulses occurring in a critical interval approximately equal to the sum of the refractory period plus the driving latency.

At the end of each recording experiment, the electrode placement was marked with an iontophoretic deposit of pontamine sky blue dye (-20 µA, continuous current for 12-15 min). To mark electrical stimulation sites, 10 µA of positive current was passed through the stimulation electrode for 1 min (Fig. 3). After the experimental procedures, the animals were deeply anesthetized with halothane (5%, vol/vol) and decapitated. Brains were removed and snap-frozen in a solution of isopentane at -70 °C. The tissue was sectioned into 25-µm-thick coronal slices, mounted, and stained with neutral red to enable histological determination of recording and stimulating electrodes sites.

Viral construct. To construct pAAV-TRE-GluA1ctmCherry-CMV-Tet-On3G, sequential subclonings were performed as described below. PCR primers and synthetic genes were purchased from Eurofins MWG Operon. TRE-GFP was PCR amplified with Phusion High Fidelity DNA Polymerase (ThermoScientific) using pTRE-GFP as template (Clontech). Product was digested with NsiI and SpeI enzyme (New England BioLabs) and cloned into NsiI/SpeI sites of the pCMV-Tet-On3G vector (Clontech) using T4 DNA ligase (Invitrogen), resulting in pTRE-GFP-CMV-Tet-On3G. GluAct DNA (was obtained by gene synthesis of mouse GluA1 nucleotides 2,425 to 2,667 corresponding to amino acids 809 to 889 (ref. 19). GluA1ct was cloned in pCMV-mCherry (kindly provided by X. Nicol, Institut de la Vision) in frame with N-terminal mCherry. GluA1ct-mCherry was then PCR amplified and digested with AgeI and EcoRV enzymes. pTRE-GFP-CMV-Tet-On3G was digested with AgeI and EcoRV enzymes and GluA1ct-mCherry was cloned in place of GFP to get pTRE-GluA1ct-mCherry-CMV-Tet-On3G. pAAV-hsyn-hChR2mcherry (Addgene) was cut with MluI and EcoRV to remove hSyn-hChR2mcherry and obtain pAAV empty vector. A linker sequence containing PpumI and AvrII sites was inserted into MluI and EcoRV sites by oligonucleotides annealing and ligation. pTRE-GluA1ct-mCherry-CMV-Tet-On3G was digested with PpumI and AvrII and the TRE-GluA1ctmCherry-CMV-Tet-On3G cassette was ligated into PpumI and AvrII sites of the pAAV vector. Then, the WPRE sequence was removed from 3′ of Tet-On3G by digestion with RsrII and ClaI, filling in and self-ligation of the vector. WPRE was finally cloned in 3′ of the GluA1ct-mCherry into NheI and SpeI sites to obtain pAAV-TRE-GluR1ctmCherry-CMV-Tet-On3G. All constructs were confirmed by restriction and sequence analysis. All of the AAV plasmids were made into recombinant AAV2/5 particles by the Vector Core Facility at the University of North Carolina, Chapel Hill, USA.

Immunoblotting. Treated and control LHb were initially microdissected and stored at -80 °C until processing. Tissue was homogenized by ten strokes in a Teflon-glass homogenizer in ice-cold 0.32 M sucrose containing 1 mM HEPES, 1 mM MgCl 2 , 1 mM NaHCO 3 , 0.5 mM NaF and 0.1 mM phenylmethylsulfonyl fluoride, in the presence of a complete set of protease inhibitors (Complete, Roche Diagnostic, Basel, Switzerland) and phosphatases inhibitors (Sigma Aldrich). Proteins were quantified using Bradford assay (Bio-Rad Laboratories) and 20 µg per sample were loaded on 7% SDS-PAGE gel and transferred to nitrocellulose membrane using Trans-Blot Turbo Transfer System (Bio-Rad Laboratories) to perform western blot analysis. Membranes were blocked with iBlock-TBS (Invitrogen, T2015) for at least 45 min and subsequently exposed to primary antibody overnight at 4 °C in iBlock-TBS. The next day, membranes were washed in Tris-buffered saline/Tween 20 (TBST) three times for at least 10 min at ~22 °C, then incubated with horseradish peroxidase (HRP)-coupled secondary antibody (Bio-Rad Laboratories) for 1 h at ~22 °C and washed again as before. For detection, Clarity Western ECL Substrates (Bio-Rad Laboratories) were used, and chemiluminescence was exposed to trans-UV (302 nm) with Chemidoc MP System (Bio-Rad Laboratories). We used antibodies to phospho-GluA1 (Ser845) (Millipore, clone EPR2148, 1:1,000), phospho-GluA1 (Ser831) (Millipore, clone N453, 1:1,000) α-Tubulin (Sigma-Aldrich, clone DM1A, 1:10,000).

Behavioral assays. All behavioral tests were conducted during the light phase (8:00-19:00). Animals were randomly assigned to the saline or cocaine group after viral injection. locomotor sensitization. Procedure was done as described earlier 22 . The locomotor activity was measured in cylindrical locomotor activity boxes (Imetronics, Pessac, France) in a low luminosity environment. Mice previously injected with the AAV-GluA1ct-mcherry in the LHb, were treated with doxycycline as described above. 1 d after the last doxycycline exposure, experimental day 1, locomotor activity was recorded 30 min before and 1 h after cocaine injection (20 mg per kg, intraperitoneal). 1 week after (day 8), each mouse was injected as on day 1, in the same activity box. Locomotor activity was measured automatically as the number of consecutive beam breaks (one beam per quadrant). conditioned place preference. Conditioned place preference was performed in two compartments of the apparatus (Imetronic) with different visual cues and different wall textures, in a low luminosity environment. On day 1, mice were placed in the center of the apparatus and allowed to explore freely both compartments for 15 min. Then mice of each group were assigned with one or the other compartment in a counterbalanced way to avoid any initial bias in preference. On days 2, 4 and 6 mice were injected with cocaine 20 mg per kg and placed immediately in the assigned closed compartment for 20 min. On days 3, 5 and 7 the mice were placed in the other closed compartment after saline injection 10 ml kg -1 . Finally on day 8, mice were tested for preference during 15 min of free exploration and the time they spent in each compartment was automatically recorded. Offline analysis was performed blind to the animal treatments.

Forced swim test. Forced swim test was conducted under normal light condition as previously described 23 . Mice were placed in a cylinder of water (23-25 °C, 14 cm in diameter, 27 cm in height for mice) for 6 min. The depth of water was set to prevent animals from touching the bottom with their hind limbs. Animal behavior was videotracked from the top (Viewpoint). The latency to the first immobility event and the immobility time of each animal spent during the test was counted online by two independent observers blind to the animal treatments. Immobility was defined as floating or remaining motionless, which means absence of all movement except motions required to maintain the head above the water. tail suspension test. The tail suspension test was used to assess behavioral despair and conducted under normal light. Mice were suspended by their tails with adhesive tape for a single session of 6 min. Animal behavior was videotracked from the top/front (Viewpoint) and video recorded. Immobility time of each animal spent during the test was counted online by two independent observer blind of the animal treatments. Mice were considered immobile only when they hung passively and motionless.

Analysis and drugs. All drugs were obtained from Abcam and Tocris and dissolved in water, except for TTX (citric acid 1%, wt/vol) and cocaine HCl (NaCl 0.9%, vol/vol). Analysis for the electrophysiology data was performed in blocks depending on the experiment. Online/offline analysis were performed using IGOR-6 (Wavemetrics) and Prism (Graphpad). Data analysis for in vivo electrophysiology was performed off-line using Spike2 (CED) software. Sample size required for the experiment was empirically tested by running pilots experiments in the laboratory. Data distribution was assumed to be normal, and single data points are always plotted. Frequency histograms and mean firing rate were determined for each recorded cells. Compiled data are expressed as mean ± s.e.m. Significance was set at P < 0.05 using Student's t test, one-or two-way ANOVA with multiple comparison when applicable.

A Supplementary methods checklist is available.

Discussion

In the results section I presented two main pieces of work that have provided new insights on 1) how mGluR-dependent synaptic plasticity controls the firing activity of LHb neurons and 2) how cocaine-evoked synaptic and cellular adaptations in a specific LHb pathway contribute to depressive-like behaviors following cocaine withdrawal. These data describe detailed mechanisms underlying activity-or experience-dependent synaptic modifications and their repercussions on LHb neuronal output. Further we demonstrate a causal relationship between cocaine withdrawal-induced synaptic and cellular modifications in the LHb and subsequent depressive-like states. However several points arise following these studies.

Which inputs undergo mGluR-eLTD and iLTD?

The LHb is embedded in a highly interconnected circuit receiving multiple feedforward and feedback glutamatergic and GABAergic inputs. Here we show that low-frequency stimulation of axons within the stria medullaris triggers mGluR1dependent LTD of excitatory synaptic transmission, while high frequency stimulation leads to mGluR1-dependent LTD of inhibitory transmission. We further show that mGluR-eLTD and iLTD bidirectionally contribute to LHb evoked firing. These findings raise the possibility that distinct inputs, capable to spike at low or high frequency ranges, may drive mGluR-eLTD or iLTD respectively. Indeed, when we monitored the effect of mGluR activation on EPSCs and IPSCs evoked from light stimulated EPN terminals we find that only inhibitory transmission undergoes mGluR-driven decrease in efficacy (Fig15C). Whether other afferents also express mGluR-iLTD, remains to be established. One plausible scenario is that other co-releasing inputs such as the VTA and potentially the LH may also be capable to trigger this form of plasticity given the close localization of excitatory and inhibitory postsynaptic structures (Fig15B) (Root et al., 2014b). Likewise HFS-induced glutamate release would trigger mGluR1 signaling in the vicinity of postsynaptic GABA A receptors allowing the interaction of PKC with the 2-GABA A receptor subunit. An electron microscopy investigation of the precise subcellular localization of mGluRs along the dendritic arborization of LHb neurons as well as the identity and specific phenotype of the opposed inputs would be required to address this possibility.

Interestingly, LFS of the stria medullaris also induced an mGluR1-independent iLTD which occurred in the absence of presynaptic modifications. An alternative scenario is that other GPCRs activation may trigger this form of plasticity, the circuit specificity, molecular mechanisms and contribution to firing modulation of which remain unexplored. Consistently with this idea, results obtained in recombinant systems and cultured neurons demonstrate that the G q -coupled muscarinic acetylcholine (mAChRs) and serotonin type 2C (5HT2C) receptors also reduce GABA transmission via PKC targeting of -GABA A and 2-GABA A receptor subunits respectively (Feng et al., 2001;Brandon et al., 2002). 5HT2C receptors and mACh receptors are also expressed in the LHb [START_REF] Pompeiano | Distribution of the serotonin 5 -H T 2 receptor family mRNAs : comparison between 5-HT2A and 5-HT2c receptors[END_REF][START_REF] Vilaró | Localization of m5 muscarinic receptor mRNA in rat brain examined by in situ hybridization histochemistry[END_REF]. These evidences raise therefore the possibility that subtypes of G q -coupled GPCRs (i.e. mGluRs, 5HT-2C, mAChRs) throughout the CNS and potentially in the LHb may reduce synaptic inhibition via PKC phosphorylation of specific GABA A receptor subunits (2, 2, 1 respectively (Fig14) (Brandon et al., 2002;Feng et al., 2001;Kittler and Moss, 2003).

Another open issue remaining to be explored is to assess which inputs express CB1 receptors and whether they undergo mGluR1-driven and CB1-dependent reduction in glutamate release (Fig15A). Notably, we have also noticed that inhibitory transmission can undergo CB1 receptor agonist-induced reduction in efficacy (data not shown). However this does not require mGluR activation since mGluR-iLTD was not prevented in presence of CB1 receptor antagonist. The inhibitory inputs as well as the precise molecular determinants of this form of plasticity would require further investigation.

The potential input-specific expression of mGluR-eLTD and iLTD suggests that under physiologically relevant circumstances the contribution of specific inputs to LHb output firing can be tuned by mGluRs. It is therefore interesting to test whether light LFS or HFS of specific ChR2-expressing inputs, capable to trigger either mGluR-eLTD or iLTD, are able to decrease or increase evoked neuronal firing respectively.

Is there any output-specificity for mGluR-eLTD and iLTD?

Despite certain variability of the expression and magnitude of mGluR-eLTD and iLTD across recordings, we did not observe apparent medio-lateral or antero-posterior distinctions. We therefore conclude that the expression of mGluR-eLTD and iLTD could occur in different LHb neuronal populations independently of their output targets. However, we performed our recordings in the sagittal plane where the assessment of the medio-lateral axis is less precise. Moreover, different outputspecific LHb populations may reside within close LHb subterritories (Fig6). A targetspecific retrograde labeling of LHb neurons could help to identify projection-specific neurons and assess their ability to express mGluR-eLTD or iLTD (Fig15D, E, F).

What is the behavioral relevance of mGluR-LTD in the LHb?

Our data suggest that mGluR plasticity in the LHb occurs in an input-and frequencyspecific manner. However we still lack information about the physiological conditions in which this might happen. Given that mGluR-eLTD contributes to decreased and mGluR-iLTD to increased neuronal output it is plausible that they may occur during persistent rewarding and aversive experience respectively (Matsumoto and Hikosaka, 2007). If this is the case one would expect that aversive experience (i.e.

foot shock, restrained stress, predator odor exposure etc) would lead to mGluR-iLTD, but not eLTD in the LHb ultimately leading to increased neuronal activity. My prediction would be that mGluR-iLTD (DHPG or HFS), but not mGluR-eLTD (DHPG or LFS) in slices from these animals would be occluded [START_REF] Pascoli | Reversal of cocaine-evoked synaptic potentiation resets drug-induced adaptive behaviour[END_REF]. In parallel the excitation/inhibition balance would be shifted towards more excitation compared to unexperienced animals. Similarly, if mGluR-eLTD and a subsequent firing decrease are triggered by reward experience (i.e. sucrose, enriched environment), LFS or DHPG application would fail to induce mGluR-eLTD in slices from these animals, without affecting the induction of mGluR-iLTD. Whether these potential scenarios hold true in the LHb, remains to be tested.

mGluR long term plasticity can be involved in learning mechanisms [START_REF] Jörntell | Synaptic Memories Upside Down: Bidirectional Plasticity at Cerebellar Parallel Fiber-Purkinje Cell Synapses[END_REF][START_REF] Riedel | Comparing the role of metabotropic glutamate receptors in long-term potentiation and in learning and memory[END_REF]. Would it be possible that mGluR adaptations in the LHb engage any form of learning related to rewarding or aversive experience?

While the activity of the LHb has been strongly associated to encoding aversive stimuli and in driving avoidance behaviors, much less is known about a potential control of the LHb on reward-related behaviors [START_REF] Lammel | Input-specific control of reward and aversion in the ventral tegmental area[END_REF]Matsumoto and Hikosaka, 2009a;Stamatakis and Stuber, 2012). In this regard, if LHb neurons undergo mGluR-dependent learning, it would be more plausible that they do so via mGluR-iLTD and increased neuronal output. A way to assess this issue would be to submit mice to aversive experience (i.e. predator odor) in a particular context and assess whether the animal learns to avoid the aversion-associated compartment. If we prevent mGluR-iLTD induction prior to experience (viral-based injection of a dominant negative peptide to block PKC and 2-GABA A receptor interaction) we would expect to abolish conditioned place avoidance.

Another alternative is that mGluR plasticity in the LHb could underlie pathological behaviors following drug exposure or stressful events leading to aberrant neuronal hyperactivity [START_REF] Li | Synaptic potentiation onto habenula neurons in the learned helplessness model of depression[END_REF]Meye et al., 2016;Shabel et al., 2014). Therefore a possibility is that also in these conditions mGluR-iLTD would be occluded. It would be interesting to examine what the role of mGluR-eLTD in such pathological conditions is and whether its induction is impaired [START_REF] Kreitzer | Endocannabinoid-mediated rescue of striatal LTD and motor deficits in Parkinson's disease models[END_REF]. These scenarios are plausible, but still hypothetical and require further investigation. This ultimately leads to reduced inhibitory control over EPN-driven LHb firing activity and is instrumental for depressive like states and cocaine relapse (Fig16) (Meye et al., 2016). This evidence together with our finding that LHb neurons activity is overall increased following cocaine withdrawal suggest that distinct synaptic adaptations occurring in parallel in the LHb increase neuronal output specifically to GABA neurons in the midbrain and are sufficient to drive negative emotional states (Fig16).

Notably, a single LHb neuron may receive different inputs that undergo cocaineinduced modifications and the relative contribution of each may depend on the specific localization on the dendritic arbor as well as on its computational properties.

Altogether these studies raise the hypothesis that regardless of the input and the synaptic mechanisms (increased excitatory transmission versus decreased inhibitory transmission) engaged after cocaine withdrawal, an increased excitability of LHb neurons is required for the establishment of depressive-like states. Although this hypothesis seems plausible, particularly in light of the evidence that LHb neurons can activate GABA neurons in the midbrain (Meye et al., 2016), it remains still to be tested whether LHb hyperactivity following cocaine withdrawal leads to dopamine neurons inhibition and reduces dopamine release (Fig16).

Synaptic adaptations after cocaine withdrawal

We have demonstrated that cocaine withdrawal induces GluA1-dependent synaptic potentiation of excitatory transmission and a reduction in potassium channels conductance. What could be the link between GluA1 trafficking and the decrease in potassium channels function? Several studies have suggested that activitydependent LTP engages GluA1 trafficking together with endocytosis of small conductance Ca 2+ -activated K + type 2 (SK2) channels or voltage-gated A-type K + channel Kv4.2 subunits [START_REF] Kim | Regulation of dendritic excitability by activity-dependent trafficking of the A-type K+ channel subunit Kv4.2 in hippocampal neurons[END_REF][START_REF] Lin | Coupled activity-dependent trafficking of synaptic SK2 channels and AMPA receptors[END_REF]. The insertion of the GluA1-containing AMPA receptors and the endocytosis of the SK channel seem to be functionally and unidirectionally coupled since selective inhibition of GluA1-containing AMPA receptors exocytosis blocks SK2-containing channel endocytosis, but the inverse manipulation does not affect AMPA receptor trafficking [START_REF] Lin | Coupled activity-dependent trafficking of synaptic SK2 channels and AMPA receptors[END_REF].

Given that SK and A-type voltage gated channels trafficking can be coupled to AMPA receptors trafficking and the fact that they strongly regulate neuronal excitability [START_REF] Adelman | Small-conductance Ca2+-activated K+ channels: form and function[END_REF][START_REF] Jerng | Modulatory mechanisms and multiple functions of somatodendritic A-type K (+) channel auxiliary subunits[END_REF] it is possible that these K + channels are the ones affected in the LHb following cocaine withdrawal. Although we did not identify the nature of these conductances their pattern of activation and voltage dependence suggest that they might be rather of the A-type. It remains still unknown what is the precise sequence of circuit synaptic and cellular events following chronic cocaine exposure and whether the plasticity of AMPA receptors and K + channels in the LHb results from homeostatic adaptations.

A previous study in our laboratory has also shown that cocaine exposure induces metaplasticity in the LHb. Indeed, an anti-Hebbian type of protocol using relatively low frequency stimulation patterns (10Hz) together with somatic hyperpolarization induces LTD of EPSCs in LHb-to-RMTg neurons in saline treated animals but LTP in cocaine treated ones (Maroteaux and Mameli, 2012). These results demonstrate that the potentiation of these synapses following cocaine does not reach a ceiling effect, but rather opens a window for further plasticity to be expressed, the function of which as well as it's synaptic mechanisms remain elusive.

LHb

Distinct synaptic adaptations converge to increase LHb neuronal and behavioral output

In our study two or five consecutive days of cocaine exposure led to persistent synaptic potentiation of AMPA-mediated transmission. Two weeks of withdrawal following chronic regiment of cocaine resulted in increased LHb-to-RMTg output, sufficient to drive behavioral despair. We propose that these synaptic and cellular adaptations in the LHb underlie the emergence of drug-induced negative symptoms which are thought to be instrumental for the establishment of compulsive drug use (Koob and Le Moal, 2008;[START_REF] Koob | Negative reinforcement in drug addiction : the darkness within[END_REF]. Indeed, it has been previously shown that prolonged cocaine administration (from 5 to 14 days) induces synaptic adaptations in VTA dopamine neurons, which persist even after long abstinence periods (up to 90 days) and are required for cocaine-seeking behaviors [START_REF] Chen | Cocaine but not natural reward self-administration nor passive cocaine infusion produces persistent LTP in the VTA[END_REF][START_REF] Mameli | Cocaine-evoked synaptic plasticity: persistence in the VTA triggers adaptations in the NAc[END_REF]. Notably the persistence of cocaine-induced synaptic changes depended on the cocaine administration procedure. Indeed, animals that self-administered cocaine presented more long-lasting plasticity compared to animals that were injected by the experimenter [START_REF] Chen | Cocaine but not natural reward self-administration nor passive cocaine infusion produces persistent LTP in the VTA[END_REF]. Whether the cocaine-induced plasticity in the LHb persists more than two weeks and whether it triggers other longterm adaptations in downstream targets, such as the dopamine system require further investigation.

Meye et al., showed recently that shifting the balance of excitation and inhibition at EPN-to-LHb synapse after cocaine withdrawal is instrumental for the susceptibility to relapse following stressful events (Meye et al., 2016). This raises the question whether AMPA receptors and K + channels plasticity in the LHb also leads to such behavioral phenotype given its contribution to depressive-like symptoms. How does the plasticity of excitatory and inhibitory synaptic transmission occurring simultaneously, but at different synapses cooperate? Preventing the establishment of GluA1-dependent synaptic adaptations in the LHb was sufficient to prevent the depressive-like state. Similarly, rescuing Vgat expression at EPN-to-LHb synapses restored the behavioral performance in the forced-swim test and precluded the stress-induced relapse. These data suggest that the induction of the two mechanisms is either interdependent or alternatively they are required to occur simultaneously in order to shift LHb neuronal activity towards a hyperexcitable state. A possible strategy to address this issue would be to test whether preventing GluA1-dependent plasticity would also prevent Vgat-dependent adaptations at EPN synapses or alternatively, whether overexpression of Vgat at these synapses would restore excitatory transmission.

Concluding remarks

In this thesis I attempted to provide an overview on lateral habenula function in motivational processing. Indeed, the LHb encodes aversive-related stimuli and drives These studies open new avenues for the investigation of the pathophysiology of the lateral habenula. In the future it would be necessary to assess the specificity of mGluR-eLTD and iLTD within distinct LHb microcircuits as well as their relative inputspecific contribution for single LHb neuronal activity. Indeed, the specific locus and integration properties of LHb neurons could impact on mGluR-dependent control on firing. Given that activity of specific LHb populations drive motivational states and that mGluRs control neuronal output, it would be further interesting to probe the behavioral relevance of this plasticity for reward and aversive behaviors or alternatively in pathological states. Following our study and others, the LHb is now emerging as a target of drugs of abuse and drug-driven behavioral maladaptations.

Therefore the data here presented raises the possibility that the LHb could represent a general substrate for drug-induced withdrawal symptoms. Future studies could lead to the identification of alternative and novel mechanisms contributing to withdrawaldependent behaviors.

These and other questions require further studies that will allow us to understand the precise circuit, synaptic and cellular substrates controlling LHb-driven behaviors. This knowledge may ultimately permit to develop interventions capable to prevent or reverse pathological plasticity and behaviors.

Publications and contributions

Finally, I want

  to thank my family, mom, dad and sis, the people who are always around me even thousands of kilometers away, for their endless love, understanding and support throughout my life. I feel lucky and extremely grateful to have such a loving and caring family. I love you! DISTINCT SYNAPTIC ADAPTATIONS CONVERGE TO INCREASE LHB NEURONAL AND BEHAVIORAL OUTPUT ....... CONCLUDING REMARKS ............................................................................................................................

Figure 1

 1 Figure 1 Simplified schematic of the meso-cortico-limbic and associated circuits in the rodent brain ................................................................................................................................................

Figure 2

 2 Figure 2 Cell-type specific modulation in the VTA for distinct motivational states........................

Figure 3

 3 Figure 3 Anatomical localization of the habenular complex ..........................................................

Figure 4

 4 Figure 4 Comparative analysis of human and rat LHb and LHb subnuclear organization ...........

Figure 5

 5 Figure 5 Afferents and their territorial distribution within the LHb .................................................

Figure 6

 6 Figure 6 LHb efferents and territorial distribution of their cell bodies throughout the LHb ...........

Figure 7

 7 Figure 7 LHb function in aversion encoding and negative prediction error ..................................

Figure 8

 8 Figure 8 Contribution of LHb output to motivated behaviors .........................................................

Figure 9

 9 Figure 9 Contribution of inputs to the LHb to different motivational states ...................................

Figure 10

 10 Figure 10 Acute effects of psychostimulants in the LHb ...............................................................

Figure 11 AMPA

 11 Figure 11 AMPA receptors trafficking and channel properties ......................................................

Figure 12

 12 Figure 12 Excitatory and inhibitory synapses in the LHb ..............................................................

Figure 13

 13 Figure 13 mGluR1/5 signaling and mechanisms of long term depression ...................................

Figure 14

 14 Figure 14 GqPCR model of inhibitory LTD ..................................................................................

Figure 15

 15 Figure 15 Input and cell type-specific model for mGluR-LTD in the LHb ....................................

Figure 16

 16 Figure 16 Effects of cocaine withdrawal in the LHb ....................................................................

Figure 2

 2 Figure 2 Cell-type specific modulation in the VTA for distinct motivational states

Figure 3

 3 Figure 3 Anatomical localization of the habenular complex

Figure 4

 4 Figure 4 Comparative analysis of human and rat LHb and LHb subnuclear organization

Figure 5

 5 Figure 5 Afferents and their territorial distribution within the LHb

Figure 6

 6 Figure 6 LHb efferents and territorial distribution of their cell bodies throughout the LHb

Figure 7

 7 Figure 7 LHb function in aversion encoding and negative prediction error

Figure 8

 8 Figure 8 Contribution of LHb output to motivated behaviorsLHb neurons are glutamatergic and are connect predominantly to GABA neurons of the midbrain(Meye et al., 2016).ChR2-driven stimulation of LHb terminals within the RMTg induces real-time and conditioned place avoidance, negatively reinforces behavior and disrupts positive reinforcement. RMTg neurons inhibiting dopamine neurons are excited by aversive stimuli and are inhibited by rewards similarly to LHb neurons. Some VTA-projecting LHb neurons target predominantly dopamine neurons projecting to the PFC. Stimulation of LHb neurons projecting to the VTA induces conditioned place avoidance.

  dopamine levels as assessed by fast-scan voltametry. Moreover, these LHb-projecting VTA TH+/CHR2+ neurons showed a reduced expression of Vmat2 (vesicular monoamine transporter 2), D2 dopamine receptor (D2R) and DAT (dopamine transporter) and exhibited different electrophysiological properties compared to classical dopamine releasing VTA-to-NAc neurons. Optogenetic activation of VTA TH+/ChR2+ axons in the LHb evoked GABAergic postsynaptic responses and decreased firing activity of LHb and

Figure 9

 9 Figure 9 Contribution of inputs to the LHb to different motivational states EPN neurons increase their firing in response to aversive stimuli and decrease their firing in response to rewards, similarly to LHb neurons. EPN-to-LHb and LH-to-LHb optogenetic stimulation increases LHb firing and drives real-time avoidance. Stimulation of the PFC-to-LHb input drives behavioral despair. Vglut2-expressing VTA terminals stimulation in the LHb predominantly inhibits LHb neurons firing and drive conditioned place avoidance, while TH-expressing VTA terminals stimulation drives conditioned place preference.

Figure 10

 10 Figure 10 Acute effects of psychostimulants in the LHb In the initial phase following acute drug administration LHb metabolic and firing activity is decreased and animals show place preference behaviors. During these initial stages monoaminergic activity is greatly increased but it is unknown to what extend the decreased LHb activity contributes to a higher dopamine neurons activity. 15-30 min following acute cocaine injection LHb firing activity increases as well as c-fos expression in LHb-to-RMTg pathway and animals display place avoidance behaviors. The activity and the subsequent release from dopamine neurons during this later stage after acute drug exposure are unknown.

Figure 11 AMPA

 11 Figure 11 AMPA receptors trafficking and channel properties

Figure 12

 12 Figure 12 Excitatory and inhibitory synapses in the LHb

  different synapses for mGluR-dependent plasticity. Whether mGluRs are functionally expressed and whether they control the efficacy of excitatory and inhibitory synaptic transmission in the LHb remains so far unexplored. If mGluRs are expressed in the LHb and based on the fact that LHb neurons express GluA2-lacking AMPA receptors it is plausible that their activation may lead to LTD of excitatory transmission via a switch of AMPA receptors subunits. Alternatively, mGluRs may modulate synaptic transmission via changes in release probability of both glutamate and GABA. However, it is still unexplored whether functional endocannabinoid signaling exists in the LHb to trigger such presynaptic changes. During my thesis I addressed the possibility that mGluRs are expressed in the LHb and assessed their function in modulating excitatory and inhibitory synaptic transmission. I further extended my interest in understanding whether or not synaptic plasticity driven by mGluR is crucial for neuronal activity.

Figure 13

 13 Figure 13 mGluR1/5 signaling and mechanisms of long term depression

  Dysfunction of the LHb leads to negative emotional states also in addictive disorders particularly following drug withdrawal. Some of the molecular mechanisms underlying pathological plasticity in the LHb after acute or prolonged exposure to psychostimulants are now beginning to be elucidated. Bath application of cocaine directly depolarizes LHb neurons and induced reversible acceleration of firing. This effect is largely reduced in presence of blockers for excitatory synaptic transmission. Moreover, acute cocaine transiently increases the frequency of spontaneous and electrically evoked EPSCs and increases the paired pulse ratio, which are all measurements assessing presynaptic release probability. These results confirm that the increase in LHb firing is partly due to increased presynaptic glutamate release. However, cocaine also depolarizes reversibly LHb neurons independently of synaptic inputs since it induces inward currents in presence of blockers for glutamatergic and GABAergic synaptic transmission as well as in presence of the voltage-gated sodium channel blocker tetradotoxin (TTX) which prevents action potential-driven neurotransmitter release. The cocaine-induced facilitation of spontaneous firing and glutamatergic transmission is blocked either by D1

  undergo long-term synaptic and cellular adaptations potentially leading to different aspects of drug-driven behaviors. Although recent insights have shed light onto some specific cellular mechanisms underlying drug-evoked behaviors, it remains to be established which specific habenular circuits and which precise cellular mechansims drive distinct negative emotional aspects of drug-taking. Given the circuit and functional specialization of the LHb different classes of drugs can act onto distinct LHb subpopulations and at different time points to produce persistent cellular adaptations. In depth studies addressing these questions are still needed. Some of the work to which I have contributed during my thesis identified specific molecular and cellular mechanisms in the LHb-to-RMTg pathway following cocaine withdrawal ultimately leading to

  Report mGluR-LTD at Excitatory and Inhibitory Synapses in the Lateral Habenula Tunes Neuronal Output Graphical Abstract Highlights d mGluR1 induces LTD of excitatory and inhibitory transmission in the LHb d PKC mediates the induction of mGluR-LTD in the LHb d Divergent expression mechanisms underlie mGluR-eLTD and -iLTD d mGluR-eLTD and -iLTD decide the direction of LHb neuronal output Authors Kristina Valentinova, Manuel Mameli Correspondence manuel.mameli@inserm.fr

Figure 1 .

 1 Figure 1. mGluR-eLTD and -iLTD in the LHb (A) Schematic depicting the LHb microdissection (hipp, hippocampus; thal, thalamus) and mGluR1 and mGluR5 expression in the LHb. MW, molecular weight; bp: base pairs. (B) Sample trace, bar graph, and scatterplot representing DHPG-evoked current (À28.5 ± 4.6 pA, 12 of 22 responding neurons). (C) Sample traces representing EPSCs at baseline (a) and 20 min following DHPG (b). The timeline represents the DHPG effect (50 mM) on EPSCs. The bar graph and scatterplot show normalized averaged EPSCs $40 min after DHPG (66.3 ± 5%, t 19 = 6.306, ***p < 0.0001). (D) The same as (C) but for IPSCs (69.9 ± 7.3%, t 17 = 4.235, ***p < 0.0001). (E) LFS-driven (1 Hz, 15 min) eLTD. The bar graph and scatterplot show normalized averaged EPSCs $40 min after the protocol (65.8 ± 6.4%, t 9 = 5.3, ***p < 0.0001). (F) HFS-driven (100 Hz, 1 s, at 0 mV) iLTD (top). The bar graph and scatterplot show normalized averaged IPSCs $40 min after the protocol (74.2 ± 3.9%, t 6 = 7.086, ***p < 0.0001). When not indicated, the timescale represents 5 ms. Error bars represent SEM. n indicates number of recorded neurons.

Figure 3 .NormalizedFigure 4 .

 34 Figure 3. mGluR-eLTD Expression via CB1-R Activation(A) Sample traces of AMPA-EPSCs at -60, 0, and +40 mV at baseline and after DHPG and average rectification index (baseline 3.6 ± 0.7 versus post-DHPG 3.5 ± 0.6, t 8 = 0.192, p > 0.05). (B) PPR of EPSCs in artificial cerebrospinal fluid (ASCF; baseline 0.62 ± 0.05 versus post-DHPG 0.9 ± 0.05, t 19 = 4.963, ***p < 0.0001); in the presence of LY367385 (baseline 0.53 ± 0.08 versus post-DHPG 0.64 ± 0.11, t 7 = 1.860, p > 0.05); of MTEP (baseline 0.59 ± 0.08 versus post-DHPG 0.79 ± 0.08, t 8 = 3.432, **p < 0.01); of PKC[19][20][21][22][23][24][25][26][27][28][29][30][31][32][33][34][35][36] in the recording pipette (baseline 0.7 ± 0.1 versus post-DHPG 0.76 ± 0.1, t 9 = 1.214, p > 0.05); after PMA and PMA + DHPG (baseline 0.57 ± 0.08 versus PMA 0.74 ± 0.07, t 6 = 2.799,*p < 0.05; PMA baseline versus PMA post-DHPG, t 6 = 0.829, p > 0.05). Shown are neurons represented in Figures1 and 2. One-way ANOVA among all baseline PPR conditions: F (9, 83) = 0.485, p > 0.05. (C) Top: sample traces for mEPSCs. Cumulative probability plots show amplitudes and inter-event intervals for mEPSCs at baseline (black) and after DHPG (red). (mEPSC amplitude: baseline 30 ± 3.8 pA versus post-DHPG 32.1 ± 4.1 pA, KS test, p > 0.05; mEPSC frequency: baseline 3.8 ± 1.6 Hz versus post-DHPG 2.5 ± 1.1 Hz, KS test, *p < 0.05). (D) Effect of WIN-55,212-2 on EPSCs (72.7 ± 3.8%, t 5 = 7.246, ***p < 0.001) and subsequent occlusion after DHPG application (68.5 ± 3.9%, t 5 = 5.559, p > 0.05). (E) PPR of EPSCs after WIN application and subsequent DHPG application (baseline 0.45 ± 0.02, post-WIN 0.67 ± 0.05, post-DHPG 0.71 ± 0.06; baseline versus post-WIN, t 5 = 3.411, *p < 0.05; post-WIN versus post-DHPG, t 5 = 1.004, p > 0.05). (F) The same as (D) but in the presence of NESS-0327 (90.79 ± 9.02%, t 5 = 1.001, p > 0.05). (G) The same as (E) but in the presence of NESS-0327 (baseline 0.54 ± 0.09 versus post-WIN 0.57 ± 0.06, t 5 = 0.672 p > 0.05). (H) Effect of DHPG on EPSCs in the presence of NESS-0327 (95.9 ± 4.3%, t 11 = 0.766, p > 0.05). (I) PPR after DHPG in the presence of NESS-0327 (baseline 0.58 ± 0.03 versus post-DHPG 0.63 ± 0.06, t 11 = 1.404, p > 0.05). Error bars represent SEM. n indicates number of recorded neurons.

Figure 5 .

 5 Figure 5. mGluR-Dependent Bidirectional Control of LHb Neuronal Output (A) DHPG effect on synaptically evoked AP numbers in ACSF (97.7 ± 19.6; t 13 = 0.13, p > 0.05). 4 of 14 recorded neurons increased in firing (blue), and 5 of 14 decreased in firing (red) following DHPG. Sample traces indicate the bidirectional nature (blue increased firing, red decreased firing) of mGluR activation. Shown are superimposed EPSPs at baseline (black) and after DHPG (blue, increased firing; red, decreased firing). (B) Correlation between normalized mGluR-driven firing and normalized PSP area (Pearson correlation, r = 0.75, **p < 0.01). (C) The same as (A) but in the presence of NESS-0327 and GABA A -b2 peptide in the internal solution (109.2 ± 8.2, t 7 = 1.12, p > 0.05). Black and gray traces represent before and after DHPG. (D) The same as (B) but in the presence of NESS-0327 in the ACSF and GABA A -b2 peptide in the internal solution (Pearson correlation, r = À0.12, p > 0.05). Fisher r-to-z transformation for (B) versus (D) correlations yielded a Z score of 2.03. *p < 0.05. (E) Schematic indicating the induction and expression mechanisms for mGluR-eLTD and -iLTD and their relative contribution to LHb neuronal output. Error bars represent SEM. n indicates number of recorded neurons.

Figure S2 .Figure 1

 S21 Figure S2. PPR analysis for EPSC and IPSC after mGluR activation. Related to Figure 1-4. (A). Sample recordings indicating the parallel modulation of EPSCs and PPR after DHPG application. (B). Same as A but for IPSCs. (C). PPR of EPSCs after LFS (baseline 0.6±0.04 vs post-LFS 0.76±0.05, t 9 =6.699, ***p<0.0001); PPR of EPSCs in the presence of intracellular GABA A -2 and 2 peptide (GABA A -2, baseline 0.52±0.07 vs post-DHPG 0.73±0.05, t 6 =4.127, **p<0.01; GABA A -2, baseline 0.47±0.08 vs post-DHPG 0.68±0.09, t 7 =4.303, **p<0.05). When not indicated time scale represents 5ms.

Figure S3 .Figure 5 .

 S35 Figure S3. Bidirectional control of firing by mGluR-LTD, unaltered input resistance and action potential properties after mGluR activation. Related to Figure 5. (A). Normalized effect of DHPG on synaptically-evoked APs number in the presence of GABA A -2 peptide in the internal solution (76.9±10.6, t 13 =2.183, *p<0.05). (B). Normalized effect of DHPG on synaptically-evoked APs number in the presence of NESS-0327 (158.2±21.8; t 10 =2.667, *p<0.05). (C). Averaged input resistance and scatter plot for recordings obtained in current clamp (baseline 495.5±63.6 vs post-DHPG 479.4±65.4, t 13 =0.49, p>0.05). (D). No correlation between mGluR-driven change in firing vs input resistance (r=0.35, p>0.05). (E). Averaged action potential traces indicating the parameters analyzed before and after mGluR activation. (F). Pooled data and scatter plots for AP amplitude (baseline 63.2±3.1 vs post-DHPG 59.8±3.1, t 11 =1.31, p>0.05) (G). Same as D but for AP width (baseline 1.06±0.1 vs post-DHPG 1.12±0.1, t 11 =0.60, p>0.05) (H). Same as D and E but for AP afterhyperpolarization amplitude (AHP, baseline 3.21±1.2 vs post-DHPG 3.22±1.2). No correlation of firing vs AP amplitude (r=0.36, p>0.05); vs width (r=-0.11, p>0.05); vs AHP (r=0.039, p>0.05). N in the figures indicates number of recorded neurons.

Figure

  Figure S4. mGluR-dependent modulation of IPSCs, but not EPSCs, at EPN inputs onto the LHb. Related to Figure 1 and Figure 5.

  (A). Experimental protocol and sample images representing stereotaxic injection of rAAV-ChR2 within the EPN, and EPN terminals within the LHb. (B). DHPG effect on EPN LHb EPSCs (109.4±6.5%, t 9 =0.414, p>0.05). (C). DHPG effect on EPN LHb IPSCs (65.4±6.5%, t 3 =5.27, *p<0.05). N in the figures indicates number of recorded neurons. mGluR-LTD at excitatory and inhibitory synapses in the lateral habenula Marie Curie 75005 Paris, France.

©

  2015 Nature America, Inc. All rights reserved. nature neuroscience advance online publication B r i e f c o m m u n i c at i o n s

Received 19

 19 September 2014; accepted 15 December 2014; published online 2 February 2015; doi:10.1038/nn.3923 © 2015 Nature America, Inc. All rights reserved. advance online publication nature neuroscience B r i e f c o m m u n i c at i o n s trafficking on LHb neuronal firing (Supplementary Fig.

Figure 1 Figure 2

 12 Figure 1 Cocaine-evoked synaptic potentiation onto LHb →RMTg neurons requires GluA1 delivery. (a) Schematic and images indicating the retrograde labeling of LHb →RMTg neurons. (b) Sample mEPSCs from LHb →RMTg neurons. (c) Scatter plot and grouped data for mEPSC frequency and amplitude (n = 10-12 cells, 4 mice of 4-5 weeks; frequency, saline versus cocaine t 20 = 1.1, P = 0.27; amplitude, saline versus cocaine t 20 = 3.7, **P = 0.0013). (d) Images indicating the AAV-GluA1ct injection site, infected LHb neurons, and GluA1ct-mCherry expression and eGFP labeling in LHb →RMTg neurons. (e) Sample mEPSCs obtained in AAV-GluA1ct-infected and uninfected LHb →RMTg neurons. (f) Scatter plot and grouped data for mEPSC frequency and amplitude (n = 6-10, 3 mice of 7-9 weeks; frequency interaction factor F 1, 36 = 0.4, P = 0.27, two-way ANOVA; amplitude interaction factor F 1, 36 = 15.5, **P = 0.0012, two-way ANOVA). (g) AMPAR EPSCs (-70 mV) and AMPAR + NMDAR EPSCs (+50 mV) obtained from LHb →RMTg neurons. (h) AMPA/NMDA ratios from sequentially recorded neighboring neurons (AAV-GluA1ct infected versus uninfected) after saline or cocaine (AMPA/NMDA; n = 9-11, 4 mice of 7-9 weeks; uninfected saline versus cocaine t 18 = 2.9, **P = 0.008; infected saline versus cocaine t 18 = 0.4, P = 0.7). Error bars represent s.e.m.

Figure 3

 3 Figure3Cocaine-driven AMPAR trafficking in LHb serves for a cocaine-evoked depressive-like state in mice. (a) FST analysis after AAV-GluA1ct and AAV-control injection in LHb (n = 11-12 mice of 8-9 weeks; AAV-Venus; latency first immobility F 1,44 = 3.6, *P = 0.038; total immobility, interaction factor F 1,44 = 5.9, **P = 0.0011). (b) Quarter turns in control and AAV-GluA1ct-injected mice (n = 10 mice of 8-9 weeks; paired t test: t 9 = 3.7 **P = 0.0044 for cocaine on day 8 versus day 1 in AAV-control and t 9 = 3.9. **P = 0.003 cocaine on day 8 versus day 1 in AAV-GluA1ct-injected mice; P = 0.26 for locomotor response to cocaine on day 8 for AAV-GluA1ct versus control-injected mice). (c) Grouped data for conditioned place preference scores in saline and cocaine-treated animals (n = 10 mice of 8-9 weeks; t 18 = 0.36 P = 0.42). Error bars represent s.e.m.

Figure 14

 14 Figure 14 GqPCR model of inhibitory LTD

Figure 15

 15 Figure 15 Input and cell type-specific model for mGluR-LTD in the LHb

Figure 16

 16 Figure 16 Effects of cocaine withdrawal in the LHb

  motivated behaviors via its connection to monoaminergic systems. Dysfunction of the LHb leads to pathologies of motivation, including addiction and depression characterized by LHb neuronal hyperexcitability. However, information about precise synaptic and cellular mechanisms controlling LHb neuronal activity and motivational states is still scarce. The work presented here tackled this general question and has provided novel mechanistic insights on how LHb synapses can undergo distinct forms of synaptic plasticity in physiological and pathological conditions. Moreover, we show that these different pre-and postsynaptic mechanisms can control LHb neuronal output. Finally we demonstrate a causal relationship between aberrant synaptic plasticity as well as neuronal activity in a discrete LHb circuit and the expression of negative emotional states. Our results could represent important cellular corollary for LHb-driven motivated behaviors in physiology and pathology.

- 13 -

 13 Tchenio A,* Valentinova K *and Mameli M Can the lateral habenula crack the serotonin code? Under Review at Front Syn Neurosci -Valentinova K and Mameli M. mGluR-LTD at excitatory and inhibitory synapses tunes lateral habenula output. Accepted at Cell Rep. -Glangetas C, Fois GR, Jalabert M, Lecca S, Valentinova K, Meye FJ, Diana M, Faure P, Mameli M, Caille S, Georges F, Ventral Subiculum Stimulation Promotes Persistent Hyperactivity of Dopamine Neurons and Facilitates Behavioral Effects of Cocaine. Cell Rep, 2015; Valentinova K, Tchenio A, Meye FJ, Lecca S, Mameli M, [Hell after the pleasure: drug-induced negative symptoms involve lateral habenula]. Med Sci (Paris), 2015; 31 -Meye FJ*, Valentinova K*, Lecca S*, Marion-Poll L, Maroteaux MJ, Musardo S, Moutkine I, Gardoni F, Huganir RL, Georges F, Mameli M, Cocaineevoked negative symptoms require AMPA receptor trafficking in the lateral habenula. Nat Neurosci, 2015; 18 -Meye FJ, Lecca S, Valentinova K, Mameli M, Synaptic and cellular profile of neurons in the lateral habenula. Front Hum Neurosci, 2013; 7 *equally contributed

This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

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Acknowledgements

ACKNOWLEDGMENTS We thank M. Carta, V. Chevaleyre, C. Bellone, N. Gervasi, and the Poncer-Levi and M.M. laboratory for helpful discussions and comments. We thank I. Moutkine for help with PCR experiments. This work was supported by Inserm ATIP-AVENIR, the City of Paris, and the European Research Council (ERC) under the European Union's Seventh Framework Program (FP7/2007-2013)/ERC grant agreement number 335333 SalienSy (to M.M.). K.V. is supported by a Ph.D. fellowship from the doctoral school ED3C Paris. AcknowledgmentS We thank C. Bellone, M.T.C. Brown, C. Lüscher and J.C. Poncer for discussions and comments on the manuscript. This work was supported by funds from the INSERM Atip-Avenir, ERC StG SalienSy 335333 and the Paris School of Neuroscience Network (ENP) to M.M., and the Agence Nationale de la Recherche (ANR-12-bsv4-0022-01) to F. Georges. F.J.M. is supported by a postdoctoral fellowship from the Fyssen Foundation and K.V. is supported by a PhD fellowship from the French Ministry of Science (ESR).

Contents

neurons. All data are expressed as mean ± SEM. Significance was set at alpha = 0.05 using paired t test.

SUPPLEMENTAL INFORMATION

Supplemental Information includes Supplemental Experimental Procedures and four figures and can be found with this article online at http://dx.doi.org/ 10.1016/j.celrep.2016.07.064. AUtHoR contRIBUtIonS F.J.M., K.V., S.L. and M.M. performed and analyzed all of the in vitro electrophysiological recordings. K.V., L.M.-P., and M.M. designed and performed the behavioral experiments. I.M. and M.J.M., prepared and tested the viral construct. S.M. and F. Gardoni performed the immunoblots. R.L.H. provided the phospho-mutant mice. F. Georges performed the in vivo recordings. M.M. designed the study and wrote the manuscript with the help of F.J.M., S.L. and K.V.

comPetIng FInAncIAl InteReStS

The authors declare no competing financial interests.

Reprints and permissions information is available online at http://www.nature.com/ reprints/index.html.

Supplementary Figure 1

Cocaine-evoked plasticity onto LHb →RMTg neurons is long-lasting and is absent in LHb →VTA neurons.