Marc Dos Santos

Emma N Cahill

Gregory Dal Bo

Peter Vanhoutte

Jocelyne Caboche

Bruno Giros

Nicolas Heck

Cocaine increases dopaminergic connectivity in the nucleus accumbens

Keywords: Dopamine, Cocaine, Dendritic spine, 3D imaging, Striatum

The development of addictive behavior is associated with functional and structural plasticity in the mesocorticolimbic pathway. Increased connectivity upon cocaine administration has been inferred from increases in dendritic spine density, but without observations of presynaptic elements. Recently, we established a method that enables analyses of both dendritic spines and glutamatergic boutons and presented evidence that cocaine induces changes in striatal connectivity. As the pharmacological and behavioral effects of cocaine directly implicate dopaminergic neurons and their afferents, a remaining question is whether dopaminergic striatal innervations also undergo structural plasticity. To address this issue, we generated transgenic mice in which the fluorophore tdTomato is expressed under the promoter of the dopamine transporter gene. In these mice, specific labeling of dopaminergic boutons was observed in the striatum. Of note, the accordance of our results for control mice with previous electron microscopy studies confirms that our method can be used to decipher the spatial organization of boutons in relation to dendritic elements. Following repeated cocaine administration that led to behavioral locomotor sensitization, an increased density of dopaminergic boutons was observed 1 day later in the nucleus accumbens shell specifically, and not in other striatal regions. Combined labeling of dopaminergic boutons and striatal dendrites showed that cocaine significantly increased the percentage of dendritic spines associated with a dopaminergic bouton. Our results show that chronic cocaine administration induces structural plasticity of dopaminergic boutons that could participate in dopaminedependent neuronal adaptations in the striatum.

Introduction

It is now widely accepted that structural plasticity is implicated in long-term adaptations that underlie several psychiatric disorders, including drug addiction (Bernardinelli et al. 2014;[START_REF] Russo | The addicted synapse: mechanisms of synaptic and structural plasticity in nucleus accumbens[END_REF]). Structural plasticity is, however, most often studied at the level of the postsynaptic structure only. Although observation of spine density along the dendrite is a valuable estimation of connectivity, the knowledge of the structural plasticity at the level of the axon is a critical issue for a proper understanding of synapse formation [START_REF] Wierenga | GABAergic synapses are formed without the involvement of dendritic protrusions[END_REF]Hu ¨bener and Bonhoeffer 2010;[START_REF] Schoonover | Comparative strength and dendritic organization of halamocortical and corticocortical synapses onto excitatory layer 4 neurons[END_REF][START_REF] Yang | Selective synaptic remodeling of amygdalocortical connections associated with fear memory[END_REF]Johnson et al. 2016). Recently, we developed a method for observing and analyzing glutamatergic pre and postsynapses (Heck et al. 2015). This approach enabled us to demonstrate that synapse formation indeed occurred after cocaine administration, for which an increase in spine density had previously been shown [START_REF] Russo | The addicted synapse: mechanisms of synaptic and structural plasticity in nucleus accumbens[END_REF]. Those spines also receive dopaminergic inputs from the mesencephalon, which encode rewardprediction error signals [START_REF] Schulz | Getting formal with dopamine and reward[END_REF]. By blocking the dopamine transporter (DAT), cocaine induces an increase in extracellular dopamine (DA) levels (Di Chiara and Imperato 1988) and thereby hijacks, the reward-driven learning circuit. This process is thought to promote a pathological form of memory leading to the addicted state (Hyman et al. 2006). The associated mechanisms include synaptic plasticity, activation of signaling pathways, gene expression changes and epigenetic modifications in striatal projection neurons (SPN), which all contribute to the longterm modifications of the network that sustains behavioral alterations induced by drugs of abuse (Lu ¨scher and Malenka 2011; [START_REF] Robison | Transcriptional and epigenetic mechanisms of addiction[END_REF]. Whether structural changes occur at the level of DA afferents onto SPN remains an open question. Among others SPN receive glutamatergic as well as DA inputs, which synergistically activate the Extracellular-signal Regulated (ERK) pathway [START_REF] Pascoli | Extracellular signal-regulated protein kinases 1 and 2 activation by addictive drugs: a signal toward pathological adaptation[END_REF]. Hence, a change in DA synaptic organization onto SPN could participate in the cellular events involved in cocaine-induced neuroplasticity. DA neurons located in the midbrain, project axons that spread their arborization in the striatum (Matsuda et al. 2009;Aransay et al. 2015;[START_REF] Rodrı ´guez-Lo ´pez | The mesoaccumbens pathway: a retrograde labeling and single-cell axon tracing analysis in the mouse[END_REF]. The released DA modulates the activity and plasticity of striatal neurons (Gerfen and Surmeier 2011;[START_REF] Tritsch | Dopaminergic modulation of synaptic transmission in cortex and striatum[END_REF]. DA boutons can form synaptic contacts on the neck of dendritic spines of SPN, but most boutons do not establish any synaptic contact (Descarries et al. 1996;[START_REF] Sesack | Cortico-basal ganglia reward network: microcircuitry[END_REF]Be ´rube ´-Carrie `re et al. 2012). DA afferents thus form a dense lattice, and the released DA acts through volume transmission within the striatum [START_REF] Moss | A dopaminergic axon lattice in the striatum and its relationship with cortical and thalamic terminals[END_REF]. Here, the density and spatial organization of DA boutons was measured in the striatum of mice that showed locomotor sensitization following repeated administration of cocaine. The present study reveals a structural plasticity of the DA innervation on the nucleus accumbens shell, which likely participates in the neuronal and behavioral adaptations induced by cocaine.

Materials and methods

Mice

Animal care was conducted in accordance with the European Commission Guidelines on the Care and Use of Laboratory Animals (Directive 86/609/EEC), and the experiments were approved by the local ethic committee C2EA-05. Mice expressing the Cre recombinase under the DAT promoter [START_REF] Turiault | Analysis of dopamine transporter gene expression pattern-generation of DAT-iCre transgenic mice[END_REF] were crossed with mice carrying a ROSA-tdTomato reporter acquired from Jackson Laboratories [Gt(ROSA)26Sortm9(CAG-tdTomato)Hze] ( Madisen et al. 2010). Male mice from the C57BL6 strain weighing 22-24 g were housed five per cage and acclimatized to the laboratory conditions (12 h light/dark cycle, 21 ± 1 °C room temperature) with ad libitum access to food and water.

Locomotor sensitization

Adult male mice were injected intraperitoneally with saline (0.9% NaCl) and placed in the activity box for 60 min on three consecutive days for habituation. The mice were then treated with either saline or cocaine (20 mg/kg in saline, Sigma-Aldrich) for five consecutive days and perfused on the sixth day for morphological analysis. Locomotor activity was measured for 30 min after the saline or cocaine injection using rectangular activity boxes (Immetronic). Locomotor activity was counted as total travel distance, measured by interruption of photocell beams located 15 mm above the floor.

Slice preparation

Mice were deeply anesthetized with an intraperitoneal injection of pentobarbital and perfused intracardially with 30 ml of paraformaldehyde (PFA) at 1.5% in 0.1 M phosphate buffer, pH 7.4 at 4 °C. The brain was removed from the skull and placed in 1.5% PFA for 1 h at 4 °C. Slices of 150 lm thickness were cut in the frontal plane using a vibratome (Leica) and stored in phosphate-buffered saline (PBS).

Immunofluorescence

Brain slices were permeabilized for 30 min with 0.1% Triton X-100 and 3% bovine serum albumin (BSA) in PBS. Rabbit polyclonal antibodies against tyrosine hydroxylase (TH) (1/1000, Sigma) or vesicular monoamine transporter-2 (VMAT2) (1/1000, C3Bf from B. Giros Lab) were incubated overnight at 4 °C in 0.01% Triton and 3% BSA in PBS. The slices were washed in PBS and incubated with the secondary antibody coupled to Alexa 488 (anti-rabbit 1/500, Invitrogen) for 2 h in 0.01% Triton 3% BSA in PBS. The slices were washed in PBS and mounted in Prolong (Invitrogen).

Image acquisition

The principles of image acquisition and deconvolution have been extensively described in Heck et al. (2012). Image stacks coded in 16-bit were taken using a confocal laser scanning microscope (SP5, Leica) equipped with a 1.4 NA objective (oil immersion, Leica) with a pinhole aperture set to 1 Airy unit, pixel size of 60 nm and z-step of 200 nm. The excitation wavelength and emission range were 488 and 500-550 for Alexa 488 and DiO, 561 and 570-650 for tdTomato. Laser intensity and PMT gain were set so that each image occupied the full dynamic range of the detector. Metrology measurements were regularly performed using fluorescent beads to confirm proper laser alignment, laser power and field homogeneity using the ImageJ-based MetroloJ plugin. Deconvolution with experimental point spread function from fluorescent beads using a maximum likelihood estimation algorithm was performed with Huygens software (Scientific Volume Imaging). One hundred fifty iterations were applied in classical mode, background intensity was averaged from the voxels with lowest intensity, and signal to noise ratio values were set to a value of 15 for Tomato signal and 20 for DiO dendritic signal.

Segmentation of synaptic objects, volume measurement, co-localization analysis

Three-dimensional segmentation and analysis of tdTomatopositive and immunolabeled boutons were performed using the procedure described in Heck et al. (2015). The procedures were run using custom ImageJ macros and the 3D functionalities of the 3D ImageJ Suite plugin. Of note, the workflow has been implemented in a user-friendly plugin called DiAna (Gilles et al. 2017). For image segmentation, a 3D watershed procedure defined the boundaries of space regions that contained objects, and local maxima were detected in the image. Histogram analysis and image inspection allowed a threshold intensity to be defined so that only local maxima from objects were retained. For each local maximum, corresponding to one object, the 3D intensity distribution centered on the local maxima was computed and fitted to a Gaussian curve. The intensity value covering 95.4% of the surface of the Gaussian curve was determined as the intensity threshold of the border of the object. Voxels located around the local maximum were then included in the object if they fulfilled the following three criteria of acceptance: intensity higher than the threshold; intensity lower than the previously included voxel; and inclusion of neighboring voxels; the last criteria was defined for acute object shape extraction). As the segmentation of each object is based on the analysis of its 3D intensity distribution, the procedure ensures that boutons of similar size but different intensity are extracted as objects of similar size. The volume of each extracted object was computed using the 3D ImageJ Suite plugin.

Object-based co-localization analysis was performed following segmentation. For each region of interest corresponding to a segmented tdTomato-positive DA bouton, the intensity in the other segmented channel was measured. Hence, co-localization was considered to occur when a TH or VMAT2 segmented object overlapped with a tdTomato-positive segmented object. For each pair of colocalizing objects, the 3D center-to-center distance between the two objects was computed using the 3D ImageJ Suite plugin.

Dendrite labeling and dendritic spine analysis

Dendrites and dendritic spines were labeled using the Diolistic technique described by Gan et al. (2000). Briefly, 50 mg of tungsten beads (Bio-Rad) was mixed with 3 mg of solid green DiO (3,3 0 -dioctadecyloxacarbocyanine perchlorate, Molecular Probes) dissolved in methylene chloride. DiI-coated beads were coated on the inner surface of a polyvinylpyrrolidone (Sigma-Aldrich)-pretreated Teflon tube. The tube was then cut in pieces, which were inserted as cartridges in a gene gun device (Bio-Rad). Helium gas pressure (150 psi) applied through the gene gun ejected the beads out of the cartridge onto the brain slice. The beads were delivered through a 3-lm pore-size filter (Isopore polycarbonate, Millipore) to avoid clusters. After labeling, the slices were kept in PBS at room temperature for at least 2 h and mounted in Prolong Gold.

Dendritic spine density was defined as the number of spines normalized to a 10 lm length of dendrite. Neuronstudio software (version 0.9.92) [START_REF] Rodriguez | Automated three-dimensional detection and shape classification of dendritic spines from fluorescence microscopy images[END_REF] was used to reconstruct the dendrite and detect dendritic spines. When necessary, manual correction was applied. For each striatal neuron, a dendritic segment of 20-70 lm in length and distant from at least 50 lm from the soma or after the first branching point was considered. The percentage of spines for which the neck was in contact with a dopaminergic bouton was manually counted by visual inspection in 3D through confocal image stacks.

Computation of 3D distance between dopaminergic boutons and potential receptor targets

A custom ImageJ macro was built, incorporating functions from the 3D ImageJ Suite plugin. For each segmented image of DA boutons, 100 dots (one voxel size) were placed at random positions within the 3D space in between DA boutons. A mask was built so that the points were never placed within cell soma. The minimal distance from each point to the border of the closest DA bouton was measured. The distributions of minimal distances for the images from the saline and cocaine conditions were then computed.

Results

Observation and identification of dopaminergic boutons in the striatum

To observe DA boutons in the striatum, a mouse line expressing the Cre recombinase under the control of the DA neuron-specific promoter of the DAT gene was crossed with a mouse line carrying a floxed gene driving the expression of the fluorescent protein tdTomato (Fig. 1a). Hence, DA neurons express tdTomato in these mice. The expression level and brightness of tdTomato fluorescence allowed for the observation of boutons in the striatum, whereas signals in the axons were not detected with the chosen image acquisition parameters. tdTomato-positive spots were segmented from 3D images (Fig. 1b). The average volume of the segmented objects was 0.297 ± 0.017 lm 3 , corresponding a sphere 0.827 lm in diameter, a value consistent with the size of DA boutons reported in studies conducted in dorsal striatum with serial electron microscopy [START_REF] Moss | A dopaminergic axon lattice in the striatum and its relationship with cortical and thalamic terminals[END_REF]. Slightly smaller diameters have been reported in the ventral striatum in a 2D electron microscopy study (Be ´rube ´-Carrie `re et al. 2012). It is noteworthy that optical microscopy has lower resolution in z-axis compared to xyaxis, and, despite restoration by deconvolution, our measurements of volumes may be over-estimated. This however would not impair the comparison of volumes objects between different conditions. The specificity of the labeling was confirmed by immunostaining against TH, which is expressed in DA axons innervating the striatum (Fig. 1c). Following segmentation, 3D co-localization analysis revealed extensive overlap, with 88.4% of tdTomato-positive objects co-localizing with TH objects (n = 1883 tdTomato-positive objects from n = 2 mice). To confirm that the tdTomato-positive objects contained dopaminergic vesicles, immunostaining against VMAT2 was performed (Fig. 1d). Of all tdTomato-positive segmented objects, 95.2% co-localized with VMAT2 segmented objects (n = 4610 tdTomato-positive objects from n = 2 mice). The center-to-center distance from tdTomato-positive objects to VMAT2 objects was computed in 3D. Measurements from n = 3775 object pairs retrieved a value of 227 ± 0.008 nm, which indicates accurate co-localization and thus confirms that the tdTomato-positive objects are DA boutons.

Chronic cocaine exposure induces structural plasticity of dopaminergic boutons in the nucleus accumbens

The possible reorganization of DA inputs upon repeated cocaine exposure was studied in a locomotor sensitization paradigm (Fig. 2a). Mice received either a single dose of cocaine or saline daily, and their locomotor activity was recorded. The same dose of cocaine (20 mg/kg) induced a progressive increase in locomotor activity compared to saline treated mice. This increased behavioral output induced by cocaine reveals that the neuronal network undergoes changes, such that the next administration of the same dose of cocaine acts on a primed network. One day after the last cocaine injection, images of the DA boutons were acquired by confocal microscopy in the different regions of the striatum: the medial and lateral part of the dorsal striatum and the shell and core of the nucleus accumbens (NAc). The sections imaged were located 0.9-1.2 mm rostral from bregma. The images were deconvolved and segmented to measure bouton density and volumes (Fig. 2b). A significant increase in DA bouton density was measured in the NAc shell, but not in the other regions of the striatum (Fig. 2c). Three-dimensional morphometric analysis revealed a decrease in the volume of the boutons in both the shell and the core of the NAc, but not in the dorsal striatum (Fig. 2d).

Many DA boutons are devoid of synapse, so the released DA diffuses to bind with its' receptors (Descarries et al. 1996;[START_REF] Moss | A dopaminergic axon lattice in the striatum and its relationship with cortical and thalamic terminals[END_REF][START_REF] Robinson | Monitoring rapid chemical communication in the brain[END_REF]. DA receptors are found on the dendritic shaft and dendritic spines of SPN as well as on glutamatergic and dopaminergic boutons [START_REF] Sesack | Ultrastructural localization of D2 receptor-like immunoreactivity in midbrain dopamine neurons and their striatal targets[END_REF][START_REF] Yung | Immunocytochemical localization of D1 and D2 dopamine receptors in the basal ganglia of the rat: light and electron microscopy[END_REF]De Mei et al. 2009;[START_REF] Uchigashima | Dopamine synapse is a neuroligin-2-mediated contact between dopaminergic presynaptic and GABAergic postsynaptic structures[END_REF]. Therefore, a change in the density and volume of DA boutons could modify the range of DA volume transmission and access to DA receptors. To address this hypothesis, a model was built whereby single points, representing putative receptor sites, were inserted at random positions in images of DA boutons obtained from saline and cocaine treated mice. The minimal distance between each point and the borders of DA boutons was measured in the three dimensions (Fig. 2e). In images from saline treated mice, 85.9% of DA boutons were less than 1 lm away from the nearest putative target (Fig. 2f). The cumulative frequency distribution of the minimal distances from the borders of DA boutons to putative DA receptors showed that in images from mice that received repeated cocaine administrations, the minimal distances were shorter than in the saline condition, with 89.8% of DA boutons located less than 1 lm away from the nearest putative target in the cocaine condition (Fig. 2f).

Taken together, these results reveal that repeated administration of cocaine induces structural plasticity of the DA afferents in the ventral part of the striatum, a cerebral structure strongly implicated in the early phase of cocaine addiction [START_REF] Steketee | Drug wanting: behavioral sensitization and relapse to drug-seeking behavior[END_REF].

Spatial relationship between dopaminergic boutons and dendritic spines of striatal projection neurons

Dopamine boutons in the striatum are not necessarily associated with a postsynaptic structure but can contact the dendritic spines of SPN [START_REF] Sesack | Cortico-basal ganglia reward network: microcircuitry[END_REF][START_REF] Moss | A dopaminergic axon lattice in the striatum and its relationship with cortical and thalamic terminals[END_REF][START_REF] Xu | Dopaminergic axons preferentially innervate dendritic spines with hyperactive glutamatergic synapses in the rat striatum[END_REF][START_REF] Uchigashima | Dopamine synapse is a neuroligin-2-mediated contact between dopaminergic presynaptic and GABAergic postsynaptic structures[END_REF]. Because repeated exposure to cocaine induces an increase in dendritic spine density [START_REF] Russo | The addicted synapse: mechanisms of synaptic and structural plasticity in nucleus accumbens[END_REF]Heck et al. 2015) and our results show an increase in DA bouton density in the NAc shell, it raises the intriguing question as to whether combined pre and post-synaptic structural plasticity could modify the spatial relationship between the dendritic spines and the DA boutons. To address this question, SPN were sparsely labeled with a green membrane-associated fluorochrome in slices of saline or cocaine treated transgenic mice, allowing the simultaneous observation of dendritic spines and DA boutons (Fig. 3a,b). Following repeated cocaine administration, which induced locomotor sensitization (see Fig. 2a), a significant increase in dendritic spine density was measured in the NAc shell compared to saline control conditions (Fig. 3c). The occurrence of contacts between DA boutons and the neck part of the dendritic spines was then monitored. In saline conditions, 22.8 ± 1.5% of dendritic spines were in direct contact with a DA bouton (Fig. 3d, n = 27 dendrites with 1257 spines). In mice treated with cocaine, the percentage of spines in contact with a DA bouton was 35.6 ± 0.7%, corresponding to an increase of 56% in comparison to saline controls (Fig. 3d, n = 21 dendrites with 783 spines). Altogether, these results highlight that cocaine induces a spatial rearrangement of the DA connectivity to favor putative synaptic contacts.

Discussion

Cocaine, by inhibiting the uptake of released DA, induces an elevated level of extracellular DA that triggers adaptations in striatal neurons leading to addictive behavior.

Whereas dendritic spine density [START_REF] Russo | The addicted synapse: mechanisms of synaptic and structural plasticity in nucleus accumbens[END_REF]) and glutamatergic connectivity (Heck et al. 2015) changes induced by cocaine have been extensively studied, a possible structural reorganization of dopaminergic afferents remained to be addressed. In the present study, we used the DAT-tdTomato transgenic mouse line that allowed the direct observation of DA boutons in the striatum. Following repeated cocaine administrations that induced locomotor sensitization, an increase in DA bouton density was observed in the NAc shell as well as an increase in the percentage of dendritic spines in contact with a DA bouton.

Our methodological approach enabled the examination of the 3D organization of DA boutons within the striatum. It is important to note that our measurements in control conditions are in accordance with results from a previous electron microscopy study [START_REF] Moss | A dopaminergic axon lattice in the striatum and its relationship with cortical and thalamic terminals[END_REF]Be ´rube ´-Carrie `re et al. 2012). Analysis of serial electron microscopy images enabled the percentage of dendritic spines in contact with a DA bouton to be estimated at 20%; and measurements of distances between randomly selected points and DA presynapses revealed that 96% of randomly selected points were located less than 1.04 lm from DA boutons [START_REF] Moss | A dopaminergic axon lattice in the striatum and its relationship with cortical and thalamic terminals[END_REF]. The spatial relationship between DA boutons and dendritic spines from striatal neurons we described, as well as the distance between DA boutons and potential receptor sites we measured, fit remarkably well with the conclusion of the study of Moss and Bolam, although it should be noted that this study was conducted in the dorsal striatum. Hence, our method, associating optical confocal microscopy with image deconvolution and 3D analysis, is an efficient strategy for the investigation of spatial organization of boutons, with the advantage of sampling large volumes of brain tissue and providing information from thousands of objects. Accordingly, recently published studies, including from our laboratory (Heck et al. 2015;[START_REF] Schoonover | Comparative strength and dendritic organization of halamocortical and corticocortical synapses onto excitatory layer 4 neurons[END_REF][START_REF] Yang | Selective synaptic remodeling of amygdalocortical connections associated with fear memory[END_REF], demonstrated that optical microscopy combined with careful analysis can successfully assess the spatial organization of boutons and dendritic spines.

The possible changes of DA innervation following cocaine administration have been previously addressed indirectly using western blotting and immunohistochemistry against TH, an enzyme involved in DA synthesis. However, those approaches either did not detect an increase in axonal TH expression (Beitner-Johnson and Nestler 1991;[START_REF] Sorg | Time course of tyrosine hydroxylase expression after behavioral sensitization to cocaine[END_REF] or led to contradictory results (Todtenkopf and Stellar 2000;Todtenkopf et al. 2000;Lee et al. 2011). Those methods likely suffered from false negatives artefacts inherent to immunostaining techniques, undersampling due to two-dimensional analysis and false positives due to the presence of noradrenergic fibers [START_REF] Sesack | Cortico-basal ganglia reward network: microcircuitry[END_REF] Here, we clearly demonstrate that 24 h following chronic administration of cocaine, the density of DA boutons is increased. One possible consequence could be an enhancement of DA transmission. The extracellular level of DA has been measured by microdialysis and voltammetry techniques in the NAc shell in a cocaine administration protocol similar to the one applied in our study. During the early phase of abstinence, the basal level of DA was found to be either unchanged (Addy et al. 2010) or decreased (Chefer and Shippenberg 2002) in sensitized animals, whereas a challenge of cocaine increased DA levels to a greater magnitude in sensitized compared to saline treated animals (Addy et al. 2010;Chefer and Shippenberg 2002). Our observation of an increase in DA terminal density seems in contradiction with the decreased or unchanged basal DA levels may seem paradoxical. One possible explanation would be that not all DA boutons are functional, as it has been shown that some boutons containing vesicle clusters do not release DA [START_REF] Pereira | Fluorescent false neurotransmitter reveals functionally silent dopamine vesicle clusters in the striatum[END_REF]. In this respect, the decrease in DA boutons volume observed in the NAc might indicate alterations in DA storage. On the other hand, the increase in DA terminal density fits with the increased DA levels upon cocaine challenge in sensitized animals. Several mechanisms could explain the changes in extracellular DA levels in the striatum of sensitized rodents, including changes of DA neuron activity, probability of DA release, regulation of DA release through DA presynaptic receptors or DAT expression level (Kuhar and Pilotte 1996;[START_REF] Robinson | Monitoring rapid chemical communication in the brain[END_REF]Kalivas and Stewart 1991). Here, we show that chronic administration of cocaine induces an increase in the number and volume of DA axonal boutons. Hence, this parameter should be taken into consideration for the global understanding of the consequences of cocaine administration on DA homeostasis in the striatum. Furthermore, it would be of interest to assess whether the changes we describe are dynamic through withdrawal period and upon cocaine challenge.

Interestingly, the increase in DA bouton density was restricted to the shell part of the NAc. Without precluding involvement of other brain structures and circuit-level mechanisms, the shell is critically involved in cocaine sensitization [START_REF] Steketee | Drug wanting: behavioral sensitization and relapse to drug-seeking behavior[END_REF], with chronic cocaine administration inducing synaptic plasticity [START_REF] Thomas | Long-term depression in the nucleus accumbens: a neural correlate of behavioral sensitization to cocaine[END_REF]) and signaling pathway activation [START_REF] Robison | Behavioral and structural responses to chronic cocaine require a feedforward loop involving DFosB and calcium/calmodulin-dependent protein kinase II in the nucleus accumbens shell[END_REF] in the shell, but not in the core of the NAc. Different DA neurons project to sub-divisions of the NAc (Lammel et al. 2014) and it has been shown that acute cocaine administration elicits increase in DA release in the shell but not in the core (Aragona et al. 2008), evokes synaptic plasticity in DA neurons projecting to the shell but not to dorsal striatum (Lammel et al. 2011), and induces increase or decrease of the bursting activity in different neurons from the ventral tegmental area (Mejias-Aponte et al. 2015). Those results provide experimental evidence that cocaine can differentially affect dopaminergic neurons projecting to different brain areas, forming a basis on which further experiments could be conducted to determine the mechanisms by which cocaine induce an increase in bouton density specifically in the shell.

The observation of DA boutons devoid of synaptic membrane specialization has led to the idea of volume transmission, which proposes that DA, following release, diffuses in the extracellular space to reach its targets (Cragg and Rice 2004;[START_REF] Robinson | Monitoring rapid chemical communication in the brain[END_REF][START_REF] Rice | Dopamine release in the basal ganglia[END_REF]. The efficacy of DA transmission would thus depend on its concentration, diffusion efficacy and on the affinity of its receptors (Cragg and Rice 2004). Because the DA D2 receptor (D2R) has a higher affinity than DA D1 receptor (D1R) [START_REF] Tritsch | Dopaminergic modulation of synaptic transmission in cortex and striatum[END_REF], it is thought that volume transmission has a smaller sphere of influence on the D1Rs [START_REF] Rice | Dopamine release in the basal ganglia[END_REF]. Our observation of a reduced distance between DA sources and their targets might indicate that following cocaine administration, the capacity of DA to also activate the D1Rs becomes facilitated.

Moss and Bolam termed the spatial distribution of DA boutons in the striatum a 'lattice', as it would correspond to a dense network of DA innervations through which volume transmission can act on all potential receptors [START_REF] Moss | A dopaminergic axon lattice in the striatum and its relationship with cortical and thalamic terminals[END_REF][START_REF] Sesack | Cortico-basal ganglia reward network: microcircuitry[END_REF]. Therefore, our results show that this lattice becomes denser after cocaine administration, which could imply an enhanced effect of DA transmission. Our previous demonstration of increased glutamatergic synapse density after repeated cocaine administrations, albeit without an increase in the density of glutamatergic boutons (Heck et al. 2015), along with the present observation of increased DA bouton density and association with dendritic spines, shows that chronic cocaine administration modifies glutamatergic and DA connectivity in the ventral striatum. Such connectivity

cFig. 1

 1 Fig. 1 Specific detection of dopaminergic boutons in the striatum. a To specifically label DA boutons in the mouse brain, the DAT::Cre mice were crossed with the tdTomato ''flex'' mice to specifically express the fluorescent protein tdTomato in DA neurons and in their striatal afferents. b Single plane from a confocal image stack showing tdTomato labeling. False-color rendering enables better visualization of the high heterogeneity in the size and fluorescence intensity of the objects. Following a custom segmentation procedure, tdTomatopositive objects are extracted. The output of the segmentation is shown on the same single plane of the confocal stack as the original image, and with 3D volume rendering. c Immunohistochemistry for TH shows co-localization with tdTomato-positive objects. d Immunohistochemistry for VMAT2 shows co-localization with tdTomatopositive objects. Scale bar in b 1 lm; c, d = 2 lm. SN substantia nigra, VTA ventral tegmental area, NAc nucleus accumbens

  and a subpopulation of THpositive striatal interneurons (Iba ´n ˜ez-Sandoval et al. 2010).

Fig. 2

 2 Fig. 2 Repeated cocaine exposure increases dopaminergic innervation in the nucleus accumbens shell. a Transgenic mice injected daily with cocaine (ip. 20 mg/kg) present locomotor sensitization, defined as a progressive increase in locomotor activity upon repeated injection of the same dose of cocaine. Two-way ANOVA, Bonferroni post hoc test: cocaine day 1 vs. day 5: p \ 0.001, saline day 1 vs. day 5: p [ 0.05; saline day 5 vs. cocaine day 5: p \ 0.001. ***p \ 0.001. b DA boutons were imaged and segmented in 3D, as illustrated by a single plane from confocal stacks imaged in the NAc shell. c The measurement of DA bouton density reveals an increase in the NAc shell but not in the other striatal regions. One-way ANOVA, Dunn's multiple comparison test, Saline vs. cocaine: NAc Shell: 152.7 ± 4.6 vs. 174.8 ± 3.9, p \ 0.05, NAc Core: 160 ± 7.6 vs. 170 ± 5.2, DM Str: 161 ± 9.2 vs. 175 ± 6.4, DL Str: 166 ± 8 vs. 170 ± 4.4. n = 6-7 mice per condition. *p \ 0.05. d Cumulative distributions of the volumes of DA boutons analyzed in the different striatal regions. Bouton size is decreased only in the ventral parts of the striatum. Kolmogorov-Smirnov test. n = 114,265-147,076 boutons from

b

  Fig. 3 Repeated cocaine exposure increases dopaminergic connectivity in the nucleus accumbens. Contacts between DA boutons (red) and the neck of dendritic spines labeled with DiO (green) were detected in the NAc shell. a Upper panel: Three-dimensional surface rendering of a confocal microscopy image stack showing the dendrite and dendritic spines and the DA boutons. Lower panel: close-ups showing single planes from the same image stack. b 3D surface rendering of contacts between DA boutons and the neck of dendritic spines. c Spine density on SPN is increased in cocaine-treated mice compared to saline condition. Spine density was measured in the NAc shell of transgenic mice. d The percentage of DA boutons in contact with the neck of a dendritic spine is increased in cocaine-treated mice compared to saline condition. t test in c and d: p \ 0.001, n = 27 and 21 dendrites with 1257 and 783 spines in saline and cocaine condition, respectively. ***p \ 0.001. Scale bar in a 1 lm

Acknowledgements We thank Francois Tronche for providing the DAT-Cre mouse line, and the Cellular Imaging facility of the IBPS (Institut de Biologie Paris-Seine) for expert assistance on microscopy and metrological control of the setup. This work was supported by Centre National pour la Recherche Scientifique (CNRS), Institut National de la Sante ´et de la Recherche Me ´dicale (INSERM), University Pierre and Marie Curie (UPMC), Agence Nationale de la Recherche (ANR), Fondation pour la Recherche Me ´dicale (FRM) and the Labex Bio-Psy cluster of excellence. M.D.S. was a recipient of a fellowship from French Ministry of Research and Labex Bio-Psy. E.N.C. was supported by the Ecole de Neuroscience de Paris (ENP) and FRM.

Compliance with ethical standards

Conflict of interest

The authors report no biomedical financial interests or potential conflicts of interest.

Addy NA, Daberkow DP, Ford JN, Garris PA, Wightman RM (2010) Sensitization of rapid dopamine signaling in the nucleus accumbens core and shell after repeated cocaine in rats. J Neurosci 35:1965J Neurosci 35: -1978


Jocelyne Caboche
email: jocelyne.caboche@upmc.fr

Marc Dos Santos

Marine Salery

Benoit Forget

Maria Alexandra

Garcia Perez

Sandrine Betuing

Thomas Boudier

Peter Vanhoutte

Nicolas Heck

Dos Santos

Rapid Synaptogenesis in the Nucleus Accumbens Is Induced by a Single Cocaine Administration and Stabilized by Mitogen-Activated Protein Kinase Interacting Kinase-1 Activity

Keywords: Cocaine, dendritic spine, synaptogenesis, MAP Kinase Interacting Kinase (MNK), striatum, Extracellular-signal Regulated Kinase (ERK)

INTRODUCTION

Cocaine administration induces long-lasting changes in behaviors that result from synaptic plasticity and morphological changes in neurons of the brain's reward circuitry [START_REF] Volkow | The brain on drugs: from reward to addiction[END_REF][START_REF] Russo | The addicted synapse: mechanisms of synaptic and structural plasticity in nucleus accumbens[END_REF]. Cocaine increases dopamine (DA) in the Nucleus Accumbens (NAc) [START_REF] Chiara | Drugs abused by humans preferentially increase synaptic dopamine concentrations in the mesolimbic system of freely moving rats[END_REF], with dendritic spines from striatal projection neurons (SPN) receiving glutamate inputs informative of contextual information, together with DA inputs that code for reward signals [START_REF] Schulz | Getting formal with dopamine and reward[END_REF][START_REF] Steinberg | A causal link between prediction errors, dopamine neurons and learning[END_REF] and modulate the efficacy of glutamatergic synapses [START_REF] Nicola | Dopaminergic modulation of neuronal excitability in the striatum and nucleus accumbens[END_REF][START_REF] Hyman | Neural mechanisms of addiction: the role of reward-related learning and memory[END_REF]. Most studies assessing structural changes in the striatum have focused on the observation of dendritic spine density changes induced by chronic cocaine in the adult striatum. However, in-depth studies of synaptogenesis and connectivity changes, which require the analysis of both pre and postsynaptic elements [START_REF] Schoonover | Comparative strength and dendritic organization of halamocortical and corticocortical synapses onto excitatory layer 4 neurons[END_REF][START_REF] Wierenga | GABAergic synapses are formed without the involvement of dendritic protrusions[END_REF], remained largely unexplored until recently. To assess this issue we have set up a method that allows for 3D automated morphological analysis of synaptic connections onto SPN [START_REF] Heck | A new automated 3D detection of synaptic contacts reveals the formation of cortico-striatal synapses upon cocaine treatment in vivo[END_REF]. Using VGLUT1 Venus mice, which express the vesicular glutamate transporter 1 (VGLUT1), a presynaptic protein present in afferents from cortices (neocortex, hippocampus and amygdala) fused to the fluorescent protein Venus [START_REF] Herzog | In vivo imaging of intersynaptic vesicle exchange using VGLUT1 Venus knock-in mice[END_REF], we showed that new spines formed after chronic exposure to cocaine were indeed connected to presynaptic cortical terminals [START_REF] Heck | A new automated 3D detection of synaptic contacts reveals the formation of cortico-striatal synapses upon cocaine treatment in vivo[END_REF]. How and when these new glutamatergic synapses are formed within the striatum still remains a key issue, which has to be addressed to disentangle the cellular bases responsible for the initiation of neuronal plasticity mediated by cocaine. It was recently shown that DA gated glutamate driven spine enlargement in the NAc in a narrow time window, thereby providing a mechanism for reward-driven learning at the level of the dendritic spine [START_REF] Yagishita | A critical time window for dopamine actions on the structural plasticity of dendritic spines[END_REF]. Furthermore, the synergistic interaction between D1R and glutamate NMDA receptor induces activation of the MAPkinase/ERK (extracellular-signal regulated kinase) pathway [START_REF] Pascoli | Cyclic adenosine monophosphate-independent tyrosine phosphorylation of NR2B mediates cocaineinduced extracellular signal-regulated kinase activation[END_REF][START_REF] Cahill | D1R/GluN1 complexes in the striatum integrate dopamine and glutamate signalling to control synaptic plasticity and cocaine-induced responses[END_REF][START_REF] Pascoli | Extracellular signalregulated protein kinases 1 and 2 activation by addictive drugs: a signal toward pathological adaptation[END_REF], which plays a key role in cocaine-induced neuronal and behavioral adaptations [START_REF] Valjent | Involvement of the extracellular signal-regulated kinase cascade for cocaine-rewarding properties[END_REF][START_REF] Valjent | Role of the ERK pathway in psychostimulant-induced locomotor sensitization[END_REF][START_REF] Miller | Molecular substrates for retrieval and reconsolidation of cocaine-associated contextual memory[END_REF][START_REF] Lu | Central amygdala ERK signaling pathway is critical to incubation of cocaine craving[END_REF]. The ERK pathway also DA receptor of type 1 (D1-SPN) [START_REF] Ren | Dopamine D1 and Nmethyl-D-aspartate receptors and extracellular signal-regulated kinase mediate neuronal morphological changes induced by repeated cocaine administration[END_REF][START_REF] Dobi | Cocaine-induced plasticity in the nucleus accumbens is cell specific and develops without prolonged withdrawal[END_REF]. Classically, these long-term adaptations are thought to rely on gene transcription and epigenetic regulation [START_REF] Robison | Transcriptional and epigenetic mechanisms of addiction[END_REF]. However, it was recently demonstrated that a single injection of cocaine was sufficient to cause an ERK-dependent synaptic plasticity and subsequent behavioral adaptations induced by a second administration of cocaine [START_REF] Pascoli | Reversal of cocaine-evoked synaptic potentiation resets drug-induced adaptive behaviour[END_REF][START_REF] Valjent | Mechanisms locomotor sensitization to drugs of abuse in a two-injection protocol[END_REF]. We therefore hypothesized that a single injection of cocaine was able to induce a rapid and stable modification of striatal connectivity. To address these issues, we investigated the changes in glutamatergic connectivity, along with the rules of synapse formation, as well as the associated molecular mechanisms. We bring evidence that an acute administration of cocaine is able to induce stable synaptic rewiring in the NAc and demonstrate that the ERK pathway plays a dual role in the growth and stabilization of new spines in D1-SPN. In vivo treatments, tissue preparation.

Intraperitoneal injection of cocaine was performed. When indicated anisomycin (100 mg/kg) or SL327 (50 mg/kg) were injected intraperitoneally 30 min and 60 min, respectively, prior to cocaine. For CGP57380 treatment, cannulae were pre-implanted in the NAc shell in anesthetized 7-8 weeks-old mice. One week later, unilateral injections of 10 mM CGP57380 were performed, along with contralateral vehicle injection, followed by cocaine administration 15 min later. Mice brains were fixed in situ after intracardiac perfusion of paraformaldehyde (PFA) and coronal sections were cut with a vibratome. Dendrites were labeled with DiI using diolistic method.

Dendritic spine analysis, segmentation of presynaptic boutons and detection of synaptic contacts from confocal images.

Dendritic spine detection was performed using the Neuronstudio software and data were imported into ImageJ for custom 3D automated spine heads segmentation. Segmentation of VGLUT1 objects and detection of synaptic contacts were performed in 3D as described previously [START_REF] Heck | A new automated 3D detection of synaptic contacts reveals the formation of cortico-striatal synapses upon cocaine treatment in vivo[END_REF]. Border to border distance between each spine head and all VGLUT1 objects was computed and the synaptic contact was decided for a distance of zero, meaning that the two objects overlap.

Viral infections and preparation of slices for two-photon microscopy.

RESULTS

A single cocaine exposure induces a rapid and stable connectivity change in the NAc shell

We investigated spine formation and potential changes of striatal connectivity induced by a single administration of cocaine. Dendritic spine density of SPNs from the NAc shell was increased one hour after a single injection of cocaine (Figure 1A-C). A similar increase was observed at a lower dose (10 mg/kg, Supplemental Figure S1A), while no change was observed either in the NAc core, or in the dorsal striatum at higher doses (Supplemental Figure S1B,C). Upon cocaine administration, activated D1-SPNs express immediate early genes, including c-Fos [START_REF] Bertran-Gonzalez | Opposing patterns of signaling activation in dopamine D1 and D2 receptorexpressing striatal neurons in response to cocaine and haloperidol[END_REF]. Spine density increase in SPNs expressing c-Fos was greater when compared to randomly picked SPN (Figure 1A-C), thus indicating that cocaine-induced spine formation occurred in activated SPNs. VGLUT1-positive boutons from glutamatergic axons were segmented, and synaptic contacts were identified by measurement of the 3D contacts between spine heads and boutons. About 60% of spines were connected to VGLUT1positive boutons in saline conditions (Figure 1A,E), a result in agreement with electron microscopic studies [START_REF] Doig | Cortical and thalamic innervation of direct and indirect pathway medium-sized spiny neurons in mouse striatum[END_REF]. One hour after cocaine administration the number of spines in contact with a VGLUT1 bouton was increased (Figure 1A, D), indicating that acute cocaine rapidly increases glutamatergic connectivity in the NAc. Of note, the percentage of spines connected to VGLUT1 boutons remained unchanged, indicating that the increased connectivity did not modify the stoichiometry of VGLUT1 connections onto SPN (Figure 1E). We next assessed whether this rapid increase in connectivity was stable over time. One week after a single injection of cocaine, the increase in spine density and synaptic contacts were still present, while the percentage of VGLUT1-positive synapses remained stable (Figure 1F-I). An increased spine density was also found 28 days after a single administration of cocaine (Supplemental Figure S1D). Following a custom 3D procedure for spine heads segmentation (Figure 2A), the measurements of spine head volume and spine length showed an increase one hour after cocaine administration, which was better revealed in c-Fos positive neurons (Figures 2B,C). One week after cocaine administration, spine head volume, but not spine length, was still larger in cocaine-treated mice as compared to saline controls (Figures 2D,E). The increased volume of spine heads corresponded to an increase in mushroomshaped spine density that was significantly increased one hour and one week after a single cocaine administration (Figures 2F,G), indicating a rapid maturation of the newly-generated spines.

Synapse formation occurs in clusters with spines connecting pre-existing presynaptic boutons

The total density of VGLUT1-positive boutons in the NAc-shell was not increased either one hour or one week after cocaine administration (Figures 3A,B, left panels). Since synaptogenesis occurred, this indicates that new spines contact pre-existing presynaptic boutons. Accordingly, the occurrence of spines connected to a common VGLUT1-positive bouton -an observation reported in electron microscopy study [START_REF] Doig | Cortical and thalamic innervation of direct and indirect pathway medium-sized spiny neurons in mouse striatum[END_REF]-was increased one hour and one week after cocaine injection (Figure 3A, B, right panels). The rules of synapse formation were further studied using live imaging in acute adult striatal slices. In order to recapitulate the effects of cocaine on SPN, we applied a co-stimulation with a low dose of glutamate together with a D1R agonist, as previously shown [START_REF] Pascoli | Cyclic adenosine monophosphate-independent tyrosine phosphorylation of NR2B mediates cocaineinduced extracellular signal-regulated kinase activation[END_REF][START_REF] Cahill | D1R/GluN1 complexes in the striatum integrate dopamine and glutamate signalling to control synaptic plasticity and cocaine-induced responses[END_REF]. As expected, the costimulation, but not the application of each agonist independently, induced ERK activation and increased spine density and synaptic contacts in the NAc-shell (Supplemental Figure S2). Time-lapse experiments were performed on D1-SPN since cocaine induces ERK activation [START_REF] Bertran-Gonzalez | Opposing patterns of signaling activation in dopamine D1 and D2 receptorexpressing striatal neurons in response to cocaine and haloperidol[END_REF] and dendritic spine increase [START_REF] Dobi | Cocaine-induced plasticity in the nucleus accumbens is cell specific and develops without prolonged withdrawal[END_REF][START_REF] Kim | Cell type-specific alterations in the nucleus accumbens by repeated exposures to cocaine[END_REF] in this subpopulation. To this end, D1-SPN were sparsely labeled by injecting in the NAc from adult mice a combination of an adenoassociated virus (AAV) carrying tdTomato sequence flanked by loxP sites and a virus expressing Cre recombinase under the promoter of preprotachykinin A (PPTA), which is specifically expressed in D1-SPN [START_REF] Yagishita | A critical time window for dopamine actions on the structural plasticity of dendritic spines[END_REF], as confirmed by immunodetection of the D1R Dendritic spine growth occurred as soon as 25 minutes after the beginning of the costimulation, and reached a plateau at 60 min (Figure 3C,D) and was absent when the selective D1R antagonist SCH23390 was applied (Supplemental Figure S3C). The amplitude of spine density increase at one hour was +21% (Fig. 3E).

A single cocaine administration occludes synaptic potentiation induced in striatal slices [START_REF] Pascoli | Reversal of cocaine-evoked synaptic potentiation resets drug-induced adaptive behaviour[END_REF].

We next tested whether previous cocaine administration could alleviate the spine growth induced by the co-stimulation in striatal slices. The spine density increase induced by cocaine in vivo was preserved in the slices before co-stimulation (Supplemental Figures S4A,B). A partial occlusion was observed in slices from mice acutely administrated with cocaine, since co-stimulation induced spine growth but the percentage of increase was not significant. On the other hand, chronic administration of cocaine led to complete occlusion (Supplemental Figures S4C-D). Those results are consistent with observation in the prefrontal cortex in vivo of progressive decline of spine density increase upon repeated injection of cocaine [START_REF] Muñoz-Cuevas | Cocaine-induced structural plasticity in frontal cortex correlates with conditioned place preference[END_REF].

The connectivity and functionality of spines observed in time-lapse were then explored. In acute slices from VGLUT1 venus mice 62.8% of spines induced by the co-stimulation were in contact with a VGLUT1-positive bouton (5 mice, n = 35 spines). Newly-generated spines entered in contact with a VGLUT1-positive bouton that was in all cases present before spine growth (5 mice, n = 22 spines) (Figure 3F), which is consistent with our in vivo observation of increased density of synaptic contacts along with constant density of VGLUT1 boutons upon acute cocaine. In order to assess the functionality of the newly formed spines, the viruses AAV-PPTA::Cre, AAV-FLEX-tdTomato and AAV-FLEX-GCaMP6S were injected in the NAc. Spine growth and calcium events were analyzed in D1-SPN from striatal slices (Fig. responded by an increase in calcium content. These functional spines had a bigger head diameter than the newly formed spines that did not respond to the stimulation (Figure 3G). This kinetics of maturation is in accordance with previous studies from other neuronal types [START_REF] Hill | LTP-induced long-term stabilization of individual nascent dendritic spines[END_REF][START_REF] Nägerl | Protracted synaptogenesis after activity-dependent spinogenesis in hippocampal neurons[END_REF][START_REF] Zito | Rapid functional maturation of nascent dendritic spines[END_REF][START_REF] Kwon | Glutamate induces de novo growth of functional spines in developing cortex[END_REF]. The fact that new spines contact axonal boutons already in place raised the question as to whether synaptogenesis could be driven by a stochastic spine growth. We generated an in silico simulation of synaptogenesis from random positions on the dendrite, and found that it did not reproduce the experimental data (Supplemental Figures S5A,B). Moreover, the analysis of spine distribution in vivo revealed a bimodal distribution with a high proportion of adjacent spine heads, which was increased after cocaine exposure (Supplemental Figures S5C,D). Hence, spine growth does not occur randomly but at locations where synapse clusters are generated. Taken together, our results indicate that new spines grow in clusters, are rapidly functional and contact pre-existing VGLUT1 boutons.

3G, left panel). Following co-stimulation, 6 out of 10 newly formed spines in the NAc

A single administration of cocaine induces the formation of thalamo-striatal synaptic contacts

Since a single administration of cocaine induced an increase in VGLUT1-positive synaptic contacts while keeping the ratio of spines connected to VGLUT1 afferents (see Figure 1), it was plausible that the unconnected spines would correspond to thalamo-striatal synapses, which are VGLUT2-positive [START_REF] Fremeau | VGLUTs define subsets of excitatory neurons and suggest novel roles for glutamate[END_REF]. D1-SPN were labeled by in vivo viral infections (AAV-PPTA::Cre and AAV-FLEX-tdTomato, and the mice were treated with a single administration of cocaine. VGLUT2-positive boutons were immunolabeled and thalamo-striatal synaptic contacts were analysed (Figure 4). The density of VGLUT2-connected spines significantly increased upon cocaine administration, thus indicating that acute cocaine also induces the formation of thalamo-striatal synapses.

Dendritic spine growth and stabilization are regulated by ERK and MNK-1

M A N U S C R I P T A C C E P T E D ACCEPTED MANUSCRIPT Dos Santos et al. 12
We next investigated the signaling pathways involved in spine formation induced in striatal slices one hour after the co-stimulation. A pretreatment with U0126, a selective inhibitor of the ERK pathway, abolished the spine density increase induced by the co-stimulation (Figure 5A), revealing that the ERK pathway is necessary for spinogenesis. Similarly, rapamycin an inhibitor of the mammalian target of rapamycin (mTOR) pathway completely blocked the increase in spine density induced by co-stimulation in slices (Figure 5B). Since the ERK and mTOR pathways converge on the control of protein synthesis via the eiF complex, we investigated the possible implication of MNK-1, a cytoplasmic substrate of ERK, which activates the eIF complex to initiate protein translation (34)(Figure 5C). An increase in phosphorylated MNK-1 (pMNK-1) positive cells was measured by immunodetection in the NAc twenty minutes after co-stimulation (Figure 5D). Slices were then pre-treated with CGP57380, a specific inhibitor of MNK-1 activation, which totally inhibited the increase in spine density induced by the co-stimulation (Figure 5E).

In order to gain further insights into the specific role of ERK and MNK-1 in spine growth and maintenance, we used time-lapse imaging from striatal slices (Figure 6A,B). D1-SPN dendrites were labeled in vivo after intra-NAc infection with AAV PPTA::Cre and AAV-FLEX-tdTomato. Spine growth was absent in the presence of U0126, the inhibitor of the ERK pathway (Figure 6C), indicating that ERK activation is mandatory for the formation of new spines. In contrast, CGP57380 did not modify growth rate, clearly indicating that MNK-1 is not required for spine growth (Figure 6B,C). However, it strongly impacted the maintenance of the newly formed spines, with a loss of 59.1% and 91% of newly-formed spines, 30 minutes and 90 minutes after growth, respectively (Figure 6B,D). At this later time point, 67% of newly-formed spines remained stable in control conditions, as previously reported [START_REF] Kim | Cell type-specific alterations in the nucleus accumbens by repeated exposures to cocaine[END_REF](Figure 6B,D). Noteworthy, the inhibition of MNK-1 activation did not affect the stability of spines present before stimulation (Supplemental Figure S6). Hence these data show that MNK-1 is mandatory for stabilization but not for spine growth. Since MNK-1 regulates protein synthesis, we interrogated the respective role of global transcription and translation for spine growth and maintenance. Inhibition of protein synthesis with anisomycin did not modify spine growth, but reduced spine stabilization, with identical kinetics and amplitude to that observed with the MNK-1 inhibitor (Figure 6B,D). By contrast, bath application of the transcription inhibitor actinomycin D did not alter either growth or stabilization of new spines (Figure 6B,D). Therefore, time-lapse analysis unraveled sequential molecular events involved in spine growth and stabilization. While ERK is essential for both steps of spine formation, MNK-1 activation and protein synthesis selectively control spine stabilization independently of transcription.

MNK-1 is involved in structural plasticity in vivo

We analyzed the impact of MNK-1 activation on long-lasting spinogenesis in vivo. An

DISCUSSION

Our data show that a single administration of cocaine is sufficient to trigger long-lasting increase in glutamatergic connectivity in the NAc, with new spines growing in clusters and contacting pre-existing presynaptic boutons. We unravel the sequence of signaling events specifically involved in spine growth and stabilization. Spine growth was transcription and translation-independent, thus supporting a key role of ERK-induced phosphorylation of preexisting proteins in this process. By contrast, spine stabilization required an ERK-mediated proteins synthesis via MNK-1, independently of transcription. Finally, we identify MNK-1 as essential for long-term connectivity changes induced by cocaine (Figure 8).

It has been long thought that persistent structural plasticity/morphology changes were selectively engaged following repeated administration of cocaine [START_REF] Heck | A new automated 3D detection of synaptic contacts reveals the formation of cortico-striatal synapses upon cocaine treatment in vivo[END_REF][START_REF] Dobi | Cocaine-induced plasticity in the nucleus accumbens is cell specific and develops without prolonged withdrawal[END_REF][START_REF] Muñoz-Cuevas | Cocaine-induced structural plasticity in frontal cortex correlates with conditioned place preference[END_REF][START_REF] Shen | Altered dendritic spine plasticity in cocaine-withdrawn rats[END_REF][START_REF] Dumitriu | Subregional, dendritic compartment, and spine subtype specificity in cocaine regulation of dendritic spines in the nucleus accumbens[END_REF]. Here we provide evidence that a single administration is sufficient to induce a rapid and persistent change in glutamatergic connectivity in the NAc. The rapid formation of mushroom-shaped spines with increased spine head volumes in vivo, and our calcium imaging in adult slices, both showed a rapid functional maturation of nascent spine. These observations fit with electrophysiological recordings performed after acute exposure to cocaine, showing an increase in the frequency of miniature currents in c-Fos positive SPN [START_REF] Koya | Silent synapses in selectively activated nucleus accumbens neurons following cocaine sensitization[END_REF], and synaptic potentiation [START_REF] Pascoli | Reversal of cocaine-evoked synaptic potentiation resets drug-induced adaptive behaviour[END_REF]. Interestingly, repeated cocaine regimen leads to an increased spine density along with unchanged [START_REF] Heck | A new automated 3D detection of synaptic contacts reveals the formation of cortico-striatal synapses upon cocaine treatment in vivo[END_REF][START_REF] Dobi | Cocaine-induced plasticity in the nucleus accumbens is cell specific and develops without prolonged withdrawal[END_REF] or decreased spine head size [START_REF] Dumitriu | Subregional, dendritic compartment, and spine subtype specificity in cocaine regulation of dendritic spines in the nucleus accumbens[END_REF][START_REF] Cahill | Structural plasticity after chronic cocaine administration occurs through Rap1 Small GTPase signaling[END_REF], and the presence of immature spines is coherent with the observation of LTD [START_REF] Kourrich | Cocaine experience controls bidirectional synaptic plasticity in the nucleus accumbens[END_REF] and generation of silent synapses [START_REF] Huang | In vivo cocaine experience generates silent synapses[END_REF][START_REF] Graziane | Opposing mechanisms mediate morphine-and cocaine-induced generation of silent synapses[END_REF], which are not induced by acute cocaine injection [START_REF] Koya | Silent synapses in selectively activated nucleus accumbens neurons following cocaine sensitization[END_REF]. The shift from potentiated mature spines to silent immature spines is an active process, since chronic regimen recruits specific molecular pathways involved in dendritic spine remodeling, such as deltaFosB accumulation [START_REF] Maze | Essential role of the histone methyltransferase G9a in cocaine-induced plasticity[END_REF] and inactivation of Rac1 [START_REF] Dietz | Rac1 is essential in cocaine-induced structural plasticity of nucleus accumbens neurons[END_REF] single cocaine injection. In this context, it is noteworthy that dendritic spine maturation and unsilencing of synapses are observed after a 3 weeks period of cocaine withdrawal [START_REF] Cahill | Structural plasticity after chronic cocaine administration occurs through Rap1 Small GTPase signaling[END_REF][START_REF] Lee | Maturation of silent synapses in amygdala-accumbens projection contributes to incubation of cocaine craving[END_REF] and following a challenge injection [START_REF] Shen | Altered dendritic spine plasticity in cocaine-withdrawn rats[END_REF][START_REF] Kourrich | Cocaine experience controls bidirectional synaptic plasticity in the nucleus accumbens[END_REF]. Hence, the morphology of spines is dynamically regulated during the different stages of cocaine administration and addiction development [START_REF] Dong | The neural rejuvenation hypothesis of cocaine addiction[END_REF], through the combined involvement of metaplasticity and homeostatic processes [START_REF] Dong | Cocaine-induced metaplasticity in the nucleus accumbens: silent synapse and beyond[END_REF][START_REF] Kourrich | Intrinsic plasticity: an emerging player in addiction[END_REF].

Whether VGLUT1 afferences arising from hippocampus, cortex or amygdala are similarly engaged onto new synapses remain to be established. On the other hand, we reveal that thalamic, VGLUT2-positive, synaptic contacts are generated after a single administration of cocaine. Along with previous work showing a plasticity of thalamo-striatal synapses upon chronic cocaine treatments [START_REF] Joffe | Cocaine Experience Enhances Thalamo-Accumbens N-Methyl-D-Aspartate Receptor Function[END_REF][START_REF] Neumann | Cocaine-Induced Synaptic Alterations in Thalamus to Nucleus Accumbens Projection[END_REF], our results pave the way for future study on the role of thalamic inputs on D1-SPN in long term adaptation induced by cocaine. In line with our results, it was previously observed that acute cocaine induces an increase in spine density in dopaminergic neurons [START_REF] Sarti | Acute cocaine exposure alters spine density and long-term potentiation in the ventral tegmental area[END_REF] and in the prefrontal cortex [START_REF] Muñoz-Cuevas | Cocaine-induced structural plasticity in frontal cortex correlates with conditioned place preference[END_REF], although those studies did not assess the connectivity of dendritic spines. Our results bring evidence that acute cocaine is a first essential step that primes the meso-cortico-limbic connectivity, which implies that subsequent cocaine action will operate on a modified network.

Our results also unravel the rules of synapse formation that leads to this connectivity change. Previous studies showed that new spines could make contact with pre-existing boutons [START_REF] Nägerl | Protracted synaptogenesis after activity-dependent spinogenesis in hippocampal neurons[END_REF][START_REF] Holtmaat | Experience-dependent structural synaptic plasticity in the mammalian brain[END_REF][START_REF] Knott | Spine growth precedes synapse formation in the adult neocortex in vivo[END_REF][START_REF] Medvedev | Multiple spine boutons are formed after long-lasting LTP in the awake rat[END_REF][START_REF] Yang | Selective synaptic remodeling of amygdalocortical connections associated with fear memory[END_REF] and our in vivo as well as time-lapse observations in slices extend this mode of formation to striatal neurons (Figure 8). This mode of formation might imply a constraint on the location of spine growth. Accordingly, our modeling of synaptogenesis revealed the occurrence of clustered spines along the dendrite, indicating that the position of spine growth along the dendrite is actively regulated. The formation of dendritic spines in clusters has been studied in other brain structures [START_REF] Roo | LTP promotes a selective long-term stabilization and clustering of dendritic spines[END_REF][START_REF] Chen | Clustered dynamics of inhibitory synapses and dendritic spines in the adult neocortex[END_REF][START_REF] Fu | Repetitive motor learning induces coordinated formation of clustered dendritic spines in vivo[END_REF] where they have implication on the integrative properties of the dendrite [START_REF] Lu | Clustered structural and functional plasticity of dendritic spines[END_REF]. Our study reinforces these observations, and further show that newly-formed spines rapidly form synapses with pre-existing glutamatergic boutons. Moreover, we observed in vivo spine clusters in contact with a single bouton, which implies a temporal and spatial synchrony of the glutamatergic inputs on these spines. In conclusion, our observations of both pre and postsynaptic elements combined with a model of spinogenesis, which includes the constraint of synaptic contact formation, allows a better understanding of the rules of connectivity changes in the adult striatum.

It has been shown that dopamine gates glutamate-mediated spine enlargement in SPN via D1R [START_REF] Yagishita | A critical time window for dopamine actions on the structural plasticity of dendritic spines[END_REF]. Along those lines, we have previously demonstrated that cocaine effects result from D1R gated NMDAR-mediated ERK activation [START_REF] Pascoli | Cyclic adenosine monophosphate-independent tyrosine phosphorylation of NR2B mediates cocaineinduced extracellular signal-regulated kinase activation[END_REF][START_REF] Cahill | D1R/GluN1 complexes in the striatum integrate dopamine and glutamate signalling to control synaptic plasticity and cocaine-induced responses[END_REF][START_REF] Pascoli | Extracellular signalregulated protein kinases 1 and 2 activation by addictive drugs: a signal toward pathological adaptation[END_REF]. Here we observed that both a D1R agonist and Glu are required for ERK-dependent synaptogenesis in SPN downstream from D1R. Accordingly, it was recently shown that a single administration of cocaine can produce an ERK-dependent occlusion of synaptic potentiation on SPN of the NAc, and optogenetic inhibition of cocaine-induced plasticity prevented the behavioral adaptations induced by cocaine [START_REF] Pascoli | Reversal of cocaine-evoked synaptic potentiation resets drug-induced adaptive behaviour[END_REF]. Altogether, these data support that ERK-induced rapid synaptogenesis might be a critical cellular mechanism involved in the induction of long-term striatal plasticity and in the development of behavioral alterations induced by cocaine. We further demonstrate that synaptogenesis can be divided in different phases, with at least two distinct molecular mechanisms downstream the ERK pathway (Figure 8). We reveal a transcription and translation-independent role of ERK in the initiation of spine formation.

While a previous study has shown that ERK activity is essential for spine morphogenesis in developing cortical neurons [START_REF] Kwon | Glutamate induces de novo growth of functional spines in developing cortex[END_REF], we make the first demonstration of a functional role of cocaine-induced ERK activation independently of nuclear targets. It may likely imply the phosphorylation of pre-existing proteins, such as spinophilin, which upon phosphorylation by ERK, can increase spine protrusion density in hippocampal neurons [START_REF] Futter | Phosphorylation of spinophilin by ERK and cyclin-dependent PK 5 (Cdk5)[END_REF].

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Dos Santos et al. [START_REF] Miller | Molecular substrates for retrieval and reconsolidation of cocaine-associated contextual memory[END_REF] We further show that spine stabilization requires MNK-1 activation and local protein synthesis independently of transcription. It is now clearly established that, downstream from ERK, MNK-1 phosphorylates initiation factors essential for the onset of local translation required for long-term plasticity [START_REF] Kelleher Rj 3rd | Translational control by MAPK signaling in long-term synaptic plasticity and memory[END_REF][START_REF] Costa-Mattioli | Translational control of long-lasting synaptic plasticity and memory[END_REF]. Moreover, cocaine administration can induce synaptic plasticity via a mechanism that involves translation independently of transcription [START_REF] Scheyer | A protein synthesis-dependent mechanism sustains calcium-permeable AMPA receptor transmission in nucleus accumbens synapses during withdrawal from cocaine self-administration[END_REF]. Of interest, local synaptic translation also involves the mTOR signaling pathway, which is activated downstream Rap1b, a small GTPase essential for spine formation upon chronic cocaine administration [START_REF] Cahill | Structural plasticity after chronic cocaine administration occurs through Rap1 Small GTPase signaling[END_REF]. Several messengers are translated in dendrites ( 63

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  regulates spine formation induced by chronic cocaine administration onto SPN expressing the M A N U S C R I P T A C C E P T E D ACCEPTED MANUSCRIPT Dos Santos et al.

  Animal care of wild-type C57BL/6 and VGLUT1-venus knock-in was conducted in accordance with standard ethical guidelines (NIH publication no. 85-23, revised 1985 and European Committee Guidelines on the Care and Use of Laboratory Animals 86/609/EEC), and the experiments were approved by the local ethic committee.

  PPTA-Cre viral construct contains an expression cassette consisting of the Cre recombinase driven by the promoter of PPTA (precursor of substance P) gene, which is specifically expressed in SPN expressing D1R (12). The virus was injected in the NAc of 4week-old mice in combination with the AAVrh9-pCAG-flex-tdTomato-WRPE virus and when stated AAV1-hSyn-flex-GCaMP6S-WPRE virus. Coronal slices of 200-250 µm thickness of brains from 6 to 8 week-old mice were cut on a vibratome and maintained in equilibrated ACSF. Slices were stimulated with glutamate 3 µM and (R)-(+)-SKF-38393 10 µM in ACSF for 20 min, followed by 40 min in ACSF alone. Pharmacological inhibitors were applied 30 min before co-stimulation and washed 30 min after the end of the co-stimulation. Slices were either imaged in time-lapse or fixed with PFA 4% at RT for 60 min, processed for DiI dendrite labelling or immunofluorescence. For time lapse studies, slice imaging was performed in ACSF with two-photon upright microscope (SP5 MPII, Leica) equipped with water-immersion objective (25x, NA 0.95, Leica) and images were acquired with hybrid detector (Leica). Image stacks were sampled with voxels size of 100 nm in xy axis and z-step of 500 nm. See Supplemental information for fully detailed experimental procedures M A N U S C R I P T A C C E P T E D ACCEPTED MANUSCRIPT Dos Santos et al. 8

10 (

 10 Supplemental FigureS3A, B). Striatal slices were performed two weeks after viralinfections, and time-lapse imaging of D1-SPN was performed by two-photon microscopy.

  M A N U S C R I P T A C C E P T E D ACCEPTED MANUSCRIPT Dos Santos et al. 13

  increase in pMNK-1-positive cells was observed in the NAc 10 minutes after a single cocaine administration, and the inhibition of ERK activation by SL327 completely abolished this increase (Figure 7A, B), thus confirming that cocaine-induced MNK-1 phosphorylation is strictly dependent on ERK activation. The MNK-1 phosphorylation state was transient, with return to basal levels 30 minutes after cocaine administration (Figure 7B). The dendritic localization of pMNK-1 in D1-SPN was shown by immunodetection of pMNK-1 in dendrites labeled with the PPTA::Cre and FLEX-tdTomato AAV. pMNK-1 positive punctates were detected in the dendritic shaft and in dendritic spines from D1-SPN (Figure 7C), further demonstrating the local activation of MNK-1. In agreement with the results from acute slices, inhibition of ERK or protein synthesis abolished spine formation one hour after cocaine administration in vivo (Figure 7D). The activation of MNK-1 was inhibited locally by intracranial injection of CGP57380 in the shell part of the NAc prior to either saline or M A N U S C R I P T A C C E P T E D ACCEPTED MANUSCRIPT Dos Santos et al. 14 cocaine single administration. The local inhibition of MNK-1 did not affect basal spine density of SPN, but abolished the spine density increase observed 1 and 24 hours after cocaine administration (Figure 7E). Taken together, these results identify MNK-1 as a cytoplasmic target downstream from ERK activated by cocaine and posit MNK-1 and protein synthesis as essential for striatal structural plasticity in vivo. M A N U S C R I P T A C C E P T E D ACCEPTED MANUSCRIPT Dos Santos et al.

  , which are not observed after M A N U S C R I P T A C C E P T E D ACCEPTED MANUSCRIPT Dos Santos et al. 16

  ) and could be involved in the stability of newly formed spines. The local translation of Arc is controlled by MNK-1 (64) and Arc proteins regulate spine density and morphology (65). Furthermore, the expression of Arc is induced in dendrites of D1R-SPN upon acute cocaine administration (66). PSD-95 is another candidate locally translated in response to intra-NAc injection of a D1R agonist (67), and involved in spine stabilization (68, 69). Importantly, we show that cocaine induces a rapid ERK-mediated phosphorylation of MNK-1 in dendrites of SPNs in the NAc, and its pharmacological inhibition leads to a loss of spine density increase observed one hour and 24 hours after a single administration of cocaine. Hence stabilization of newlyformed spines via MNK-1 is essential for long-term changes in glutamatergic striatal connectivity in the NAc. M, Kenney JW, Johnston HE, Manousopoulou A, Garbis SD, Proud CG (2015): BDNF stimulation of protein synthesis in cortical neurons requires the MAP kinaseinteracting kinase MNK1. J. Neurosci. 35: 972-984. 64. Panja D, Kenney JW, D'Andrea L, Zalfa F, Vedeler A, Wibrand K, et al. (2014): Twostage translational control of dentate gyrus LTP consolidation is mediated by sustained BDNF-TrkB signaling to MNK. Cell Rep. 9: 1430-1445. 65. Peebles CL, Yoo J, Thwin MT, Palop JJ, Noebels JL, Finkbeiner S (2010): Arc regulates spine morphology and maintains network stability in vivo. Proc. Natl. Acad. Sci. U.S.A. 107: 18173-18178. 66. Salery M, Dos Santos M, Saint-Jour E, Moumné L, Pagès C, Kappès V, et al. (2016): Activity-regulated cytoskeleton-associated protein accumulates in the nucleus in response to cocaine and acts as a brake on chromatin remodeling and long-term behavioral alterations. Biol. Psychiatry doi:10.1016/j.biopsych.2016.05.025. 67. Beckley JT, Laguesse S, Phamluong K, Morisot N, Wegner SA, Ron D (2016): The first alcohol drink triggers mTORC1-dependent synaptic plasticity in nucleus accumbens dopamine D1 receptor neurons. J. Neurosci. 36: 701-713. 68. Cane M, Maco B, Knott G, Holtmaat A (2014): The relationship between PSD-95 clustering and spine stability in vivo. J. Neurosci. 34: 2075-2086. D, Bonhoeffer T, Scheuss V (2014): Balance and stability of synaptic structures during synaptic plasticity. Neuron 82: 430-443.

Figure 1 .

 1 Figure 1. A single administration of cocaine induces the rapid formation of stable

Figure 2 .

 2 Figure 2. Effect of a single cocaine exposure on spine morphology of SPN from NAc

  spines analyzed in n = 29 to 59 neurons from N = 4 mice in each condition. B-E: Kolmogorov-Smirnov test of distribution, F: One-way ANOVA and Bonferroni post-test, G: T-test. *: p < 0.05; **: p < 0.01, ***: p < 0.001.

Figure 3 .

 3 Figure 3. Synapse formation through contact with pre-existing presynaptic boutons (A-B) Density of VGLUT1 Venus boutons in the NAc, in a volume corresponding to a cube of

  Time-lapse analysis of synaptogenesis shows that new spines contact pre-existing VGLUT1-positive boutons. Single plane from 3D two-photon microscopy images. (G) left panel : Nascent spines in D1-SPN are identified with tdTomato labeling and their synaptic activity is visualized using GCaMP6S calcium sensor during co-stimulation. To avoid fluorescence fluctuations independent of calcium variations, GCaMP6S signal is normalized using tdTomato signal. Nascent spines are considered as active if they present GCaMP6 fluorescent peaks (∆F/F) exceeding 0.3, compared to baseline. Maximum projections show active (green arrow) and silent (red arrow) nascent spines. The spine head diameter was measured from non-active and active spines (Fig. 3G, right panel). Data show values and mean ± SEM, n = 10 spines from 3 independent neurons and N = 3 mice, T-test. *: p < 0.05, **: p < 0.01, ***: p < 0.001. See supplemental figures S3 and S4. Scale bar in A, B = 5 microns, C = 4 microns, F and G = 1 micron.

Figure 4 .

 4 Figure 4. A single administration of cocaine induces the rapid formation of thalamo-

  2 microns. Histograms represent mean ± SEM with n=number of neurons analyzed from N = 4 mice in each condition. B-D: T-test. **: p < 0.01, ***: p < 0.001.

Figure 5 .

 5 Figure 5. Molecular mechanisms involved in spine density increase

Figure 6 .

 6 Figure 6. Time lapse imaging and molecular dissociation of spine growth and

ACKNOWLEDGEMENTS AND DISCLOSURES

We would like to thank Jean-Antoine Girault (INSERM, Paris, France) for critical reading of the manuscript. We wish to thank Dr. Haruo Kasai (Tokyo University, Japan) for providing the plasmid coding PPTA-Cre and Alexis Bemelmans (Molecular Imaging Research Center, CEA, France) for producing the AAV virus. The VGLUT2 antibody was a kind gift from Dr.

Salah El Mestikawy (NPS, Paris, France). The tdTomato expressing virus was made available by Dr. Hongkui Zeng, of the Allen Institute for Brain Science. We wish to thank the Cellular Imaging facility of the IBPS (Institut de Biologie Paris-Seine) for expert assistance on