Introduction article 2

La première partie des résultat obtenus au cours de ma thèse (article 1) a permis de montrer que les injections répétées de cocaïne chez la souris VGLUT1^venus induisait une augmentation de la densité en synapses VGLUT1-positives dans le NAc associée à la sensibilisation locomotrice. D’après les résultats de Ren et al. (Ren et al., 2010) cette spinogénèse est dépendante de la voie des ERK et est bloquée par les inhibiteurs des récepteurs D1 ou NMDA. Ces mécanismes cellulaires et moléculaires avaient été montrés dans la littérature et au sein du laboratoire comme essentiels aux adaptations comportementales et neuronales à la cocaïne (Cahill et al., 2014a; Girault et al., 2007; Pascoli et al., 2014b).

Au début de mon M2 est parue une élégante étude de Pascoli et al. montrant qu’une seule injection de cocaïne induisait une LTP des SPN cruciale dans le développement de la sensibilisation locomotrice en deux injections, elle-aussi dépendante de ERK. L’équipe d’accueil avait observé, peu de temps avant mon arrivée, qu’une seule injection de cocaïne induisait une pousse d’épines dans les SPN du NAc en seulement une heure.

Deux questions importantes se sont alors posées : 1) ces épines néoformées en réponse à une injection unique de cocaïne forment-elles des connexions avec des boutons pré- synaptiques ? 2) ces épines sont-elles stables dans le temps ?

Enfin, nous nous sommes intéressés aux mécanismes moléculaires qui présidaient la formation rapide d’épines au sein des SPN du NAc. La pharmaco-dynamique de la pousse et du maintien des épines sur les D1-SPN a été étudiée sur des tranches de striatum stimulées par des traitements mimant, ex vivo, les effets de la cocaïne in vivo. Nous avons attaché une attention particulière aux voies de signalisation ERK et mTOR qui régulent la formation des épines en réponse à des traitements chroniques à la cocaïne (Cahill et al., 2016; Ren et al., 2010), ainsi qu’aux mécanismes cellulaires dépendants de la transcription et de la traduction protéique. La spinogénèse a été étudiée en microscopie biphotonique afin d’élucider en temps réel les signatures moléculaires impliquées dans les phases rapides de formation et de maintien des épines. Les modes de régulations de la spinogénèse, établis sur ce modèle, ont été vérifiés in vivo après administration aiguë de cocaïne chez la souris. Ils nous ont permis de

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mettre en évidence, de façon originale, le rôle essentiel de MNK-1, une cible cytoplasmique de ERK impliquée dans la synthèse protéique et la formation d’épines, indépendamment de la transcription.

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Extracellular Signal-regulated Kinase exerts a dual role on dendritic spine growth and stabilization of glutamatergic

clustered-synaptogenesis induced by cocaine

Marc Dos Santos

, Marine Salery

, Benoit Forget

, Maria Alexandra Garcia Perez

, Thomas Boudier

, Peter Vanhoutte

, Nicolas Heck

*

& Jocelyne Caboche

*

1Sorbonne Universités, UPMC Univ Paris 06, INSERM, CNRS, Neurosciences Paris Seine - Institut de Biologie Paris Seine (NPS - IBPS), 75005 Paris, France

2Sorbonne Universités, UPMC Univ Paris 06, IPAL, CNRS, 75005 Paris France

3Bioinformatics Institute, Agency for science, technology and research (A*STAR), Singapore

* Those authors contributed equally

+ Corresponding author: nicolas.heck@upmc.fr; phone : +33 1 44 27 53 26; fax : +33 44 27 25 08.

# Lead contact: jocelyne.caboche@upmc.fr; phone : +33 1 44 27 53 52; fax : +33 44 27 25 08

Key words: Cocaine, dendritic spine, synaptogenesis, MAP Kinase Interacting Kinase (MNK), striatum, Extracellular-signal Regulated Kinase (ERK), addiction.

113 Summary

Brief experience-driven events induce long-term changes at the level of neuronal networks. A single administration of cocaine induces reward-driven learning and leads to stable behavioral adaptations. Using a new method for 3D morphometric analysis of both presynaptic boutons and dendritic spines, we show that acute cocaine induces rapid synaptogenesis and persistent increase in striatal connectivity. We observe that new spines grow in clusters and form synapses by contacting preexisting glutamatergic boutons. Using two-photon imaging on slices, we further unravel that spine growth and stabilization are driven by different molecular mechanisms downstream the Extracellular signal-Regulated Kinase pathway (ERK). Spine growth is dissociated from stabilization, which is regulated by transcription-independent protein synthesis regulated by MAP Kinase Interacting Kinase-1 (MNK-1). Finally, we show that MNK-1 activity induced after a single exposure of cocaine, is implicated in long-term increase of dendritic spine density in vivo.

114 Introduction

In the adult brain, the neuronal network offers adaptive properties, and experience-induced synaptic plasticity occurs at both functional and structural levels, with persistent reorganization of synaptic connections and modulation of synaptic strength (Holtmaat and Svoboda, 2009; Hübener and Bonhoeffer, 2010). It is now admitted that structural plasticity is implicated in long-term adaptations that underlie psychiatric disorders, including drug addiction (Bernardinelli et al., 2014; Russo et al., 2010). Addiction induces long-lasting changes in behaviors that rely on neuronal plasticity and morphological changes in neurons of the brain’s reward circuitry, more specifically in the ventral part of the striatum (nucleus accumbens, NAc) (Hyman et al., 2006). Within the NAc, striatal projection neurons (SPN) receive on the one hand glutamatergic inputs, which bring contextual information, and dopaminergic (DA) inputs that code for reward signals. Those inputs converge onto the dendritic spines of SPN, which are key cellular elements for the integration of reward and sensory cues, and are likely involved in the mechanisms that sustain mnesic processes (Pascoli et al., 2014; Cahill et al., 2014a). In this way, it was recently shown that DA gates spine enlargement induced by glutamate in SPN, providing a mechanism for reward-driven learning at the level of the dendritic spine (Yagishita et al., 2014). Classically, long lasting changes in neuronal plasticity and spine formation induced by cocaine have been associated with alterations in gene transcription, via several transcription factors and epigenetic mechanisms in SPN expressing the DA receptor of type 1 (D1-SPN) (Robison and Nestler, 2011). Those events depend on the MAPkinase/ERK (Extracellular-signal Regulated) pathway, which is activated upon synergistic interaction between D1R and NMDA glutamatergic receptor (Cahill et al., 2014b). Similarly, chronic administration of cocaine induces spine enlargement and spine formation in the D1-SPN (Shen et al., 2009; Dobi et al., 2011), which has been shown to rely on the activation of the ERK pathway (Ren et al., 2010).

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Those studies focused on the observation of dendritic spines, but in-depth understanding of synaptogenesis and connectivity changes requires the analysis of both pre and postsynaptic elements (Schoonover et al., 2014, Wierenga et al., 2008). In this way, we have recently established a new method, which allows for 3D automated morphological analysis of synaptic connections and confirmed that the increase in spine density upon chronic cocaine indeed corresponds to connectivity changes (Heck et al., 2015). As stated above, most studies have focused on the association between transcription and new spine formation induced by chronic cocaine administration. Since a single injection of cocaine is sufficient to cause long-term synaptic plasticity and behavioral adaptations to a second administration (Pascoli et al., 2011a), we hypothesized that a single injection of cocaine could modify striatal connectivity.

We bring evidence that acute administration of cocaine is able to induce stable synaptogenesis in the NAc. We unravel the mode of formation of glutamatergic synapses and present evidence that ERK activation is involved in a multi-step process for this rapid synaptogenesis in D1-SPN.

Results

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

We hypothesized that connectivity changes could occur rapidly after a single cocaine injection. Dendritic spine density of SPN was increased in the NAc one hour after a single injection of cocaine (20 mg/kg)(Figures 1A-C). A similar increase was observed at a lower dose (10mg/kg, Figure S1A), while no change was observed in the dorsal part of the striatum (20 mg/kg, Figure S1B). Upon acute administration, cocaine activates intracellular signaling pathways and induces the expression of Immediate Early Genes, including c-Fos, in D1-SPN (Bertrand-Gonzales et al., 2008). We therefore measured spine density in SPN expressing c-

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Fos and found a larger increase when compared to randomly picked SPN (Figures 1A-C).

Following a custom 3D procedure for headspine segmentation (Figure S2A), the measurements of headspines volumes and spine lengths showed an increase, which was better revealed in c-Fos positive neurons (Figures S2B and S2C). The rapidity of this phenomenon prompted us to assess whether the increase in spine density could correspond to an increase in connectivity. To address this issue, dendritic labeling was performed in VGLUT1^venus mice, which express the vesicular transporter of glutamate (VGLUT1), a presynaptic protein expressed in afferents from cortices (neocortex, hippocampus and amygdala), fused to the fluorescent protein venus (Herzog et al., 2011). Following an automated 3D method that we recently set up (Heck et al., 2015), all single VGLUT1-positive boutons were segmented, and synaptic contacts were identified by measurement of the 3D contacts between headspines and boutons. We detected that 60% of spines were connected to VGLUT1-positive boutons in saline conditions (Figures 1A and 1E), a result in agreement with electron microscopic studies showing that half of SPN spines make synapse with VGLUT1-positive boutons, while the other half contact VGLUT2 afferences (Doig et al., 2010). One hour after cocaine administration the number of spines in contact with a VGLUT1 bouton was increased (Figures 1A and 1D), which indicates that the glutamatergic connectivity is rapidly modified in the ventral striatum in vivo. Of note, the percentage of spines connected to VGLUT1 boutons remained unchanged (Figure 1E). We next assessed whether the rapid increase in connectivity was stable over time. One week after a single injection of cocaine, a spine density increase was still observed when compared to the saline group (Figures 1F-G).

Headspine volume, but not spine length, was still larger in cocaine-treated mice as compared to saline controls (Figures S2D and S2E). The detection of spines in contact with a VGLUT1 bouton confirmed that the connectivity increase rapidly induced by a single cocaine exposure was maintained over one week (Figure 1H), with an equal percentage of VGLUT1-positive

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synapses along the dendrite in both saline and cocaine conditions (Figure 1I). Altogether, these results show that a single injection of cocaine induces a rapid synaptogenesis leading to a persistent increase in striatal connectivity, while keeping the stoechiometry of the VGLUT1 afferents onto SPN.

Synapse formation occurs in clusters with spines connecting pre-existing presynaptic boutons The total density of VGLUT1-positive boutons in the NAc was not increased either one hour or one week after cocaine administration (Figures 2A and 2B, left panels). Since glutamatergic synaptogenesis occurred (see Figures 1D and 1H), we hypothesized that synapse formation corresponded to a mode by which new spines contact pre-existing presynaptic boutons. Accordingly, the occurrence of spines connected to a common VGLUT1-positive presynapse was increased one hour and one week after cocaine injection (Figures 2A and 2B, right panels). In order to further test our hypothesis on the mode of synapse formation, we used live imaging on acute striatal slices by two-photon microscopy.

We first set up the model system using stimulation with low doses of glutamate together with a D1R agonist, a co-stimulation known to mimic cocaine effects in primary cultures of SPN (Pascoli et al., 2011b). As expected, the co-stimulation, but not the application of each agonist independently, induced ERK activation in SPN from the NAc (Figures S3A-C), a hallmark of cocaine-induced SPN plasticity (Pascoli et al., 2014). The co-stimulation induced an increase in spine density and spines that contacted VGLUT1 boutons (Figures S3E-S3G), as observed in vivo after cocaine administration (see Figures 1A-1E). Time-lapse experiments were performed on D1-SPN specifically labeled by combining a virus expressing Cre recombinase under the promoter of preprotachykinin A (PPTA) gene and a virus carrying tdTomato sequence flanked by loxP sites (Yagishita et al., 2014)(Figure S4A). The specificity of the

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viral expression was confirmed by immunodetection of the D1R (Figure S4B). Two-photon microscopy stack images allowed the visualization of dendritic spines growth, which was observed as soon as ten minutes after the beginning of the co-stimulation and continued thirty minutes after the onset of stimulation (Figures 2C and 2D). The increase in spine density measured in slice one hour after the co-stimulation exhibited a similar amplitude to what measured in vivo (Figure 2E). Finally, the spine growth was absent when the selective D1R antagonist SCH23390 was bath applied (Figure S4C). In acute slices from VGLUT1^venus mice 62.8% of spines induced by the co-stimulation in D1-SPN expressing tdTomato were in contact with a VGLUT1-positive bouton (N = 5 mice, n = 35 spines). When considering the newly generated spines that ended in contact with a VGLUT1 presynapse, we observed that, in all of cases, the bouton was present before spine growth (N = 5 mice, n = 22 spines) (Figure 2F). Taken together, our results obtained both in vivo on fixed striatal tissue and in time-lapse on striatal slices indicate that synaptogenesis occurs via a mode of formation by which new spines contact pre-existing VGLUT1 boutons. This raised the question as to whether synaptogenesis could be driven by a stochastic spine growth. We generated a model to examine whether spine growth at random positions combined with synapse formation by contact with preexisting boutons could account for the experimental results. Images taken from saline conditions containing the dendrite with headspines and all VGLUT1 boutons were used as a frame for the model. Virtual spines grew from random position at the dendrite surface until they either contacted a VGLUT1 bouton or reached a maximum distance from the dendrite, whilst avoiding crossing headspines (Figure 3A). The modeling of random spine growth yielded an increase in synapse density, which was lower than the measurements from experimental data (Figure 3B). The discrepancy between the experimental results and the model could result from a non-random distribution of the location of spinogenesis along the dendrite. The spine distribution was thus quantified on dendrites imaged in vivo by

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measurement of the shortest border-border distance in between headspines. This revealed a bimodal distribution with a strong proportion of adjacent headspines, which was increased after cocaine exposure (Figures 3C and 3D). Contrasting with this observation, the in silico model retrieved a decrease in the percentage of adjacent spines (Figure 3D), which indicates that the increase of the occurrence of adjacent spines in vivo was not a simple consequence of the increase in spine density. In conclusion, a model of uniform spine growth along the dendrite did reproduce neither the enhancement of synapse density, nor the increased proportion of adjacent spines observed in vivo following acute cocaine. Hence, this model suggests that spine growth does not occur randomly but at locations where synapses clusters will be generated.

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

We next investigated the signaling pathways involved in spine formation induced in striatal slices one hour after the co-stimulation. It is well established that cocaine activates the extracellular signal-regulated kinase (ERK) pathway in D1-SPN (Valjent et al., 2000; Bertran- Gonzales et al., 2008), which controls spine density increase in the case of chronic cocaine administrations (Ren et al., 2010). The involvement of the ERK pathway in spinogenesis was thus assessed using U0126, a selective inhibitor of MEK, the kinase upstream of ERK.

Pretreatment with U0126 abolished the increase in spine density induced by the co- stimulation (Figure 4A), indicating that the activation of the ERK pathway is necessary for spinogenesis. Recently, the Akt and mammalian Target of Rapamycin (mTOR) pathway was shown to be involved in spine density increase induced by chronic cocaine injections (Cahill et al., 2016). We tested the implication of this pathway using rapamycin and found that it completely blocked the increase in spine density induced by co-stimulation in slices (Figure

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4B). Since both ERK and mTOR pathways were mandatory for spinogenesis, we turned towards possible common molecular mechanisms activated downstream these pathways. The ERK and mTOR pathways indeed converge on the control of protein translation (Figure 4C), via the Eukaryotic Inititation Factors complex. In this way ERK-dependent activation of MAP Kinase Interacting Kinase-1 (MNK-1) activates this complex and regulates its binding to mRNA (Bkahar et al., 2012). We thus hypothesized that MNK-1 could be involved in spine formation. We first assessed the activation of MNK-1 using immunodetection of its phosphorylated form (pMNK-1). Twenty minutes after co-stimulation, an increase in pMNK- 1 positive cells was detected in the NAc (Figure 4D). Slices were then pre-treated with CGP 57380, a specific inhibitor of MNK-1 activation, which totally inhibited the increase in spine density induced by the co- stimulation (Figure 4E). We next reasoned that the abolishment of the stimulation-induced increase in spine density by ERK or MNK-1 inhibition could result from either inhibition of spine growth or lack of their stabilization (Figure 5A). In order to gain insights into these two possibilities, we used time-lapse imaging of spine growth and maintenance from D1-SPN dendrites (Figure 5B) labeled in vivo using intra-NAc infection with AAV PPTA::Cre/AAV TdTomato (see Figure S4). Spine growth was absent in the presence of U0126, the inhibitor of the ERK pathway (Figure 5C), indicating that ERK activation is mandatory for the formation of new spines. In contrast, CGP 57380 did not modify growth rate, clearly indicating that MNK-1 is not required for spine growth (Figures 5B and 5C). However, it strongly impacted the maintenance of the newly formed spines, with only 9% of the newly formed spines remaining 90 minutes after growth, while 67% were stable at that later time point in control conditions (Figures 5B and 5D). The requirement of MNK-1 for stabilization was observed 30 minutes after spine growth, since 59.1% of the spines were lost at this time point under conditions of MNK-1 inhibition. Noteworthy, the inhibition of MNK-1 activation did not affect the stability of spines before stimulation (Figure

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S5). Hence these data show for the first time that MNK-1 is mandatory for stabilization but not formation of spines upon stimulation. 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 to that observed with the MNK-1 inhibitor (Figures 5B-5D). Moreover, bath application of the transcription inhibitor actinomycin D did alter neither growth nor stabilization of new spines (Figures 5B-5D).

Therefore, time-lapse analysis unraveled sequential ERK-dependent steps for spinogenesis.

Upon stimulation, ERK is essential for both spine growth and maintenance, whilst ERK- induced MNK-1 phosphorylation and protein synthesis specifically control spine stabilization independently of transcription.

Parsing the role of MNK-1 in spine formation in vivo

Finally, we analyzed the impact of MNK-1 activation on long-lasting spinogenesis in vivo. An 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 6A and 6B). The dendritic localization of pMNK-1 in D1-SPN was confirmed by combining immunodetection of pMNK-1 and labeling of dendrites with the PPTA-Cre and fl/fl tdTomato-AAV viruses. pMNK-1 positive punctates were detected in the dendritic shaft and in dendritic spines from D1-SPN (Figure 6C). In agreement with the results from acute slices, inhibition of ERK as well as protein synthesis abolished spine formation one hour after cocaine administration (Figures S6A and S6B). The activation of MNK-1 was inhibited locally by intracranial injection of CGP in the NAc prior to either saline or cocaine single administration (Figure 6D). The inhibition of MNK-1 did not affect basal