David De

Sa Nogueira

Romain Bourdy

Dominique Filliol

Pascal Romieu

LNCA Katia Befort
email: katia.befort@unistra.fr

Hippocampal mu opioid receptors are modulated following cocaine self-administration in rat

Keywords: mu opioid receptor, cocaine, hippocampus, gene expression, epigenetics cocaine self-administration; DAMGO: [D-Ala2, N-MePhe4, Gly-ol]-

de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Addiction is a multi-factorial disease involving genetic, psychological and environmental factors, which has detrimental consequences for individuals and society. It is characterized by excessive intake over time, craving, continued drug use despite negative consequences and relapse [START_REF] Koob | Neurobiology of addiction: a neurocircuitry analysis[END_REF]. The progression of addiction is associated with neuroplastic changes [START_REF] Robinson | Alterations in the morphology of dendrites and dendritic spines in the nucleus accumbens and prefrontal cortex following repeated treatment with amphetamine or cocaine[END_REF][START_REF] Ron | The "Ups and Downs" of Signaling Cascades in Addiction[END_REF], whereby altered gene expression impacts neuronal function and behavior [START_REF] Scherrer | The delta agonists DPDPE and deltorphin II recruit predominantly mu receptors to produce thermal analgesia: a parallel study of mu, delta and combinatorial opioid receptor knockout mice[END_REF][START_REF] Przewlocki | Opioid abuse and brain gene expression[END_REF][START_REF] Rhodes | Gene expression induced by drugs of abuse[END_REF][START_REF] Mcclung | Neuroplasticity Mediated by Altered Gene Expression[END_REF]. In the case of cocaine these gene expression changes are widespread, but are particularly prominent in reward-related brain structures such as the prefrontal cortex (PFC) [START_REF] Kalivas | Unmanageable motivation in addiction: a pathology in prefrontal-accumbens glutamate transmission[END_REF], dorsal striatum (DS) (Ahmed & Kenny, 2011), hippocampus (HPC) (Zhou et al., 2014;Walker et al., 2018) and nucleus accumbens (NAc) [START_REF] Russo | The addicted synapse: mechanisms of synaptic and structural plasticity in nucleus accumbens[END_REF]. Many of these changes involve the opioid system, possibly linked with its role in reward and motivation [START_REF] Merrer | Reward processing by the opioid system in the brain[END_REF]. Indeed, the mu opioid receptors (MOP) have been described as a gateway to addiction to several drugs, including cocaine [START_REF] Scherrer | The delta agonists DPDPE and deltorphin II recruit predominantly mu receptors to produce thermal analgesia: a parallel study of mu, delta and combinatorial opioid receptor knockout mice[END_REF]Charbogne et al, 2014). Chronic cocaine induces molecular neuroadaptations of MOP in striatal regions, including increased MOP gene expression in both NAc (Azaryan et al., 1998;[START_REF] Leri | Effects of High-Dose Methadone Maintenance on Cocaine Place Conditioning, Cocaine Self-Administration, and Mu-Opioid Receptor mRNA Expression in the Rat[END_REF] and DS [START_REF] Valenza | Strain and cocaine-induced differential opioid gene expression may predispose Lewis but not Fischer rats to escalate cocaine self-administration[END_REF], MOP-agonist binding [START_REF] Unterwald | Chronic cocaine alters brain mu opioid receptors[END_REF][START_REF] Unterwald | Repeated cocaine administration upregulates kappa and mu, but not delta, opioid receptors[END_REF][START_REF] Unterwald | The frequency of cocaine administration impacts cocaine-induced receptor alterations[END_REF], and GTPS binding [START_REF] Schroeder | Cocaine alters mu but not delta or kappa opioid receptor-stimulated in situ [35S]GTP?S binding in rat brain[END_REF]. Noticeably, cocaine regulates gene expression through epigenetic processes, including histone modification, mostly in the NAc [START_REF] Nestler | Epigenetic mechanisms of drug addiction[END_REF]De Sa Nogueira et al., 2019). For example, cocaine increased the enrichment level of the trimethylated lysine 4 of histone 3 (H3K4Me3) in NAc following chronic administration which resulted in an enhancement of associated gene expression, such as FosB (Cates et al., 2019).

Whether histone modifications could occur at MOP gene level following cocaine treatment is not yet known.

In this study, we investigated MOP gene expression and functionality following voluntary cocaine intake in rat, in brain reward regions (PFC, NAc, DS and HPC). To further characterize MOP transcript alterations in the HPC, we examined H3K4Me3 levels at MOP promoter region and also measured the enrichment of acetylated lysine 27 of histone 3 (H3K27Ac), a histone mark of active enhancers promoting gene expression (Achour et al., 2015).

Materials and Methods

Subjects

Male Wistar rats (n=34, Janvier, France) weighting 250-300 g at the beginning of the experiments were habituated for two weeks to housing conditions in a temperature and humidity-controlled environment with a reverse 12 h light/dark cycle (lights on at 7:00 PM).

Rats were grouped housed (5/cage) with ad libitum access to food and water until surgery and then single-housed. All experimental procedures were performed in agreement with the EU Directive 2010/63/EU for animal experiments and approved by the institutional ethics committee CREMEAS (Comité d'Éthique pour l'Expérimentation Animale de Strasbourg, France, APAFIS#2015012716049550). All efforts were made to minimize animal suffering and to reduce the number of animals used.

Drugs

Cocaine hydrochloride was obtained from Cooper (#1164500, Melun, France) and dissolved in sterile NaCl 0.9%. The MOP agonist DAMGO was obtained from Sigma Aldrich (E7384, St Louis, USA).

Surgery

An intravenous catheterization procedure was performed as previously described (Fonteneau et al., 2017). Briefly, rats were anesthetized by i.p. injection (1 ml/kg) of a mixture containing 90 mg/kg of ketamine (Imalgene 1000®, Centravet, France) and 10 mg/kg of xylazine (Rompun®, Centravet). Surgery was performed to implant a chronic indwelling catheter in the right jugular vein. Catheters were flushed daily with 150 μl saline solution containing 100 U/ml heparin and 50 mg/ml ampicillin to prevent catheter clotting and potential infection, respectively.

Cocaine self-administration procedure

Seven days after surgery, drug self-administration was performed as previously described [START_REF] Romieu | The inhibition of histone deacetylases reduces the reinstatement of cocaine-seeking behavior in rats[END_REF]Fonteneau et al., 2017). Briefly, a computer driven syringe pump (Imetronic, Pessac, France) activated a 10 ml syringe and pushed fluid into Silastic® tubing connected to the rat through its externalized 23-gauge guide cannula. Each operant chamber was equipped with two 2.5 cm-diameter holes on the same wall, 4 cm above the floor.

Nose pokes into the inactive hole had no programmed consequence. Nose pokes into the active hole triggered the i.v. infusion of 60 l of cocaine hydrochloride (0.33 mg/kg, Cocaine-SA) or of the same volume of saline in control animals (NaCl 0.9%, Saline-SA), which were ran concurrently. The volume infused was adjusted according to the rat body weight every other session. A 40 s-time-out period began simultaneous to the cocaine infusion. A fixed-ratio 1 (FR1) paradigm was carried out for 10 days (2h daily sessions). Animals were given an overdose of pentobarbital (100 mg/kg, i.v) 24h after the last session followed by brain extraction to collect PFC, NAc, DS, and HPC (dorsal part).

Quantitative real-time PCR

Samples (n=5-9/group) were processed to extract total RNA using Ribozol (VWR, Fontenay-sous-bois, France) which was reverse transcribed (750 ng) with iScript (iScript TM cDNA Synthesis Kit, Biorad, France). Real-time PCR was performed in triplicate (CFX96 Touch TM apparatus, Biorad, France) in Sso Advanced TM Universal SYBR Green supermix (Biorad, France). Thermal cycling parameters were 30 sec at 95°C followed by 40 amplification cycles of 5 sec at 95° and 45 sec at 60°C. Primer sequences were the following: MOP, ACAGCCTGTGCCCTCAGACC (forward) and CAGGAAGTTTCCGAAGAGGCC (reverse) and rplp0: CTGCCCGAGCCGGTGCCATC (forward) and TTCAATGGTACCTCTGGAG (reverse). Expression levels were normalized to rplp0 housekeeping gene levels [START_REF] Saad | Regulation of Brain DNA Methylation Factors and of the Orexinergic System by Cocaine and Food Self-Administration[END_REF] and compared between Saline-SA and Cocaine-SA samples using the 2 -ΔΔ Ct method [START_REF] Livak | Analysis of relative gene expression data using realtime quantitative PCR and[END_REF].

Agonist-stimulated [ 35 S]-GTPγS binding assay

The [ 35 S]-GTPγS binding assay was used to measure G protein activation following MOP receptor stimulation with DAMGO, as previously described [START_REF] Scherrer | The delta agonists DPDPE and deltorphin II recruit predominantly mu receptors to produce thermal analgesia: a parallel study of mu, delta and combinatorial opioid receptor knockout mice[END_REF].

Membranes (50 μg) were incubated in the assay buffer (50mM Tris-HCl pH 7.4; 3 mM MgCl2; 0.2mM EGTA; 100mM NaCl) containing 0.1nM [ 35 S]-GTPγS (NEG030H, PerkinElmer, Courtaboeuf, France), 30 μM GDP and DAMGO (10 -9 M to 10 -4 M) for 1 h at 25 °C.

Radioactivity was detected using a Top-Count scintillation counter (PerkinElmer, Billerica, MA, USA). Basal [ 35 S]-GTPγS binding was determined in the absence of agonist, and nonspecific binding by replacing [ 35 S]-GTPγS by 10 μM non-radiolabeled GTPγS. Stimulated specific binding was converted in percentage of basal specific binding, defined as 100%. Data were analyzed using Prism 6 GraphPad software. Four to seven independent assays were performed in triplicate. Stimulation (%), EC50s and IC50s were calculated for each experiment and averaged.

Chromatin immunoprecipitation

Samples were prepared as previously described (Caputi et al., 2014) with minor modifications. Frozen tissues were ground on carbonic ice before fixation (1% formaldehyde)

and quenched with glycine (0.125M). Tissue fragments were washed in presence of protease inhibitors (#4693132001, Roche, France). Tissues were homogenized (0.5% SDS, 10mM

EDTA, 50mM Tris-HCL, pH8) and lysates were sheered using a diogenode bioruptor XL at 4°C, to obtain DNA fragments <600 bp. Protein A magnetic beads (Dynabeads, Invitrogen)

were coated (4°C overnight) with the respective antibody of interest (H3K4Me3, ab8580;

H3K27Ac, ab4729, Abcam, France), washed and added to sheared chromatin for histone ChIPs (4°C overnight). A sample of each sheared chromatin (10%) was used as input control. Samples were washed with RIPA buffer (HEPES-KOH 50mM pH7.5, LiCl 500mM, EDTA 1mM, NP-40 1%, Na-Deoxycolate 0.7%) and elution buffer (SDS 1%, NaHCO3 100mM). Reverse crosslinking was performed at 65°C overnight, proteins and RNA removed with proteinase K and RNAse A (ThermoFisher, USA) respectively and DNA purified (Qiagen, France). A negative control without IgG was performed to test for nonspecific binding. PCR of MOP genomic sequence around the promoter region associated with the immunoprecipitated proteins was performed. Primers targeting MOP promoter region were the following: (forward)

GGCCAGAGATGCATCGATCA and (reverse) GAGTCAGCCTGGCCAAGATT.

Statistical analysis

All results are expressed as mean  S.E.M. Behavioral data were analyzed using a repeated-measures two-way ANOVA followed by post-hoc Sidak tests when requested (GraphPad Prism V.7). Other data were analyzed using student t-test. Significance was set at p ≤ .05.

Results

Acquisition of cocaine self-administration

Rats were trained for 10 days during 2h daily sessions of cocaine self-administration under the FR1 schedule of reinforcement (Figure 1A). The difference between cocaine and saline infusions was highly significant across all sessions [group effect, F(1, 32)=76.78, p <

.0001] (Figure 1A, top panel 1B).

Transcriptional changes of opioid receptors induced by cocaine self-administration

We measured MOP gene expression in several brain-reward related structures. The analysis was performed 24h after the last cocaine session in PFC, NAc, DS, and HPC, in order to explore mRNA expression in the absence of an acute effect of cocaine. We found that MOP gene expression was significantly decreased by cocaine-SA in DS [p = .03] and, in contrast, increased 1.85 fold in HPC [p = .045] (Figure 2A). No significant changes could be detected in the PFC or NAc. The level of expression was not correlated with the amount of cocaine intake (rPFC= -.54; rNAc= -.009; rDS= .1; rHPC= -.09) (Figure 2B).

Cocaine self-administration modulated MOP functional activity

To further characterize MOP receptor alterations, we investigated MOP functional activity by [ 35 S]-GTPγS binding assay using MOP agonist DAMGO, in three brain regions of interest, PFC, DS and HPC. Not enough material was available from NAc samples to perform this experiment on single animals. Interestingly, a significant effect was observed in HPC, with an increased efficacy (Emax) in cocaine-SA rats compared to saline controls (Emax: Saline-SA = 148.7 ± 3.4; Cocaine-SA = 170 ± 5.6; HPC: p = 0.015) (Figure 3). The potency (EC50)

was not altered in this region (EC50: Saline-SA = 1,465.10 -7 M ± 0.11; Cocaine-SA = 1,863.10 -

Histone modifications at MOP promoter region

To further investigate MOP transcriptional changes in HPC, we examined whether epigenetic modifications occurred at MOP promoter region in this structure. We performed a chromatin-immunoprecipitation experiment followed by a qPCR (ChIP-qPCR) using specific primers directed towards MOP promoter. No enrichment was obtained in the absence of antibodies targeting the histone tails. In contrast, we obtained an enrichment of both marks at the MOP promoter, but the enrichment percentage compared to input in both conditions remained unchanged [H3K4Me3: p = .165 H3K27Ac: p = .126], indicating no significant change in histone modification in HPC following cocaine intake (Figure 4).

Discussion

Role of hippocampal MOP in HPC following cocaine-SA. We investigated MOP transcript expression in several reward-related brain structures and observed, for the first time, a marked increase of MOP transcript in the HPC, a region essential for forming cocaine-context associations (Grant et al., 1996). Interestingly, this regulation was associated with increased functionality of the receptor in the same brain structure, as revealed by GTPγS binding.

Whether the observed gene up-regulation is specific to cocaine self-administration or would be observed with passive cocaine intake is not known. Also, we did not observe similar MOP gene regulation in the HPC following a binge sucrose [START_REF] Smail-Crevier | Binge-like intake of sucrose reduces the rewarding value of sucrose in adult rats[END_REF] paradigm (data not shown), suggesting that natural rewards may not modify MOP expression in this brain region. Moreover, there are scarce data on MOP gene alterations following drug-SA in HPC.

An increase of MOP receptor (binding and GTPS studies) was observed in HPC following heroin-SA, with a low but constant drug intake during a short time (Fattore et al., 2007).

Opposite results, with decreased MOP activity and efficiency, were reported following paradigms using high doses of heroin and escalating intake during a prolonged period [START_REF] Sim-Selley | Chronic heroin self-administration desensitizes μ opioid receptoractivated G-proteins in specific regions of rat brain[END_REF][START_REF] Maher | Mechanisms of mu opioid receptor/Gprotein desensitization in brain by chronic heroin administration[END_REF]. MOP hippocampal alteration was also reported following cannabinoid-SA with increased MOR binding and GTPS (Fattore et al., 2007).

Also, MOP regulation may result from or lead to endogenous peptide regulations in HPC, which could directly impact neurochemical responses during cocaine intake [START_REF] Jacobs | Neuroadaptive effects of active versus passive drug administration in addiction research[END_REF]. Altogether, this may reflect a key role for hippocampal MOP in associative learning related to drug administration.

HPC is a crucial structure for the formation and maintenance of cocaine-context associations [START_REF] Kilts | Neural Activity Related to Drug Craving in Cocaine Addiction[END_REF]Wexler et al., 2001). In particular, the dorsal HPC controls context-induced reinstatement. Its inhibition (tetrodotoxin or muscimol) attenuated contextinduced, but not cue-induced reinstatement of cocaine seeking (Fuchs et al., 2005(Fuchs et al., , 2007)). A recent study showed that chemogenetic inhibition of dorsal, but not ventral hippocampal inputs to lateral septum specifically attenuated context-induced reinstatement of cocaine [START_REF] Mcglinchey | Dorsal Hippocampus Drives Context-Induced Cocaine Seeking via Inputs to Lateral Septum[END_REF]. Alterations in MOP gene expression may underlie cocaine-induced neuroplasticity in the brain. MOP knockout mice exhibited decreased cocaine-SA [START_REF] Mathon | Increased gabaergic input to ventral tegmental area dopaminergic neurons associated with decreased cocaine reinforcement in μ-opioid receptor knockout mice[END_REF] and decreased cue-induced relapse associated with a decrease in HPC neuronal activity [START_REF] Gutiérrez-Cuesta | Effects of Genetic Deletion of Endogenous Opioid System Components on the Reinstatement of Cocaine-Seeking Behavior in Mice[END_REF]. Cocaine-SA induced long term potentiation (LTP) in CA1 region of the HPC has been reported after short withdrawal [START_REF] Thompson | Modulation of long-term potentiation in the rat hippocampus following cocaine self-administration[END_REF]. MOP activation is partly responsible for mossy fiber LTP in hippocampal CA3 (Derrick & Martinez, 1994) and MOP deficient mice exhibited an impairment of spatial learning, that may be associated with LTP deficit observed in mossy fibers in CA3 of these mice [START_REF] Jamot | Differential involvement of the mu and kappa opioid receptors in spatial learning[END_REF]. Noteworthy, recent data indicate MOP expression in CA1 astrocytes and demonstrate astroglial MOP-mediated LTP in HPC [START_REF] Nam | Expression of µ-Opioid Receptor in CA1 Hippocampal Astrocytes[END_REF][START_REF] Nam | Activation of Astrocytic μ-Opioid Receptor Causes Conditioned Place Preference[END_REF]. Altogether, even though little information is yet available, the behavioral and neurochemical data suggest that MOP is involved in cocaine responses by modifying the neuronal activity in this brain area closely related to memory processing.

MOP regulation in reward-related brain regions following cocaine-SA.

In our conditions, no detectable modifications could be observed in the PFC or NAc of rats selfadministering cocaine. Reports investigating MOP regulations extensively focus on striatal regions with the vast majority relying on passive cocaine intake (for a review see Yoo et al., 2012). MOP up-regulations in the NAc were previously observed in passive paradigms [START_REF] Unterwald | Chronic cocaine alters brain mu opioid receptors[END_REF][START_REF] Unterwald | Repeated cocaine administration upregulates kappa and mu, but not delta, opioid receptors[END_REF][START_REF] Izenwasser | Continuous cocaine administration enhances μ-but not δ-opioid receptor-mediated inhibition of adenylyl cyclase activity in nucleus accumbens[END_REF]Azaryan et al., 1998;Eipper-Mains et al., 2013) whereas no changes were observed in a voluntary paradigm [START_REF] Valenza | Strain and cocaine-induced differential opioid gene expression may predispose Lewis but not Fischer rats to escalate cocaine self-administration[END_REF], similarly to our findings. This suggests that the cognitive processes involved in voluntary intake do not implicate MOP gene expression regulation in the NAc. A yoked control-operant group would assess whether the associative learning is specific to instrumental learning [START_REF] Jacobs | Neuroadaptive effects of active versus passive drug administration in addiction research[END_REF]. No report in the literature was found for MOP regulation by cocaine in the PFC, and our results indicate no effect of cocaine-SA on MOP gene expression, suggesting a modest role for MOP in prefrontal functions.

In contrast, we detected decreased MOP gene expression in the DS. This data is in contradiction with a previous work reporting a two-fold increase of MOP gene expression in Lewis strain with escalating cocaine-SA consumption and no change in Fischer strain showing a stable intake across sessions [START_REF] Valenza | Strain and cocaine-induced differential opioid gene expression may predispose Lewis but not Fischer rats to escalate cocaine self-administration[END_REF]. MOP binding was up-regulated in DS of cocaine users, a modification that lasted following long term abstinence (Zubieta et al., 1996), and MOP up-regulation was also observed in rat DS at mRNA [START_REF] Unterwald | Chronic cocaine alters brain mu opioid receptors[END_REF][START_REF] Unterwald | Repeated cocaine administration upregulates kappa and mu, but not delta, opioid receptors[END_REF][START_REF] Unterwald | The frequency of cocaine administration impacts cocaine-induced receptor alterations[END_REF] or functional level (Bailey et al., 2007;[START_REF] Schroeder | Cocaine alters mu but not delta or kappa opioid receptor-stimulated in situ [35S]GTP?S binding in rat brain[END_REF] in passive chronic cocaine paradigms. Noticeably, few reports indicate the absence of MOP regulation in similar passive cocaine administration (Azaryan et al., 1996;Yuferov et al., 1999). Altogether, these results

indicate that the rodent strain and/or the pattern of cocaine consumption represent specific factors that alter differentially the opioid system in DS. and CB1 in HPC (Fattore et al., 2007). We therefore hypothesize that similar adaptations could arise at MOP/DOP or MOP/CB1 levels following cocaine-SA in HPC. Further work will therefore investigate cellular and molecular consequences of cocaine-SA, and our preliminary results indicate an up-regulation of CB1 transcript in HPC following the same paradigm (DN, personal communication).

Molecular mechanisms involved in cocaine induced

It is now well described that cocaine can induce epigenetic changes in the brain [START_REF] Nestler | Epigenetic mechanisms of drug addiction[END_REF]De Sa Nogueira et al., 2019). To investigate whether increased gene expression of MOP in HPC was under the control of such modifications, we analyzed H3K4me3 and H3K27ac

activating marks at the level of MOP gene promotor. The absence of specific changes of H3K4me3 and H3K27ac levels in our conditions indicates that these marks were not associated with MOP gene regulation observed in the HPC. Previous work has shown H3K4Me3 regulation following cocaine intake in the HPC of postmortem cocaine abuser brains (Zhou et al., 2011), and this mark has been proposed as an opioid system element regulator in striatum (Caputi et al., 2014), indicating that the opioid system can be under the control of epigenetic regulations upon cocaine. Among other epigenetic changes, DNA methylation has been described at MOP promotor region in mouse brain and in heroin addict brain [START_REF] Hwang | Evidence of endogenous mu opioid receptor regulation by epigenetic control of the promoters[END_REF][START_REF] Hwang | Upregulation of the mu-opioid receptor gene is mediated through chromatin remodeling and transcriptional factors in differentiated neuronal cells[END_REF][START_REF] Nielsen | Increased OPRM1 DNA methylation in lymphocytes of methadone-maintained former heroin addicts[END_REF][START_REF] Nielsen | Ethnic diversity of DNA methylation in the OPRM1 promoter region in lymphocytes of heroin addicts[END_REF]Chorbov et al., 2011). Whether DNA methylation occurs at the opioid system level upon cocaine-SA is not yet know. Further examination of the role of the opioid system in the HPC and associated epigenetic changes, will be crucial for the development of therapeutic treatments.

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 4 Figure4: Histone modifications at the MOP promoter following cocaine selfadministration. Chromatin was extracted from HPC samples, immunoprecipitated with antibodies recognizing H3K4Me3 or H3K27Ac, two activating histones marks. Samples were further processed for qPCR using specific primers targeting the MOP promoter. Results were normalized according to the input (see Methods). The enrichment of histone marks at MOP promoter region was not modulated by cocaine self-administration. Enrichment was specific to both antibodies as no enrichment could be detected without antibody. Histograms represent the mean + S.E.M. n= 5-7/group for Saline-SA and Cocaine-SA groups.

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M ± 0.12). No significant changes of either Emax or EC50 were observed in PFC and DS.

Conflicts of Interest: None

Legends to figures


David De

Sa Nogueira

Romain Bourdy

Dominique Filliol

Cyril Quessada

Isabelle Mccort

Francine Acher

Jean Zwiller

Pascal Romieu

Katia Befort
email: katia.befort@unistra.fr

David De Sa Nogueira

Isabelle Mccort-Tranchepain

LSP2-9166, an orthosteric mGlu4 and mGlu7 receptor agonist, reduces cocaine self-administration under a progressive ratio schedule in rats

Keywords: operant self-administration, LSP2-9166, Grm4, Grm7, cocaine, motivation

come

Introduction

Cocaine addiction is a chronic brain disease characterised by a high level of motivation for the drug and relapse in cocaine users [START_REF] Kalivas | The neural basis of addiction: a pathology of motivation and choice[END_REF]. Given the multifaceted health issue that results from this, including mortality and morbidity, research aimed at identifying the neurobiological mechanisms underlying cocaine's rewarding effects may lead to a better understanding of the processes involved in addiction and potential therapeutic strategies. It is well established that the dopamine (DA) neurotransmission system, particularly the mesolimbic pathway connecting the ventral tegmental area (VTA) to the nucleus accumbens (NAc), is crucial for the motivational component of behavior. This is classically measured in rodents using a progressive ratio (PR) schedule of cocaine self-administration (SA) [START_REF] Gardner | What we have learned about addiction from animal models of drug selfadministration[END_REF]. In addition, emerging evidence points to a major role for glutamate in the progressive elaboration of cocaine-seeking behaviors [START_REF] Scofield | The Nucleus Accumbens: Mechanisms of Addiction across Drug Classes Reflect the Importance of Glutamate Homeostasis[END_REF] and glutamate levels are modified during relapse [START_REF] Knackstedt | Glutamate and reinstatement[END_REF]. Glutamate acts on fast-acting ligand-gated ion channels, the ionotropic receptors, and slow-acting G-protein coupled metabotropic glutamate (mGlu) receptors. Three groups of mGlu receptors exist in the brain. Group I receptors (mGlu1/5 receptors) were the first to be demonstrated playing a role in drug dependence [START_REF] Mihov | Negative Allosteric Modulators of Metabotropic Glutamate Receptors Subtype 5 in Addiction: a Therapeutic Window[END_REF]. However, mnesic processes were also altered by these compounds [START_REF] Gass | Glutamatergic substrates of drug addiction and alcoholism[END_REF]. Also, activation of group II mGlu receptors (mGlu2/3) reduces both seeking and taking behavior towards distinct drugs of abuse in rodents, through normalization of an altered glutamatergic neurotransmission induced by drug exposure [START_REF] Knackstedt | Glutamate and reinstatement[END_REF].

However, activation of mGlu2/3 produced reduced food-seeking behaviors [START_REF] Peters | The group II metabotropic glutamate receptor agonist, LY379268, inhibits both cocaine-and food-seeking behavior in rats[END_REF] and a decrease in cognitive performance [START_REF] Aultman | Distinct contributions of glutamate and dopamine receptors to temporal aspects of rodent working memory using a clinically relevant task[END_REF][START_REF] Higgins | Pharmacological manipulation of mGlu2 receptors influences cognitive performance in the rodent[END_REF]. Among receptors of group III, mGlu6 and mGlu8 receptors lack precise behavioral function, whereas mGlu4/7 receptors negatively control glutamate transmission and have been recently proposed as targets for several neurodegenerative and neuropsychiatric conditions [START_REF] Crupi | Role of Metabotropic Glutamate Receptors in Neurological Disorders[END_REF][START_REF] Nicoletti | Targeting mGlu Receptors for Optimization of Antipsychotic Activity and Disease-Modifying Effect in Schizophrenia[END_REF].

The mGlu4 and mGlu7 receptors are expressed in the basal ganglia and structures of the reward circuit [START_REF] Corti | Distribution and synaptic localisation of the metabotropic glutamate receptor 4 (mGluR4) in the rodent CNS[END_REF][START_REF] Kosinski | Localization of metabotropic glutamate receptor 7 mRNA and mGluR7a protein in the rat basal ganglia[END_REF]. L-AP4, an agonist of group III mGlu receptors, inhibits Glu and/or GABA release in key structures of the basal ganglia, such as striatum and globus pallidus [START_REF] Macinnes | Group III metabotropic glutamate receptors act as heteroreceptors modulating evoked GABA release in the globus pallidus in vivo[END_REF][START_REF] Valenti | Group III metabotropic glutamate receptor-mediated modulation of the striatopallidal synapse[END_REF], therefore blocking cocaine-induced hyperlocomotion in rats [START_REF] Mao | Distinct inhibition of acute cocaine-stimulated motor activity following microinjection of a group III metabotropic glutamate receptor agonist into the dorsal striatum of rats[END_REF]. Interestingly, a selective mGlu4 agonist, LSP1-2111, reduced the expression of cocaine sensitization [START_REF] Zaniewska | Inhibitory actions of mGlu4 receptor ligands on cocaine-, but not nicotine-, induced sensitizing and conditioning locomotor responses in rats[END_REF]. A selective allosteric agonist of mGlu7 receptors, AMN082 [START_REF] Mitsukawa | From The Cover: A selective metabotropic glutamate receptor 7 agonist: Activation of receptor signaling via an allosteric site modulates stress parameters in vivo[END_REF], reduced cocaine SA [START_REF] Li | Metabotropic Glutamate Receptor 7 Modulates the Rewarding Effects of Cocaine in Rats: Involvement of a Ventral Pallidal GABAergic Mechanism[END_REF] and altered both alcohol preference and intake [START_REF] Bahi | The selective metabotropic glutamate receptor 7 allosteric agonist AMN082 prevents reinstatement of extinguished ethanol-induced conditioned place preference in mice[END_REF]. Side-effects of this compound were observed, with a reduction of sucrose intake [START_REF] Salling | Nonselective suppression of operant ethanol and sucrose self-administration by the mGluR7 positive allosteric modulator AMN082[END_REF], and decreased locomotor activity at high doses [START_REF] Palucha | Activation of the mGlu7 receptor elicits antidepressant-like effects in mice[END_REF].

Anti-depressive and anxyolitic effects were also observed and proposed to be linked to a rapid internalization process induced by AMN082 [START_REF] O'connor | The effects of mGlu₇ receptor modulation in behavioural models sensitive to antidepressant action in two mouse strains[END_REF][START_REF] Pelkey | mGluR7 undergoes rapid internalization in response to activation by the allosteric agonist AMN082[END_REF].

The design of an orthosteric brain permeant subtype-selective ligand has facilitated new investigations. Indeed, LSP2-9166, a mGlu4/7 receptor orthosteric agonist [START_REF] Acher | Hypophosphorous acid derivatives having antihyperalgic activity and biological applications thereof[END_REF],

represents the most powerful orthosteric compound synthezised so far. Remarkably, it was recently shown to inhibit both morphine-induced place preference and reinstatement after extinction in mice [START_REF] Hajasova | Role of mGlu7 receptor in morphine rewarding effects is uncovered by a novel orthosteric agonist[END_REF], and ethanol intake in rats [START_REF] Lebourgeois | Pharmacological activation of mGlu4 and mGlu7 receptors, by LSP2-9166, reduces ethanol consumption and relapse in rat[END_REF]. To complete this work on drug abuse, we first examined brain transcript levels for both mGlu4 and mGlu7 receptors following voluntary cocaine intake. We then examined whether the LSP2-9166 could modulate cocaine motivation in an intravenous (i.v.) SA procedure in rats.

Materials and methods

Chemical compound

LSP2-9166 was prepared according to an improved synthesis [START_REF] Acher | Hypophosphorous acid derivatives having antihyperalgic activity and biological applications thereof[END_REF]. Briefly, Figure 1 illustrates these improvements from recently reported preparation of derivatives [START_REF] Habrian | Conformational pathway provides unique sensitivity to a synaptic mGluR[END_REF][START_REF] Selvam | Increased Potency and Selectivity for Group III Metabotropic Glutamate Receptor Agonists Binding at Dual sites[END_REF] to avoid L-AP4 formation, a side product with selective and potent group III mGluR agonist activity. We first improved a straightforward synthesis of the N-Boc-(S)-vinylglycine-O-tertbutyl ester from Boc-Asp-OtBu in two steps without intermediate purification affording a scale-up [START_REF] Kastrinsky | A convergent synthesis of chiral diaminopimelic acid derived substrates for mycobacterial L,D-transpeptidases[END_REF][START_REF] Siebum | Access to any site-directed isotopomer of methionine, selenomethionine, cysteine, and selenocysteine -Use of simple, efficient modular synthetic reaction schemes for isotope Incorporation[END_REF]. Purity was checked by NMR ( 31 P), with no detectable L-AP4, and confirmed before use by a pharmacological approach (ARPEGE Platform, Montpellier https://www.igf.cnrs.fr/index.php/fr/research-fr/facility-fr). Final deprotection under milder conditions led to the final product LSP2-9166 in higher yield, with a high purity.

Subjects

Male Wistar rats (Janvier Labs, France), aged five weeks and weighting 150-175 g upon arrival, were housed in standard home cages (5 rats/cage), under a reversed 12h light/dark cycle (lights on at 7pm), with water and food ad libitum. All procedures involving animal care were conducted in compliance with current laws and policies, validated by a Comité d'Ethique en Expérimentation Animale (CREMEAS) and authorised by the Ministère de l'Enseignement Supérieur, de la Rechercher et de l'Innovation (APAFIS#7050-2016093016163350). Eighteen rats were used for the molecular study and 69 for the SA procedure and locomotor activity.

Pharmacological treatment

Cocaine hydrochloride (Cooper, France) solution was adjusted with 0.9% NaCl to infuse an i.v. dose of 0.33 or 0.5 mg/kg/injection for SA and 1.5 mg/kg for locomotor activity. LSP2-9166 was first diluted into water at 20 mg/ml, pH was adjusted to 7.4 and final concentration was set at 2 or 5 mg/ml using phosphate-buffered-saline (PBS). LSP2-9166 (2 or 5 mg/kg, i.v.) or vehicle (PBS) was administered 15 min before each SA session.

Surgery and cocaine operant self-administration

All rats went through an i.v. catheterization procedure for operant SA, performed as described in detail previously [START_REF] Romieu | Histone deacetylase inhibitors decrease cocaine but not sucrose self-administration in rats[END_REF]. Rats were anesthetized by an intraperitoneal injection (2 ml/kg) of a mixture containing ketamine (Imalgene 1000, 90 mg/kg, Centravet) and xylazine (Rompun, 10 mg/kg; Centravet) to perform surgical implantation of a chronic indwelling catheter in the right jugular vein. Catheters were flushed daily with heparin (100 U/mL) and ampicillin (50 mg/mL) to prevent clotting and infection. Animals were then housed individually. Following a 7-day recovery period, rats were submitted to behavioral experiments conducted during the dark period. Each operant chamber was equipped with two 2.5 cm-diameter holes, 4 cm above the floor. Holes were selected as active (delivering cocaine), and inactive (without programmed consequence), and counterbalanced across groups. When the required number of active nose-pokes was reached, cocaine 60 µL was delivered [START_REF] Romieu | Histone deacetylase inhibitors decrease cocaine but not sucrose self-administration in rats[END_REF]. For molecular studies, rats followed a fixed ratio (FR) 1 schedule of reinforcement (cocaine, 0.33 mg/kg/injection, 2h/session/10d) concurrently with saline control rats. For the pharmacological study, rats were first trained for 2 h daily sessions under FR1 (3d) and FR5 (6d) and then submitted to a PR schedule, in which the number of nosepokes required to earn an injection escalated within the session. Rats were trained for 6 consecutive daily sessions before the pharmacological treatment with LSP2-9166 or vehicle (5d). Each session lasted for 5h or until animals did not achieve the ratio for delivery of an injection within 1h. The breaking point to extinguish SA behavior was determined in each animal.

Horizontal locomotor activity

Locomotor activity was measured in individual dedicated home-cages with two infrared light beams. Numbers of longitudinal crossings were counted in 5 min bins. Three groups of rats implanted with catheters were processed (vehicle/NaCl; vehicle /Cocaine; LSP/Cocaine). Rats were first habituated to the actimetry cages and injections (vehicle) (3d).

Following habituation to the cages (1h), rats received either LSP2-9166 (5 mg/kg, i.v.) or vehicle, and locomotor activity was recorded for 15 min. Then rats received cocaine (1.5 mg/kg, iv) or NaCl 0.9%, and activity was recorded for 1h.

Brain sample dissection and quantitative real-time PCR

Animals were given an overdose of pentobarbital (182 mg/kg, i.p.), 24h following the last SA session, brains were removed and sliced (1-mm coronal brain matrix, Harvard apparatus, MA, USA). Structures of interest were collected [START_REF] Paxinos | The rat brain in stereotaxic coordinates[END_REF] (see Figure 2). Samples were frozen on dry ice and kept at -80°C. Total RNA was extracted using Ribozol (VWR, France).

Reverse transcription was performed on 750 ng of total RNA, with iScript (iScript TM cDNA Synthesis Kit, Biorad, France). Real-time PCR was performed in triplicate using a CFX96 Touch TM apparatus and Sso Advanced TM Universal SYBR Green supermix (Biorad, France).

Thermal cycling parameters were 30sec at 95°C followed by 40 cycles of 5sec at 95°C and 45sec at 60°C. Primer sequences: Rplp0 Fw CTGCCCGAGCCGGTGCCATC, Rv TTCAATGGTACCTCTGGAG ; Grm4, Fw TCCAGGACCAACGGACACTT Rv ACGTGACCATCAGCAGCATG ; Grm7 Fw AGACACAGAAGGGAACGCCT, Rv TCGGTTCTCATTGGGCCTCT. Expression levels were normalized to rplp0 housekeeping gene levels and compared between saline and treated samples using the 2 -ΔΔCt method [START_REF] Livak | Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method[END_REF].

Statistical analysis

All results are expressed as mean  sem. Data from qPCR were analyzed using unpaired student's t-test. Data from SA experiments were analyzed using repeated measures ANOVAs with group as between-subject factor and treatment and session as within-subject factors. Data from locomotor activity experiments were analyzed using ANOVA with group as between-subject factor and bin as within-subject factor. The ANOVAs were followed by a Tukey HSD posthoc test when required. Significance was set at p ≤ 0.05 (Statistica v13).

Results

Effect of cocaine on mGlu4 and mGlu7 receptor gene expression

We first examined whether voluntary cocaine intake would modulate mGlu4 and mGlu7 receptor gene expression in reward-related brain structures. Following 10 days of cocaine SA (2h/d/FR1), rats displayed a stable level of cocaine intake (>100 injections/session) and directed more than 90% of the nose-pokes onto the active hole (data not shown). Rats receiving saline did not differentiate between holes (37.8±1.34% in the active hole). Microdissected brain samples were processed to measure gene expression levels by qPCR (Figure 2). Both mGlu4 and mGlu7 transcripts were detectable, with a higher expression (> 2-fold) of mGlu7 compared to mGlu4 in NAc, PFC and HPC, as previously described [START_REF] Corti | Distribution and synaptic localisation of the metabotropic glutamate receptor 4 (mGluR4) in the rodent CNS[END_REF][START_REF] Kosinski | Localization of metabotropic glutamate receptor 7 mRNA and mGluR7a protein in the rat basal ganglia[END_REF] (data not shown). Expression of both transcripts were significantly higher in cocaine-compared to saline-treated animals in NAc (Figure 2B) (Grm4 p=0.0011; Grm7 p=0.0005) and HPC (Figure 2D) (Grm4 p=0.0023; Grm7 p=0.0047). A significant decrease was observed for mGlu4 in Amy (Figure 2E) (Grm4 p=0.0024) while no regulations were observed for mGlu7 in Amy and for both receptors in PFC (Figure 2A) and DS (Figure 2C).

Effect of LSP-9166 on cocaine motivation

We performed cocaine SA under a PR schedule of reinforcement (see methods). Treatment with LSP2-9166 (0, 2 or 5 mg/kg i.v.) began 15 min before each session, for 5 days. The choice of the doses was based on preliminary experiments and literature [START_REF] Hajasova | Role of mGlu7 receptor in morphine rewarding effects is uncovered by a novel orthosteric agonist[END_REF][START_REF] Lebourgeois | Pharmacological activation of mGlu4 and mGlu7 receptors, by LSP2-9166, reduces ethanol consumption and relapse in rat[END_REF][START_REF] Girard | The mGlu7 receptor provides protective effects against epileptogenesis and epileptic seizures[END_REF]. Several SA sessions were necessary for the rats to display stable behavior, the ANOVA analysis showing no statistical difference between sessions 5 and 6 [F(1, 38)=5.36, p=0.97] (Figure 3A). Therefore, statistical analysis was performed on data from the last two sessions of each treatment (sessions 5/6 and sessions 10/11). The ANOVA (treatment, group, session) revealed a significant treatment effect [F(1, 38)=6.97, p=0.012] and a significant treatment x group interaction [F(2, 38)=3.66, p=0.035)], without significant effect of the sessions nor interaction between session and the other factors. Tukey HSD posthoc test showed that rats receiving vehicle or LSP2-9166 2 mg/kg did not display any behavioral changes in comparison to baseline (vehicle, p=0.97 and LSP2-9166 2 mg/kg p=0.99). In contrast, administration of LSP2-9166 5 mg/kg resulted in a significant reduction in the breaking point (p=0.004). We noted similar results for the active nose-pokes (treatment x group interaction, F(2,38)=5, p=0.012), with significant difference between baseline and LSP2-9166 5 mg/kg (p<0.001). A global decrease was observed for the inactive nose-poke (significant treatment effect, F(1,38)=5, p=0.016) but without any significant group effect (p=0.69), suggesting a motivational rather than a motor effect of the compound (Figure 3B).

Effect of LSP-9166 on locomotor activity

We then evaluated the effect of LSP2-9166 (5 mg/kg) on spontaneous locomotor activity (first injection) and on cocaine-induced hyperlocomotion (second injection) (Figure 3C).

Following the first injection (bin 60-75), the ANOVA revealed a significant interaction (bin x treatment, F(6, 72) = 2.24, p=0.049). Locomotor activity increased only in vehicle/NaCl and vehicle/Cocaine groups (bin 60-65, p<0.0002; LSP/Cocaine, p=0.75) (Tukey HSD test). No significant differences were observed between the 3 groups for all the bins (60-75). This indicates that LSP2-9166 reduced spontaneous activity-induced by the injection. For the second injection (bin 75-135) a significant interaction (bin x group F(24, 288) = 3.03, p= 0.000006) was observed. The Tukey HSD posthoc analysis showed a significant increase of locomotor activity in all three groups compared to their baseline (bin 75-80 p<0.0004). A significant difference was observed at bin 80 between vehicle/Cocaine and the two other groups (p<0.002), suggesting a reduction of cocaine-induced locomotor activity in presence of LSP2-9166. No significant differences were observed between the 3 groups for the other bins of the analyzed period. These results indicated a global hypolocomotor activity induced by LSP2-9166.

Discussion

In this study, we show that voluntary cocaine intake induces an increase of mGlu4 and mGlu7 receptor transcripts in NAc and HPC. The orthosteric agonist LSP2-9166 inhibits the motivation to obtain the drug in a PR schedule of reinforcement, although this ligand induces a hypolocomotor effect. An increasing interest has emerged in the addiction field directed to mGlu receptors, which are well known to regulate glutamatergic neurotransmission [START_REF] Cartmell | Regulation of Neurotransmitter Release by Metabotropic Glutamate Receptors[END_REF][START_REF] Souza | Glutamatergic transmission in drug reward: implications for drug addiction[END_REF].

Group III mGlu receptor ligands were developped, with LSP1-2111 [START_REF] Selvam | Increased Potency and Selectivity for Group III Metabotropic Glutamate Receptor Agonists Binding at Dual sites[END_REF], an agonist for mGlu4, and AMN802 [START_REF] Mitsukawa | From The Cover: A selective metabotropic glutamate receptor 7 agonist: Activation of receptor signaling via an allosteric site modulates stress parameters in vivo[END_REF], acting as the first allosteric mGlu7 agonist. Recently, the orthosteric LSP2-9166 efficaciously decreased expression and reinstatement of a morphine CPP in mice [START_REF] Hajasova | Role of mGlu7 receptor in morphine rewarding effects is uncovered by a novel orthosteric agonist[END_REF] as well as ethanol consumption, motivation for ethanol and reacquisition of ethanol SA following abstinence in rats [START_REF] Lebourgeois | Pharmacological activation of mGlu4 and mGlu7 receptors, by LSP2-9166, reduces ethanol consumption and relapse in rat[END_REF]. Our hypothesis that such modulatory effects may be expanded to cocaine behavioral responses has been tested here and we confirm a reduced motivation for drug taking in an operant paradigm.

Our results on locomotor activity have to be taken into account in the interpretation of the effects of LSP2-9166 on the motivation for cocaine. We observed a decreased spontaneous locomotor activity following a single injection of LSP2-9166 and a reduction in cocaine-induced hyperlocomotion. Interestingly, hypolocomotor effect of acute LSP2-9166 was previously reported in rats [START_REF] Lebourgeois | Pharmacological activation of mGlu4 and mGlu7 receptors, by LSP2-9166, reduces ethanol consumption and relapse in rat[END_REF], at high doses in mice [START_REF] Hajasova | Role of mGlu7 receptor in morphine rewarding effects is uncovered by a novel orthosteric agonist[END_REF], with a small transient inactive state resembling catalepsy [START_REF] Girard | The mGlu7 receptor provides protective effects against epileptogenesis and epileptic seizures[END_REF], with no effect on motor coordination in this latter study. Also no difference was detected on circular locomotor activity or rotarod test following systemic injection of LSP2-9166 (10 mg/kg ip,14 d) [START_REF] Girard | The mGlu7 receptor provides protective effects against epileptogenesis and epileptic seizures[END_REF]. In addition, it did not impact other behaviors like sucrose intake or spatial memory [START_REF] Hajasova | Role of mGlu7 receptor in morphine rewarding effects is uncovered by a novel orthosteric agonist[END_REF][START_REF] Lebourgeois | Pharmacological activation of mGlu4 and mGlu7 receptors, by LSP2-9166, reduces ethanol consumption and relapse in rat[END_REF], suggesting a moderate locomotor impact on behavior. Moreover, mice deficient for mGlu4 receptor showed a higher basal locomotor activity [START_REF] Blednov | Mice lacking metabotropic glutamate receptor 4 do not show the motor stimulatory effect of ethanol[END_REF], and mGlu7 receptor knockout mice presented a significant reduction of amphetamine-induced hyperlocomotor activity [START_REF] Fisher | Phenotypic profiling of mGlu7 knockout mice reveals new implications for neurodevelopmental disorders[END_REF], compared with wild-type animals. Also, activation of mGlu7 receptors by AMN802 decreased activity in rodents [START_REF] Salling | Nonselective suppression of operant ethanol and sucrose self-administration by the mGluR7 positive allosteric modulator AMN082[END_REF][START_REF] Palucha | Activation of the mGlu7 receptor elicits antidepressant-like effects in mice[END_REF] whereas no effect on locomotor activity was reported following LSP1-2111, a mGlu4 receptor orthostetic agonist, used at a lower dose [START_REF] Zaniewska | Inhibitory actions of mGlu4 receptor ligands on cocaine-, but not nicotine-, induced sensitizing and conditioning locomotor responses in rats[END_REF]. In addition, anxiety may play a role in the effect of LSP2-9166 on cocaine-induced behavior, as it is known that a higher level of anxiety may decrease the motivation for cocaine in a PR paradigm [START_REF] Bush | Individual differences in elevated plus-maze exploration predicted progressive-ratio cocaine self-administration break points in Wistar rats[END_REF]. Both anxiogenic and anxiolytic effects of LSP2-9166 have been described, at a high dose in mice [START_REF] Hajasova | Role of mGlu7 receptor in morphine rewarding effects is uncovered by a novel orthosteric agonist[END_REF], and following chronic LSP2-9166 treatment in rats [START_REF] Girard | The mGlu7 receptor provides protective effects against epileptogenesis and epileptic seizures[END_REF], respectively. Altogether, such effect may depend on the dose and the species under study and we can not exclude any impact in our paradigm.

We show here that voluntary cocaine intake increased both Grm4 and Grm7 in NAc and HPC, whereas a decrease was observed for Grm4 in the Amy. These regulations may play a direct role on motivation (NAc) counterbalanced by an increase in anxiety (Amy). Such adaptations may participate to a global homeostatic regulation following cocaine intake, that involves complex neuronal circuitry.These regulations are region-specific as no modification were detected in the PFC or DS. Also, they were observed following 10 d of cocaine intake, 24 h after the last drug exposure, suggesting that they do not result from short term effects of cocaine. It would be of crucial interest to evaluate whether these regulations are long-lasting, reversible and whether they occur at distinct stages of addiction (withdrawal, seeking events), to highlight changes that could specifically mediate active drug seeking. No data are yet available on the regulation of these receptors by other drugs of abuse, at the protein level.

In our conditions, the breaking point under a PR schedule was reduced, indicating that the LSP2-9166 compound was able to reduce the motivational properties of cocaine. Our results are in accordance with previous publications showing that AMN082 reduces cocaine intake under a PR schedule of reinforcement [START_REF] Li | Metabotropic Glutamate Receptor 7 Modulates the Rewarding Effects of Cocaine in Rats: Involvement of a Ventral Pallidal GABAergic Mechanism[END_REF]. Nevertheless, the neurobiological mechanisms involved in these effects are still not clear. The LSP2-9166 reduced cocaine reward-related effects probably through attenuation, via group III mGlu receptors activation, of the hyperglutamatergic state. Indeed, LSP2-9166 was demonstrated to be highly potent at mGlu4 (EC50 = 0.06 µM) and mGlu7 (EC50 = 2.2 µM) [START_REF] Acher | Hypophosphorous acid derivatives having antihyperalgic activity and biological applications thereof[END_REF] receptors. By increasing the activity of mGlu4/7 located in presynaptic terminals of glutamatergic afferents coming from the PFC, Amy and/or HPC, LSP2-9166 may lower glutamate release. Consequently, this would decrease the strength of goal-directed behavior to obtain the drug. Another hypothesis would be that the effects of LSP2-9166 are mediated by a reduction of GABA release on NAc neuronal targets, such as ventral pallidum and/or pars reticulata of the substantia nigra. Indeed, activation of group III mGlu receptors is able to weaken GABA neurotransmission, for instance in globus pallidum [START_REF] Macinnes | Group III metabotropic glutamate receptors act as heteroreceptors modulating evoked GABA release in the globus pallidus in vivo[END_REF]. Future electrophysiological or neurochemical studies will investigate these effects on cocaine-induced glutamate and/or GABA neurotransmission.

The LSP2-9166 ligand also blocked morphine CPP expression and reinstatement after extinction [START_REF] Hajasova | Role of mGlu7 receptor in morphine rewarding effects is uncovered by a novel orthosteric agonist[END_REF]. Noticeably, it was more efficient in Hajasova et al.'s study since effects were already observed at lower doses (0.5 mg/kg, ip). The explanations for such discrepancies are still unclear and may be related to differences in species, behavioral paradigms, and/or the nature of the drug studied. Another explanation could be that mGlu receptors form heterodimers that may strongly modify the ligand affinity, as very recently described for mGlu2/7 receptors [START_REF] Habrian | Conformational pathway provides unique sensitivity to a synaptic mGluR[END_REF], or their constitutive activity, for mGlu4/7 [START_REF] Habrian | Conformational pathway provides unique sensitivity to a synaptic mGluR[END_REF][START_REF] Kammermeier | Constitutive activity of metabotropic glutamate receptor 7[END_REF]. Such molecular interactions could occur between mGlu4/7 in specific reward-related brain structures, in a distinct manner depending on the drugs of abuse, and thus participate to the different effective doses observed between the studies.

Aknowledgments

This project was supported by the Université de Strasbourg, the Centre National de la Recherche Scientifique (CNRS) and by the « Innovation thérapeutique pour les maladies mentales » (ITMM CNRS). DN and CC received a PhD fellowship from the French Ministère de l'Enseignement Supérieur et de la Recherche". DN received a 4th year PhD funding from

Figure 1 .

 1 Figure 1. Optimization of LSP2-9166 synthesis. Reagents and conditions: (i) NH4H2PO2 (2.5 eq), Et3B (1 eq), O2, MeOH, RT, 30 min, 2 times; (ii) tert-butyl 2-(4-formyl-2-(trifluoromethoxy)phenoxy) acetate (1 eq), N,O-bis(trimethylsilyl) acetamide (BSA) (4 eq), degassed CH2Cl2, RT, 24 h; (iii) a) 3 M aqueous HCl, THF, RT, 48 h; b) C-18 flash chromatography. Bu: butyl; Boc; tert-butoxycarbonyl; BSA: N,O-bis(trimethylsilyl)acetamide; eq: equivalent; RT: room temperature.

Figure 2 .

 2 Figure 2. Cocaine effect on mGlu group III receptor gene expression. Transcript levels for Grm4 and Grm7 genes were examined by qPCR in PFC (A, 7-8/group), NAc (B, 7-9/group), DS (C, 6-8/group), HPC (D, 5-10/group) and Amy (E, 6-8/group) following cocaine SA. Bars represent mean ± SEM fold change vs saline group for each receptor. **p < 0.01; ***p < 0.001. Samples were dissected with large (3 mm) or small (1.9 mm) punchers as indicated.

Figure 3 .

 3 Figure 3. Effect of LSP2-9166 on cocaine-self administration and locomotor activity. Effect of LSP2-9166 (2 and 5 mg/kg i.v., 15 min before the operant session) on cocaine SA in a progressive ratio (PR) schedule of reinforcement. Number of injections (A) and number of nose-pokes (B) are presented across all sessions. Results are expressed as mean ± SEM for each group (vehicle, n=18; LSP2-9166, 2mg/kg, n=8; LSP2-9166, 5mg/kg, n=15). **p < 0.01, baseline (S5/S6) vs treatment (S10/S11) of LSP2-9166 5mg/kg. Locomotor activity (C) was recorded following habituation (1h), i.v. injection of LSP2-9166 (5 mg/kg, iv) or vehicle (bin 60-75), and then cocaine (1.5 mg/kg) or NaCl 0.9% (bin 75-135); (vehicle/NaCl n=8; vehicle/cocaine n=10; LSP2-9166/cocaine n=9). ***p<0.001 vehicle/cocaine vs LSP/cocaine at bin 80.

Abbreviations