Gilberto Fisone
email: gilberto.fisone@ki.se

Emmanuel Valjent

Jesus Bertran-Gonzalez

Heather Bowling

Sébastien Lopez

Emanuela Santini

Miriam Matamales

Alessandra Bonito-Oliva

Denis Hervé

Charles Hoeffer

Eric Klann

Jean-Antoine Girault

Haloperidol regulates the state of phosphorylation of ribosomal protein S6 via activation of PKA and phosphorylation of DARPP-32

Keywords: antipsychotics, adenosine A2A receptor, dopamine D2 receptor, medium spiny neurons, protein phosphatase-1, striatum

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Introduction

The ability to antagonize neurotransmission at dopamine D2 receptors (D2Rs) is generally regarded as an important feature of drugs used in the treatment of schizophrenia and other psychotic disorders [START_REF] Creese | Dopamine receptor binding predicts clinical and pharmacological potencies of anti-schizophrenic drugs[END_REF]. However, blockade of D2Rs leads to the development of irreversible side-effects, including tardive dyskinesia and parkinsonism, which seriously hamper the use of effective medications. These motor disorders have been proposed to depend on the ability of antipsychotic drugs to affect neurotransmission in the dorsal striatum, a major component of the basal ganglia [START_REF] Robertson | Neuroleptics increase c-fos expression in the forebrain: contrasting effects of haloperidol and clozapine[END_REF]Wan et al, 1995). In this region, D2Rs are selectively expressed by a large group of GABAergic medium spiny neurons (MSNs), which project to the external segment of the globus pallidus [START_REF] Gerfen | The neostriatal mosaic: multiple levels of compartmental organization in the basal ganglia[END_REF]. Acting through a polysynaptic circuit, these cells modulate the activity of thalamo-cortical neurons involved in the control of motor function [START_REF] Albin | The functional anatomy of basal ganglia disorders[END_REF][START_REF] Gerfen | The neostriatal mosaic: multiple levels of compartmental organization in the basal ganglia[END_REF]. For these reasons, striatopallidal MSNs are thought to play a critical role in the motor side effects associated to the use of antipsychotic drugs.

One of the primary actions exerted in the brain by conventional antipsychotics, such as haloperidol, is to promote cAMP-dependent signaling, which is normally inhibited by D2Rs through activation of Gi/o proteins [START_REF] Stoof | Opposing roles for D-1 and D-2 dopamine receptors in efflux of cyclic AMP from rat neostriatum[END_REF]. In striatopallidal MSNs, haloperidol-induced activation of cAMP-dependent protein kinase (PKA) and of the protein phosphatase-1 (PP-1) inhibitor, dopamine-and cAMP-regulated phosphoprotein of 32 kDa (DARPP-32), has been proposed to affect gene transcription by acting on nuclear targets, including the cAMP response element-binding protein and histone H3 [START_REF] Bertran-Gonzalez | Histone H3 phosphorylation is under the opposite tonic control of dopamine D2 and adenosine A2A receptors in striatopallidal neurons[END_REF][START_REF] Konradi | Haloperidol-induced Fos expression in striatum is dependent upon transcription factor cyclic AMP response element binding protein[END_REF][START_REF] Li | Dopamine D2-like antagonists induce chromatin remodeling in striatal neurons through cyclic AMP-protein kinase A and NMDA receptor signaling[END_REF][START_REF] Pozzi | Opposite regulation by typical and atypical anti-psychotics of ERK1/2, CREB and Elk-1 phosphorylation in mouse dorsal striatum[END_REF]. Much less is known about the ability of antipsychotic drugs and of the PKA/DARPP-32 cascade to affect signaling cascades involved in translational control.

The ribosomal protein S6 (rpS6) is a component of the small 40S ribosomal subunit implicated in mRNA decoding [START_REF] Ruvinsky | Ribosomal S6 phosphorylation: from protein synthesis to cell size[END_REF]. rpS6 is phosphorylated at multiple sites, comprised between Ser235 and Ser247, by the p70 rpS6 kinase (S6K) 1 [START_REF] Bandi | Identification of 40 S ribosomal protein S6 phosphorylation sites in Swiss mouse 3T3 fibroblasts stimulated with serum[END_REF][START_REF] Krieg | Identification of the 40 S ribosomal protein S6 phosphorylation sites induced by cycloheximide[END_REF], which is a major downstream effector of the mammalian target of rapamycin complex 1 (mTORC1) [START_REF] Costa-Mattioli | Translational control of long-lasting synaptic plasticity and memory[END_REF]. Phosphorylation of rpS6 at the dual site Ser235/236 occurs also independently of mTORC1, via the p90 rpS6 kinases (RSK), which are activated by the extracellular signal-regulated kinases (ERK) (Roux et al, 2007;[START_REF] Sturgill | Insulin-stimulated MAP-2 kinase phosphorylates and activates ribosomal protein S6 kinase II[END_REF]. Recent studies performed in pancreatic β-cells identified PKA as an additional rpS6 kinase, specifically involved in the phosphorylation of Ser235/236 [START_REF] Moore | Identification of cAMP-dependent kinase as a third in vivo ribosomal protein S6 kinase in pancreatic beta-cells[END_REF]. S6K-dependent phosphorylation of rpS6 plays an important role in the control of cell size [START_REF] Pende | S6K1(-/-)/S6K2(-/-) mice exhibit perinatal lethality and rapamycin-sensitive 5'-terminal oligopyrimidine mRNA translation and reveal a mitogen-activated protein kinasedependent S6 kinase pathway[END_REF][START_REF] Ruvinsky | Ribosomal protein S6 phosphorylation is a determinant of cell size and glucose homeostasis[END_REF] and protein synthesis-dependent plasticity, such as long-term potentiation [START_REF] Antion | Removal of S6K1 and S6K2 leads to divergent alterations in learning, memory, and synaptic plasticity[END_REF]. In addition, phosphorylation at Ser235/236 has been proposed to promote protein synthesis by facilitating rpS6 recruitment to the 5'cap complex, during the formation of the preinitiation translation complex (Roux et al, 2007). In this study, we examined the ability of haloperidol to regulate rpS6 phosphorylation in the MSNs of the dorsal striatum. We show that haloperidol increases the phosphorylation of rpS6 at Ser235/236, and that this effect is independent of ERK and mTORC1-mediated activation of S6K1, but requires intact PKA/DARPP-32 signaling.

Materials and Methods

Animals

Male C57BL/6J mice (25-30 g) were purchased from Taconic (Tornbjerg, Denmark).

Bacterial artificial chromosomes transgenic mice expressing EGFP under the control of the promoter for the D2R (Drd2-EGFP) or the dopamine D1 receptor (D1R; Drd1a-EGFP) were generated by the GENSAT (Gene Expression Nervous System Atlas) program at the Rockefeller University [START_REF] Gong | A gene expression atlas of the central nervous system based on bacterial artificial chromosomes[END_REF] and were crossed on a C57BL/6 background for at least 3 generations. Knock-in mice expressing a mutated form of DARPP-32, in which Thr34 is replaced by Ala (DARPP-32 T34A mutant mice), and Gnal +/-(Gα olf ) heterozygous mice were generated as described in previous studies [START_REF] Belluscio | Mice deficient in G(olf) are anosmic[END_REF][START_REF] Svenningsson | Diverse psychotomimetics act through a common signaling pathway[END_REF] and were backcrossed for at least 10 generations on a C57BL/6 background. S6K1 knockout mice were generated via direct gene replacement with a neomycin cassette (previously described in [START_REF] Shima | Disruption of the p70(s6k)/p85(s6k) gene reveals a small mouse phenotype and a new functional S6 kinase[END_REF] and were then outcrossed as described in [START_REF] Antion | Removal of S6K1 and S6K2 leads to divergent alterations in learning, memory, and synaptic plasticity[END_REF]. Animals were maintained in a 12-h light-dark cycle, in stable conditions of temperature (22°C), with food and water ad libitum. All experiments were carried out in accordance with the guidelines of the Swedish Animal Welfare Agency, the French Agriculture and Forestry Ministry (decree 87849, license A75-05-22), or the guidelines of the New York University Institutional Animal Care and Use Committee.

Drugs

Haloperidol (0.5 mg/kg, Sigma-Aldrich, Sweden) was dissolved in saline containing 0.05% (vol/vol) acetic acid and the pH was adjusted to 6.0 with 1 M NaOH.

Clozapine (5 mg/kg, Sigma-Aldrich, France) was dissolved in 0.9% NaCl. KW6002

(3 mg/kg) and SL327 (50 mg/kg), gifts from Dr. Edilio Borroni (Hoffmann-La Roche, Basel, Switzerland), were suspended by sonication in a solution of 5% (vol/vol) Tween 80 in saline and administered 5 and 30 min before haloperidol, respectively. Rapamycin (LC Laboratories, Woburn, MA, USA) (5 mg/kg) was dissolved in a solution of 5% dimethylsulfoxide (DMSO), 15% PEG-400 and 5% Tween-20, and was administered (once per day) in a volume of 5 ml/kg body weight, starting 3 days prior to administration of haloperidol. All drugs were injected intraperitoneally (i.p.).

Mice were habituated to handling and saline injection for three consecutive days before the experiment. For the chronic experiment, mice were injected once daily with vehicle or haloperidol (0.5 mg/kg) for a period of 15 days and perfused 15 min after a challenge injection with vehicle or haloperidol.

Primary culture of striatal neurons

Striata were dissected from embryos (E14) of OF1 mice (Charles River Laboratories France) and mechanically dissociated. Striatal cells were suspended in Neurobasal medium (Invitrogen) supplemented with B27 (Invitrogen), 0.5 mM L-glutamine, 100 units/mL penicillin, and 100 µg/mL streptomycin and then plated onto 35 cm culture dishes coated with poly-L-lysine, at 37°C in 5% CO2. Every 3-4 days, half of the medium was removed and replaced with fresh medium. After 16 days of culture, neurons were treated with drugs and incubations were stopped by replacing the medium with 0.2 mL of boiling SDS (1%, w/v). Protein concentration was determined by the BCA (bicinchoninic acid) assay kit (Pierce). Samples containing 30 µg of protein were analyzed by electrophoresis on 12% polyacrylamide gels and were subjected to separation and western blotting as described below for brain samples.

Immunofluorescence

Mice were rapidly anaesthetized with pentobarbital (500 mg/kg, i.p., Sanofi-Aventis, France) and transcardially perfused with 4% (weight/vol) paraformaldehyde in 0.1 M sodium phosphate buffer (pH 7.5) (Valjent et al, 2000). Brains were post-fixed overnight in the same solution and stored at 4°C. Thirty µm-thick sections were cut with a vibratome (Leica, France) and stored at -20°C in a solution containing 30% (vol/vol) ethylene glycol, 30% (vol/vol) glycerol and 0.1 M sodium phosphate buffer, until they were processed for immunofluorescence. Brain regions corresponding to the dorsal striatum were identified using a mouse brain atlas [START_REF] Franklin | The Mouse Brain in Stereotaxic Coordinates Academic Press[END_REF]) and sections at 1.10 mm from bregma were taken. Free-floating sections were rinsed in Tris-buffered saline (TBS; 0.25 M Tris and 0.5 M NaCl, pH 7.5), incubated for 5 min in TBS containing 3% H 2 O 2 and 10% methanol (vol/vol), and then rinsed three times for 10 min each in TBS. After 20 min incubation in 0.2% Triton X-100 in TBS, sections were rinsed three times in TBS again. Finally, rpS6 phosphorylation was analyzed by incubating sections overnight with rabbit polyclonal antibodies against phospho-Ser235/236-rpS6 (1:500, Cell Signalling Technology, Beverly, MA). NaF (0.1 mM) was included in all buffers and incubation solutions. In some experiments, the expression of Gα olf protein in wild type and Gnal +/-mice was assessed using rabbit polyclonal antibodies (1:500) [START_REF] Herve | Galpha(olf) levels are regulated by receptor usage and control dopamine and adenosine action in the striatum[END_REF]. Sections were then rinsed three times for 10 min in TBS and incubated for 45 minutes with goat Cy3-coupled (1:400, Jackson Laboratory, Bar Harbor, ME) secondary antibody.

Sections were rinsed for 10 min twice in TBS and twice in TB (0.25 M Tris) before mounting in Vectashield (Vector Laboratories, Burlingame, CA) or 1,4-diazabicyclo--[2. 2. 2]-octane (DABCO, Sigma-Aldrich Sweden AB, Stockholm). Single-and double-labeled images from each region of interest were obtained bilaterally using sequential laser scanning confocal microscopy (Leica SP2 and Zeiss LSM510).

Neuronal quantification was performed in 375 x 375 μm images by counting Cy3immunofluorescent nuclei (for phospho-Ser235/236-rpS6 immunolabeling). Cell counts were done by an observer unaware of the treatment received by the mice. A home-written program based on Metamorph software (Molecular Devices, Sunnyvale, CA) was used to compute all parameters.

Western blotting

Mice were killed by decapitation, their heads were cooled in liquid nitrogen for 6 seconds and the brains were removed. The striata were dissected out on an ice-cold surface, sonicated in 750 μl of 1% SDS and boiled for 10 min. Aliquots (5 μl) of the homogenate were used for protein determination using a BCA (bicinchoninic acid) assay kit (Pierce Europe, Oud Beijerland, the Netherlands). Equal amounts of protein (30 μg) for each sample were loaded onto 10% polyacrylamide gels. Proteins were separated by sodium dodecylsulfate-polyacrylamide gel electrophoresis and transferred overnight to PVDF membranes (Amersham Pharmacia Biotech, Uppsala, Sweden) [START_REF] Towbin | Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications[END_REF]. The membranes were immunoblotted using antibodies against phospho-Ser235/236-rpS6, phospho-Thr389-S6K, phospho-Thr202/Tyr204-ERK1/2 (Cell Signaling Technology, Beverly, MA, USA) and phospho-Thr34-DARPP-32 [START_REF] Snyder | Phosphorylation of DARPP-32 and protein phosphatase inhibitor-1 in rat choroid plexus: regulation by factors other than dopamine[END_REF]. Antibodies against rpS6, S6K (Cell Signaling Technology, Beverly, MA, USA) and DARPP-32 [START_REF] Hemmings | DARPP-32, a dopamine-and adenosine 3':5'monophosphate-regulated phosphoprotein: regional, tissue, and phylogenetic distribution[END_REF] that are not phosphorylation state specific were used to estimate the total amount of proteins. Detection was based on fluorescent secondary antibody binding and quantified using a Li-Cor Odyssey infrared fluorescent detection system (Li-Cor, Lincoln, NE) or with enhanced chemiluminescence reagent (GE Healthcare). The levels of each phosphoprotein were normalized for the amount of the corresponding total protein detected in the sample.

Statistical Analysis

Data were analyzed using one-or two-way ANOVA, and post hoc comparisons between groups were made using Bonferroni, or Newman-Keuls post hoc multiple comparison tests and T-tests with equal variances for groups of 2, when relevant.

Results

Haloperidol increases rpS6 phosphorylation selectively in D2R-expressing neurons

Systemic administration of haloperidol at the dose of 0.5 mg/kg, which has been previously shown to promote the phosphorylation of several proteins including PKA substrates [START_REF] Bertran-Gonzalez | Histone H3 phosphorylation is under the opposite tonic control of dopamine D2 and adenosine A2A receptors in striatopallidal neurons[END_REF][START_REF] Håkansson | Regulation of phosphorylation of the GluR1 AMPA receptor by dopamine D2 receptors[END_REF], produced a two-fold increase in the levels of phospho-Ser235/236-rpS6, as determined by western blotting analysis of striatal tissue (Fig. 1A). This effect peaked at 15 min post-injection and declined by 60 min (Fig. 1A). Immunofluorescence analysis revealed a strong increase in the number of phospho-Ser235/236 rpS6-positive neurons in the dorsal striatum following haloperidol administration (Fig. 1B).

In the dorsal striatum, D2Rs are selectively expressed by striatopallidal MSNs, whereas dopamine D1 receptors (D1Rs) are present in the other large group of projection neurons, which form the striatonigral pathway [START_REF] Gerfen | The neostriatal mosaic: multiple levels of compartmental organization in the basal ganglia[END_REF]. To examine the effects of haloperidol on rpS6 phosphorylation in these two neuronal populations, we used transgenic mice expressing EGFP under the control of the promoter for the D2R or D1R (Drd2-EGFP and Drd1a-EGFP mice) [START_REF] Gong | A gene expression atlas of the central nervous system based on bacterial artificial chromosomes[END_REF][START_REF] Valjent | Looking BAC at striatal signaling: cell-specific analysis in new transgenic mice[END_REF]. We found that the increase in rpS6 phosphorylation induced by haloperidol (0.5 mg/kg) occurred specifically in D2R containing neurons (Fig. 1C andD).

Overall, these results are in line with the ability of this antipsychotic to act specifically at dopamine D2-type receptors and, therefore, to affect preferentially striatopallidal MSNs [START_REF] Bateup | Cell type-specific regulation of DARPP-32 phosphorylation by psychostimulant and antipsychotic drugs[END_REF][START_REF] Bertran-Gonzalez | Opposing patterns of signaling activation in dopamine D1 and D2 receptor-expressing striatal neurons in response to cocaine and haloperidol[END_REF][START_REF] Bertran-Gonzalez | Histone H3 phosphorylation is under the opposite tonic control of dopamine D2 and adenosine A2A receptors in striatopallidal neurons[END_REF]. Interestingly, when administered chronically (one injection per day during two weeks) haloperidol lost the ability to increase rpS6 phosphorylation (Fig. 1E andF). Therefore, rpS6 phosphorylation is induced selectively in striatopallidal MSNs in response to acute blockade of D2-like receptor, but this effect appears to subside when the duration of treatment is extended.

We also examined the effect on rpS6 phosphorylation of clozapine, an atypical antipsychotic with low affinity for D2Rs. Clozapine (5 mg/kg) increased rpS6 phosphorylation at Ser235/236, selectively in D2R-expressing MSNs (Figure S1).

However, this effect was much lower and less sustained than that produced by haloperidol (cf. Fig. 1C andD).

Haloperidol-induced phosphorylation at Ser235/236 does not require mTORC1 and S6K1 and is independent of ERK S6Ks are primarily involved in the regulation of rpS6 [START_REF] Bandi | Identification of 40 S ribosomal protein S6 phosphorylation sites in Swiss mouse 3T3 fibroblasts stimulated with serum[END_REF][START_REF] Krieg | Identification of the 40 S ribosomal protein S6 phosphorylation sites induced by cycloheximide[END_REF][START_REF] Pende | S6K1(-/-)/S6K2(-/-) mice exhibit perinatal lethality and rapamycin-sensitive 5'-terminal oligopyrimidine mRNA translation and reveal a mitogen-activated protein kinasedependent S6 kinase pathway[END_REF][START_REF] Ruvinsky | Ribosomal protein S6 phosphorylation is a determinant of cell size and glucose homeostasis[END_REF]. Therefore, we examined their involvement in haloperidol-induced phosphorylation at Ser235/236. Administration of the mTORC1 inhibitor, rapamycin, prevented phosphorylation of S6K at Thr389 (Fig. 2C), but did not affect phosphorylation of rpS6 at Ser235/236 (Fig. 2A andB). The effect of haloperidol was also tested in mice deficient for S6K1 [START_REF] Pende | Hypoinsulinaemia, glucose intolerance and diminished beta-cell size in S6K1-deficient mice[END_REF] (Fig. 2E). In line with the results obtained with rapamycin, we found that, in these animals, the ability of haloperidol to increase rpS6 phosphorylation at Ser235/236 was preserved (Fig. 2D).

Other protein kinases that phosphorylate rpS6 on Ser235/236 are the p90 ribosomal S6 kinases (RSK), which are activated by ERK (Roux et al, 2007). The ERK/RSK cascade accounts for the remaining phosphorylation of rpS6 on Ser235/236 observed in S6K1/S6K2 double knock out mice [START_REF] Pende | S6K1(-/-)/S6K2(-/-) mice exhibit perinatal lethality and rapamycin-sensitive 5'-terminal oligopyrimidine mRNA translation and reveal a mitogen-activated protein kinasedependent S6 kinase pathway[END_REF]. We therefore examined the involvement of this pathway in rpS6 phosphorylation. We found that blockade of ERK (Fig. 3C), achieved using SL327 [an inhibitor of the mitogen-activated protein kinase/ERK kinase (MEK)], did not affect the increase in rpS6 phosphorylation produced by haloperidol (Fig. 3A andB).

Haloperidol-induced phosphorylation of rpS6 depends on adenosine A2A receptor-mediated activation of Gα olf

The data presented above indicate that, in striatal neurons, the two major pathways known to control rpS6 phosphorylation at Ser235/236 are not involved in its regulation in response to haloperidol. Therefore, we examined the possible role of the cAMP-dependent cascade. In addition to D2Rs, striatopallidal MSNs express high levels of the A2A receptor subtype (A2AR) for adenosine. A2ARs are coupled to a Gα olf protein, which increases cAMP synthesis via activation of adenylyl cyclase [START_REF] Corvol | Galpha(olf) is necessary for coupling D1 and A2a receptors to adenylyl cyclase in the striatum[END_REF][START_REF] Kull | Adenosine A(2A) receptors are colocalized with and activate g(olf) in rat striatum[END_REF]. The ability of D2R antagonists to promote cAMP signaling has been previously shown to depend in large part on intact A2ARmediated transmission [START_REF] Bertran-Gonzalez | Histone H3 phosphorylation is under the opposite tonic control of dopamine D2 and adenosine A2A receptors in striatopallidal neurons[END_REF][START_REF] Borgkvist | Psychoactive drugs and regulation of the cAMP/PKA/DARPP-32 cascade in striatal medium spiny neurons[END_REF][START_REF] Håkansson | Regulation of phosphorylation of the GluR1 AMPA receptor by dopamine D2 receptors[END_REF][START_REF] Svenningsson | Regulation of the phosphorylation of the dopamine-and cAMP-regulated phosphoprotein of 32 kDa in vivo by dopamine D1, dopamine D2, and adenosine A2A receptors[END_REF]. Therefore we examined the effect produced by the selective A2AR antagonist, KW6002, on haloperidol-induced rpS6 phosphorylation. We found that blockade of A2ARs with 3 mg/kg of KW6002 reduced the effect of haloperidol on phosphoSer235/236-rpS6 (Fig. 4A andB).

A similar decrease in haloperidol-induced rpS6 phosphorylation was observed in Gnal +/-mice carrying a heterozygous mutation of the gene encoding for Gα olf , which reduces the levels of this protein in the striatum [START_REF] Corvol | Quantitative changes in Gαolf protein levels, but not D1 receptor, alter specifically acute responses to psychostimulants[END_REF] (Fig. 5). This observation was particularly interesting, since in these mice the levels of Gα olf are decreased by approximately 50% (cf. Fig. 5A), which results in a large reduction of cAMP signaling [START_REF] Corvol | Quantitative changes in Gαolf protein levels, but not D1 receptor, alter specifically acute responses to psychostimulants[END_REF]. Taken together, these results indicate that haloperidol increases rpS6 phosphorylation by removing the inhibitory tone normally exerted by D2Rs on A2AR-mediated cAMP production.

We used striatal neurons in culture to further explore whether activation of A2ARs could regulate rpS6 phosphorylation on Ser235/236 through cAMP signaling.

Application of the A2AR agonist CPCA (200 µM) induced the phosphorylation of rpS6 on Ser235/236 in striatal neurons (Fig. 6A). This effect was mediated by PKA, since it was abolished in the presence of the PKA inhibitor H-89 (50 µM) (Fig. 6A).

Moreover, Sp-5,6-DCl-cBIMPS (10 µM), a cAMP analog and PKA activator, induced a marked increase in rpS6 phosphorylation on Ser235/236 (Fig. 6B). Thus, our results in vivo and in vitro provide converging evidence that the effect of haloperidol on rpS6 Ser235/236 phosphorylation is mediated by activation of the cAMP pathway.

Haloperidol-induced phosphorylation of rpS6 depends on PKA-mediated activation of DARPP-32

In the striatum, a large part of the effects produced by activation of cAMP signaling depends on DARPP-32 [START_REF] Nairn | The role of DARPP-32 in the actions of drugs of abuse[END_REF]. PKA-mediated phosphorylation at Thr34 converts DARPP-32 into an inhibitor of PP-1, thereby suppressing the dephosphorylation of numerous effector targets acting downstream of the cAMP/PKA cascade [START_REF] Greengard | The neurobiology of slow synaptic transmission[END_REF]. Haloperidol is known to increase DARPP-32 phosphorylation at Thr34 in vivo [START_REF] Pozzi | Opposite regulation by typical and atypical anti-psychotics of ERK1/2, CREB and Elk-1 phosphorylation in mouse dorsal striatum[END_REF]. Western blot analysis revealed a significant increase in the levels of phospho-Thr34-DARPP-32, determined in our experimental conditions 15 and 60 min after administration of haloperidol (Fig. 7A).

In order to assess the involvement of DARPP-32 in the haloperidol-mediated phosphorylation of rpS6, we used DARPP-32 T34A mutant mice, in which replacement of Thr34 by Ala prevents PKA-mediated phosphorylation (Fig. 7B). We found that, in these mice, haloperidol did not produce any change in the state of phosphorylation of rpS6 (Fig. 7C andD). These data point to the PKA/DARPP-32 signaling pathway as a major modulator of rpS6 in striatal neurons and indicate that phosphorylation at Ser235/236 is under the control of PP-1.

Discussion

This study demonstrates that administration of the antipsychotic drug and D2R antagonist, haloperidol, promotes phosphorylation of rpS6 at Ser235/236, specifically at the level of the MSNs of the indirect, striatopallidal pathway. They also show that this effect is exerted by promoting A2AR-mediated activation of the cAMP/DARPP-32 signaling cascade and that it does not involve activation of S6K, or ERK/RSK.

In striatopallidal neurons, blockade of D2Rs increases the state of phosphorylation of several substrates of PKA [START_REF] Bertran-Gonzalez | Opposing patterns of signaling activation in dopamine D1 and D2 receptor-expressing striatal neurons in response to cocaine and haloperidol[END_REF][START_REF] Bertran-Gonzalez | Histone H3 phosphorylation is under the opposite tonic control of dopamine D2 and adenosine A2A receptors in striatopallidal neurons[END_REF][START_REF] Håkansson | Regulation of phosphorylation of the GluR1 AMPA receptor by dopamine D2 receptors[END_REF][START_REF] Li | Dopamine D2-like antagonists induce chromatin remodeling in striatal neurons through cyclic AMP-protein kinase A and NMDA receptor signaling[END_REF][START_REF] Pozzi | Opposite regulation by typical and atypical anti-psychotics of ERK1/2, CREB and Elk-1 phosphorylation in mouse dorsal striatum[END_REF]. This effect has been attributed to the removal of the inhibitory tone exerted by D2Rs on adenylyl cyclase, which leads to the accumulation of cAMP and activation of PKA. The ability of D2R antagonists, including haloperidol, to promote cAMP/PKA signaling depends on A2ARs [START_REF] Bertran-Gonzalez | Histone H3 phosphorylation is under the opposite tonic control of dopamine D2 and adenosine A2A receptors in striatopallidal neurons[END_REF][START_REF] Borgkvist | Psychoactive drugs and regulation of the cAMP/PKA/DARPP-32 cascade in striatal medium spiny neurons[END_REF][START_REF] Håkansson | Regulation of phosphorylation of the GluR1 AMPA receptor by dopamine D2 receptors[END_REF][START_REF] Svenningsson | Regulation of the phosphorylation of the dopamine-and cAMP-regulated phosphoprotein of 32 kDa in vivo by dopamine D1, dopamine D2, and adenosine A2A receptors[END_REF], which maintain basal adenylyl cyclase activity via activation of Gα olf [START_REF] Corvol | Galpha(olf) is necessary for coupling D1 and A2a receptors to adenylyl cyclase in the striatum[END_REF]. The present results, showing that blockade of A2ARs or reduction of the levels of Gα olf prevent the haloperidol-induced increase in Ser235/236, indicate the importance of PKA in this phenomenon. This idea is further supported by the observation that, in cultured striatal neurons, A2AR-mediated increase in Ser235/236 phosphorylation is blocked by PKA inhibition and is mimicked by PKA activation. These observations are in line with the recent identification of PKA as a third in vivo rpS6 kinase in pancreatic β-cells [START_REF] Moore | Identification of cAMP-dependent kinase as a third in vivo ribosomal protein S6 kinase in pancreatic beta-cells[END_REF].

The ability of the cAMP/PKA signaling pathway to regulate the activity of downstream target proteins depends on concomitant phosphorylation of DARPP-32 at Thr34, which suppresses protein dephosphorylation via inhibition of PP-1 [START_REF] Greengard | The neurobiology of slow synaptic transmission[END_REF]. Thus, mutation of DARPP-32 at the PKA phosphorylation site, Thr34, prevents the ability of haloperidol and other D2R antagonists to increase the state of phosphorylation of the GluR1 subunit of glutamate AMPA receptors and of histone H3 [START_REF] Bertran-Gonzalez | Histone H3 phosphorylation is under the opposite tonic control of dopamine D2 and adenosine A2A receptors in striatopallidal neurons[END_REF][START_REF] Håkansson | Regulation of phosphorylation of the GluR1 AMPA receptor by dopamine D2 receptors[END_REF]. The present data indicate that a similar mechanism is involved in the regulation of rpS6 phosphorylation exerted by haloperidol at Ser235/236. Thus, the effect of haloperidol depends not only on increased PKA-catalyzed phosphorylation, but also on decreased dephosphorylation exerted by phospho-Thr34-DARPP-32, via inhibition of PP-1. In support of this type of regulation, it has been previously proposed that PP-1 acts as an S6 phosphatase [START_REF] Belandia | Attenuation of ribosomal protein S6 phosphatase activity in chicken embryo fibroblasts transformed by Rous sarcoma virus[END_REF].

The effect of haloperidol on rpS6 phosphorylation at Ser235/236 is exerted independently of S6K. Thus, administration of rapamycin, which suppresses S6K activity, or deletion of S6K1, does not prevent the ability of the D2R antagonist to promote rpS6 phosphorylation.

It has been shown that the ERK/RSK signaling pathway regulates rpS6 phosphorylation via activation of mTORC1 and S6K [START_REF] Ma | Phosphorylation and functional inactivation of TSC2 by Erk implications for tuberous sclerosis and cancer pathogenesis[END_REF][START_REF] Roux | Tumor-promoting phorbol esters and activated Ras inactivate the tuberous sclerosis tumor suppressor complex via p90 ribosomal S6 kinase[END_REF]. Moreover, ERK has been proposed to regulate rpS6 phosphorylation independently of mTORC1, via RSK-catalyzed phosphorylation at Ser235/236 (Roux et al, 2007). Our data show that blockade of ERK, achieved with the MEK inhibitor SL327, does not modify the ability of haloperidol to increase rpS6 phosphorylation. This shows that the modest increase of ERK phosphorylation produced by haloperidol in striatopallidal MSNs [START_REF] Bertran-Gonzalez | Opposing patterns of signaling activation in dopamine D1 and D2 receptor-expressing striatal neurons in response to cocaine and haloperidol[END_REF][START_REF] Bertran-Gonzalez | Histone H3 phosphorylation is under the opposite tonic control of dopamine D2 and adenosine A2A receptors in striatopallidal neurons[END_REF] is not involved in the regulation of rpS6.

Work performed in a mouse model of Parkinson's disease, shows that activation of D1Rs increases the phosphorylation of rpS6 at Ser235/236. This effect, which is exerted selectively in the MSNs of the direct pathway, depends on concomitant activation of ERK [START_REF] Santini | Inhibition of mTOR signaling in Parkinson's disease prevents L-DOPA-induced dyskinesia[END_REF]. Therefore, it appears that, in the striatum, the state of phosphorylation of rpS6 at the level of the two major neuronal populations (i.e. the MSNs of the direct and indirect pathway) is controlled by distinct signaling mechanisms.

The ability of haloperidol to increase the state of phosphorylation of rpS6 in the dorsal striatum represents a novel mechanism potentially implicated in the action of typical antipsychotic drugs. In particular, the present results suggest that the motor side-effects produced by these substances may be linked to changes in the efficiency of mRNA translation occurring in striatopallidal MSNs. In this regard, it is interesting to note that clozapine, an atypical antipsychotic with low liability for motor complications, produces a much more modest effect on rpS6 phosphorylation in comparison to haloperidol. In line with this observation, it has been shown that haloperidol, but not clozapine, increases the expression of c-fos in the dorsal striatum [START_REF] Robertson | Neuroleptics increase c-fos expression in the forebrain: contrasting effects of haloperidol and clozapine[END_REF]Wan et al, 1995). It is therefore possible that the emergence of tardive dyskinesia and parkinsonism in response to administration of typical antipsychotic drugs is related to modifications in the activity of striatal MSNs produced through a coordinated action on transcriptional and translational processes.

In conclusion, this study shows that the typical antipsychotic, haloperidol, increases the state of phosphorylation of rpS6 in striatopallidal MSNs independently of mTORC1/S6K and ERK/RSK signaling, through activation of cAMP signaling.

The data presented support a model in which blockade of D2Rs promotes PKAcatalyzed phosphorylation of Ser235/236 and suppresses PP-1-mediated Western blot analysis of phospho-Thr34-DARPP-32 (P-DARPP-32) in the striata of wildtype mice treated with haloperidol (0.5 mg/kg) and killed after 15 or 60 min. Top panels shows representative autoradiograms obtained using antibodies against P-DARPP-32 (upper) and total DARPP-32 (lower). Bottom panel is a summary of data

Figure 3 .

 3 Figure 3. Effect of MEK inhibition on haloperidol-induced phosphorylation of rpS6. Mice were treated with vehicle (Veh), haloperidol (Hal; 0.5 mg/kg), or Hal plus SL327 (50 mg/kg; administered 45 min prior to administration of Hal) and perfused 15 min later. (A) Phospho-Ser235/236-rpS6 (P-235/236-rpS6) immunoreactivity in single confocal sections of the dorsal striata of mice treated with Hal or Hal plus SL327. Scale bar: 30 μm. (B) Quantification of P-235/236-rpS6 immunoreactive neurons in the dorsal striata of mice treated with Veh, Hal, or Hal plus SL327. (** p < 0.01 vs. Veh). (C) Mice were treated with Veh, Hal (0.5 mg/kg), or Hal plus SL327 (50 mg/kg; see above) and killed after 15 min. Representative autoradiogram showing the reduction of phospho-P-ERK immunoreactivity in the striatum of a mouse treated with the MEK inhibitor.

Figure 4 .Figure 5 .Figure 6 .

 456 Figure 4. Effect of KW6002 on haloperidol-induced phosphorylation of rpS6. Mice were treated with vehicle (Veh), haloperidol (Hal), or Hal plus KW6002 (3 mg/kg; administered 5 min prior to administration of Hal) and perfused 15 or 60 min later. (A) Phospho-Ser235/236-rpS6 (P-235/236-rpS6) immunoreactivity in single confocal sections of the dorsal striatum from mice treated with Hal or Hal plus KW6002. Scale bar: 30 μm. (B) Quantification of P-235/236-rpS6 immunoreactive neurons in single confocal sections of the dorsal striata of mice treated with Veh, Hal, or Hal plus KW6002 (** p < 0.01 vs. Veh, °° p < 0.01 vs. Hal)

Figure 7 .

 7 Figure 7. Mutation of Thr34 to Ala in DARPP-32 prevents haloperidol-induced histone H3 phosphorylation. Wildtype and T34A mutant mice were treated with vehicle (Veh) or haloperidol (Hal; 0.5 mg/kg) and perfused 15 and 60 min later. (A)

dephosphorylation via DARPP-32. They also raise the possibility that antipsychotic drugs and D2R antagonists affect striatal transmission not only through control of transcriptional process but also via changes in translational efficiency.

Acknowledgments

This work was supported by Swedish Research Council grants 20715 and 13482, the Wenner-Gren Foundations and the Agence Nationale de la Recherche (ANR) in the frame of Era-Net NEURON (project ANR-08-NEUR-006-01) (to G.F.), the Fondation pour la Recherche Médicale (FRM) and grant ANR-08-BLAN-0287 (to J.A.G.) the National Institutes of Health grants NS034007 and NS047384 (to E.K.). S.L. was a recipient of a postdoctoral fellowship from the FEBS. A.B.O. was supported by the foundation Blanceflor Boncompagni-Ludovisi, née Bildt. The authors thank Dr. Paul Greengard for providing the T34A DARPP-32 mutant mice. Valjent E, Corvol JC, Pages C, Besson MJ, Maldonado R, Caboche J (2000). Involvement of the extracellular signal-regulated kinase cascade for cocainerewarding properties. J Neurosci 20(23): 8701-8709. Wan W, Ennulat DJ, Cohen BM (1995). Acute administration of typical and atypical antipsychotic drugs induces distinctive patterns of Fos expression in the rat forebrain. Brain Res 688(1-2): 95-104.

Conflict of interest

The authors declare no conflict of interest.

Legends to figures


Nicolas Narboux-Nême

Corinne Sagné

Stephane Doly

Cédric Silvina L Diaz

Bp Martin

Gaelle Angenard

Marie-Pascale Martres

Bruno Giros

Michel Hamon

Laurence Lanfumey

Cédric B P Martin

Patricia Gaspar
email: patricia.gaspar@inserm.fr

Raymond Mongeau

Severe serotonin depletion after conditional deletion of the vesicular monoamine transporter 2 gene in serotonin neurons: neural and behavioral consequences

Keywords: Knockout mice, vesicular monoamine transporter, serotonin transporter, depression, anxiety, 5-HT 1A receptors, raphe, antidepressant drugs, defense behaviors VMAT vesicular monoamine transporter protein, Vmat2: Vesicular monoamine transporter 2 gene, 5-HT: serotonin, SERT: serotonin transporter, MAOI: monoamine oxidase inhibitor, TPH2, tryptophane hydroxylase 2

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

Introduction

Serotonin (5-HT) has a central importance in the control of mood and anxiety states [START_REF] Lucki | The spectrum of behaviors influenced by serotonin[END_REF]. Dysfunctions of 5-HT neurotransmission can result from alterations at several critical points of monoamine metabolism such as synthesis, release, reuptake, catabolism, or serotonin receptors [START_REF] Morilak | Antidepressants and brain monoaminergic systems: a dimensional approach to understanding their behavioural effects in depression and anxiety disorders[END_REF]. All these constitute entry points to pharmacological therapeutic approaches [START_REF] Berton | New approaches to antidepressant drug discovery: beyond monoamines[END_REF] and to genetic variations that impact disease predisposition. Vesicular monoamine transporters (VMAT) appear as important targets in regard to the neurobiology of mood. Indeed, the initial formulation of the monoamine theory of depression was derived from studies with reserpine, an irreversible VMAT blocker, prescribed as an antihypertensive agent, subsequently found to cause depression in humans [START_REF] Freis | Mental depression in hypertensive patients treated for long periods with large doses of reserpine[END_REF]. Moreover, there is evidence for a link of depression with genetic polymorphism of the Vmat2 gene [START_REF] Christiansen | Candidate gene polymorphisms in the serotonergic pathway: influence on depression symptomatology in an elderly population[END_REF][START_REF] Gutierrez | Identification of two risk haplotypes for schizophrenia and bipolar disorder in the synaptic vesicle monoamine transporter gene (SVMT)[END_REF]).

The main function of VMAT is to concentrate biogenic amines into intracellular storage organelles such as synaptic vesicles, shielding them from degradation, and concentrating them for release into the synaptic cleft [START_REF] Henry | The vesicular monoamine transporter: from chromaffin granule to brain[END_REF]). Nevertheless, 5-HT can also function as a paracrine transmitter by acting on receptors distant from synaptic release sites [START_REF] Bunin | Paracrine neurotransmission in the CNS: involvement of 5-HT[END_REF], and non-vesicular release of neurotransmitters is known to occur in some physiological conditions [START_REF] Attwell | Nonvesicular release of neurotransmitter[END_REF][START_REF] Wu | Nonvesicular inhibitory neurotransmission via reversal of the GABA transporter GAT-1[END_REF].

VMATs exist as two different isoforms: VMAT1 mainly expressed in chromaffin and enterochromaffin cells, and VMAT2 essentially expressed in monoaminergic neurons [START_REF] Erickson | Distinct pharmacological properties and distribution in neurons and endocrine cells of two isoforms of the human vesicular monoamine transporter[END_REF][START_REF] Erickson | Functional identification and molecular cloning of a human brain vesicle monoamine transporter[END_REF]. Microdialysis (Adell and Artigas 1998) and voltametry [START_REF] Bunin | Release and uptake rates of 5-hydroxytryptamine in the dorsal raphe and substantia nigra reticulata of the rat brain[END_REF][START_REF] Connor | Fast cyclic voltammetry can be used to measure stimulated endogenous 5-hydroxytryptamine release in untreated rat brain slices[END_REF] experiments showed that tetrabenazine, a selective VMAT2 blocker, prevents the release of 5-HT in the raphe and in axon terminal fields while Vmat2-KO mice showed no release of amines [START_REF] Fon | Vesicular transport regulates monoamine storage and release but is not essential for amphetamine action[END_REF][START_REF] Wang | Knockout of the vesicular monoamine transporter 2 gene results in neonatal death and supersensitivity to cocaine and amphetamine[END_REF]). Vmat2-KO mice do not survive beyond the first postnatal days (Alvarez, et al. 2002;[START_REF] Fon | Vesicular transport regulates monoamine storage and release but is not essential for amphetamine action[END_REF][START_REF] Wang | Knockout of the vesicular monoamine transporter 2 gene results in neonatal death and supersensitivity to cocaine and amphetamine[END_REF] preventing the long-term evaluation of the consequences of a lack of monoamine/5-HT release. VMAT2 heterozygous mice with rather small (20-30%) reductions in brain amines showed no change in anxiety-like behaviors, but indications of a "depression-like" phenotype [START_REF] Fukui | Vmat2 heterozygous mutant mice display a depressive-like phenotype[END_REF]). However, it was unclear whether this phenotype was linked to a reduced release of 5-HT or of other amines (DA, NA, histamine) that also depend on VMAT2 for vesicular storage.

To overcome this limitation, we generated a conditional deletion of VMAT2 that allowed investigating the role of this transporter specifically in 5-HT neurons. Specific ablation of VMAT2 in raphe 5-HT neurons was obtained by Cre recombinase expressed under the control of the 5-HT transporter gene (SERT, slc6a4) promoter. We report that VMAT2 sert- cre mice have a near complete depletion of 5-HT in the brain, consequent to abolished vesicular 5-HT uptake. Raphe neurons develop normally and show a normal innervation of target areas. A consequence of the sustained 5-HT depletion was to increase the coupling of 5-HT 1A autoreceptors with its G protein, a known sensitive index of chronic changes in 5-HT levels. Behavioral observations in adults indicated that abolished synaptic 5-HT release caused anxiolytic-like phenotype, while increasing reactivity to innately aversive stimuli. The latter alteration was reversed by a 3-week treatment with pargyline, a monoamine oxidase inhibitor (MAOI), which markedly increased brain 5-HT levels. These observations are coherent with the notion that elevated levels of endogenous 5-HT in stressful situations can inhibit prepotent behaviors and is linked with anxiety [START_REF] Gray | The neuropsychology of anxiety[END_REF]. This study established VMAT2 sert-cre mice as a powerful model to analyze the role of 5-HT in otherwise normally developed raphe neurons.

Materials and methods

Animals

Procedures involving animals and their care were conducted in accordance with directives of the European Community (Council directive 86/609) and the French Agriculture and Forestry Ministry (Council directive 87-848, October 19, 1987, permissions 75-977 to L.L. and 00782 to P.G).

The floxed Vmat2 mouse line was produced at the Mouse Clinical Institute (Institut Clinique de la Souris, MCI/ICS, Illkirch, France) in coordination with unit Inserm U952 (Bruno Giros, to whom correspondence concerning the mouse line should be addressed: bruno.giros@inserm.fr ). A 9 kb fragment of the Vmat2 gene encompassing exons 1-3 was subcloned into the targeting vector and the two loxP sites and the neomycin-selectable cassette flanked by two FRT sites were introduced in the introns flanking the first coding exon. Embryonic stem cells were electroporated with the targeting vector and cell clones resistant to selection were screened by PCR and Southern analysis to identify clones that resulted from a correct targeting event (2 /744 clones analyzed). Excision of the neomycin cassette was performed in vivo by crossing chimeric mice with transgenic mice expressing the Flp recombinase under the control of the early beta-actin promoter. The presence of the floxed allele in the progeny was screened by PCR. VMAT2 lox/lox mice were maintained on a C57BL/6J background.

The SERT cre mouse line has previously been described [START_REF] Zhuang | Targeted gene expression in dopamine and serotonin neurons of the mouse brain[END_REF]. This is a gene trap construction, which replaces exon 14 of the serotonin transporter gene (sert) with a gene sequence encoding cre-recombinase. Previous studies showed that recombination occurs by E12 in the raphe neurons [START_REF] Narboux-Neme | Serotonin transporter transgenic (SERTcre) mouse line reveals developmental targets of serotonin specific reuptake inhibitors (SSRIs)[END_REF] and that virtually all TPH2 neurons in the raphe have recombined in postnatal life [START_REF] Zhuang | Targeted gene expression in dopamine and serotonin neurons of the mouse brain[END_REF]. Male VMAT2 lox/+ /SERT cre/+ or VMAT2 lox/lox /SERT cre/+ mice were mated with female VMAT2 lox/lox to generate the 3 genotypes that were analyzed: VMAT2 lox/lox /SERT cre/+ (recombined); VMAT2 lox/lox (control 1); VMAT2 lox/+ /SERT cre/+ (control 2). After weaning and sexing, males and females were housed separately in groups of 6-8 animals per cage and maintained under standard laboratory conditions (22 ± 1°C, 60% relative humidity, 12-12 hours light-dark cycle, food and water ad libitum). Male mice were used at 2-3 months of age when their body weight in each genotype equally ranged between 20 and 25 g [START_REF] Narboux-Nême | Severe serotonin depletion, growth and behavioral abnormalities in conditional vesicular monoamine transporter 2 knockout mouse[END_REF]. Some histological assays (VMAT and 5-HT immunohistochemistry) were performed on P7 mice for early detection of the phenotype, which was found the same at adult ages. In addition, some C57BL/6J mice (purchased from Janvier, France) were used for pharmacological studies.

Neurotransmission studies

Tissue levels of 5-HT, 5-hydroxyindolacetic acid (5-HIAA), noradrenaline (NA) and dopamine (DA) were determined using high pressure liquid chromatography with electrochemical detection (HPLC-ED) as previously described [START_REF] Mongeau | 5-HT(2c) receptor activation prevents stress-induced enhancement of brain 5-HT turnover and extracellular levels in the mouse brain: modulation by chronic paroxetine treatment[END_REF]. Crude synaptosomes from VMAT2 sert-cre and control mice, prepared as previously described [START_REF] Gray | The isolation of nerve endings from brain: an electronmicroscopic study of cell fragments derived by homogenization and centrifugation[END_REF], were used in experiments for the measurements of [ 3 H]5-HT uptake and release induced with the releasing agent 3,4-methylenedioxymethamphetamine (MDMA: 0.1 nM -10 µM) or potassium (3 -100 mM). Quantitative autoradiography of 5-HT 1A receptormediated [ 35 S]GTP-γ-S binding using the non-selective agonist 5-carboxamido-tryptamine (5-CT; 10-1000 nM; non-specific binding defined by WAY 100635) was done as previously described [START_REF] Fabre | Altered expression and functions of serotonin 5-HT1A and 5-HT1B receptors in knock-out mice lacking the 5-HT transporter[END_REF]. See the supplementary online information for further details.

Histological analyses

Mice were perfused transcardiacally with 4% paraformaldehyde, and brains were postfixed overnight in 4% paraformaldehyde, cryoprotected in 30% sucrose and serially sectioned (60 µm thick sections) on a freezing microtome. Alternate series for immunohistochemistry, in situ hybridization and counterstaining were used. Immunohistochemistry was performed using specific antibodies against VMAT2 (1:10000, Phoenix), 5-HT (1:50000, Sigma), SERT (1:1000, Calbiochem) as described previously (Alvarez, et al. 2002). Secondary antibodies used were biotinylated anti-rabbit IgG (1:300, Jackson Laboratories) followed by avidinbiotin-peroxidase complex (1:400, Amersham). Peroxidase activity was detected with 3,3'diaminobenzidine peroxide (DAB/H 2 0 2 ). In situ hybridization experiments were performed as previously described [START_REF] Bally-Cuif | Ectopic induction and reorganization of Wnt-1 expression in quail/chick chimeras[END_REF]. The TPH2 c-DNA plasmid previously described [START_REF] Cote | Disruption of the nonneuronal tph1 gene demonstrates the importance of peripheral serotonin in cardiac function[END_REF]) was donated by P. Ravassard (CRICM, UPMC, Paris).

Images were captured with a Cool Snap FX camera fitted to a Leica DM RD microscope under consistent light conditions using 20x/0.70 objectives. Images were copied to 8-bit RGB digital format and analyzed with ImageJ software. The density of SERTimmunoreactive fibers was estimated by counting the number of intersecting fibers with a grid composed of hemicycloids, as previously described [START_REF] Gaspar | Alterations of dopaminergic and noradrenergic innervations in motor cortex in Parkinson's disease[END_REF]. The density of TPH2 labeled neurons was estimated in two sections through the rostral portion of the dorsal raphe nucleus. All neurons contained in a grid of 250x250 um were counted. The grid was placed over 3 different areas of the dorsal raphe, for each case.

Behavioral testing

Experiments were carried out in adult (2-4 months) VMAT2 sert-cre and controls from the same litters that included both VMAT2 lox/lox and VMAT2 lox/+ SERT cre/+ male mice.

Elevated plus maze (EPM)

The maze was made of polyvinylchloride with two lit open arms (27×5 cm) and two opaque closed arms (27×5×15 cm). The arms radiated from a central platform (5×5 cm) and the apparatus was 38.5 cm above the floor. To initiate the 5-min test session, the mouse was placed on the central platform, facing an open arm. The mouse was considered to be on the central platform whenever two paws were on it, and in one of the arms when all four paws were inside. Behavioral analysis was done using a video recording using ODlog (Macropod Software) by an observer unaware of genotype. Results are reported as the time spent and the number of entries in the open arms to assess anxiety-like behavior, and total number of entries into both open and closed arms to assess locomotor activity.

Spontaneous locomotor activity

Locomotor activity was measured using a computer-based photobeam apparatus (Actisystem II, Panlab, Barcelona, Spain). Actimeter box (area: 300 x 150 mm; height 180 mm; with plexiglass wall and grid floor) detected mouse movements by means of two infrared light beams. Mice were placed in the testing room at least 2 hours before the experiment.

Novelty suppressed feeding (NSF)

Animals were placed one per cage for at least one week, and their bedding was changed just before food deprivation. All mice were deprived of their regular food for 48 hours and placed in the testing room for at least 1 hour before the test. They were then placed into an unfamiliar arena (area: 400 x 400 mm; height 180 mm; containing bedding) with a small plate containing food at the center of the field. The latency to feed was measured using a video recording, from the time the mouse is placed in the periphery of the arena until the animal began feeding. The latency to feed was also measured in the home cage, to assess if changes in latency might be accounted for by alteration in the feeding drive rather than reaction to the anxiogenic environment. The delay was measured from the time the mouse was placed at the periphery of the cage containing the food in the center.

Food consumption

Animals were placed one per cage for one week before measuring daily food intake over one week. The pellets consumed were measured by weighing the amount of remaining food each day.

Tail suspension test

The apparatus consists of three suspension units divided by walls (ID-Tech-Bioseb, Chaville, France). Mice were suspended by the tail, using an adhesive tape attached to a strain gauge transducing movements into a signal transmitted to a central unit for signal digitalization.

Although VMAT2 ser-cre mouse weights differ from controls during postnatal life [START_REF] Narboux-Nême | Severe serotonin depletion, growth and behavioral abnormalities in conditional vesicular monoamine transporter 2 knockout mouse[END_REF]), weight differences were no longer observed at adult age which eliminates any bias in measurements with this test. The duration of immobility was measured automatically by the software over a 6-min period. Struggling duration was analyzed from the traces generated by the software, and was defined as the total time the mouse spent in activity burst (i.e. excluding all the small movements), during the 6 min test period. Activity burst was defined as a force exerted on the transducer greater than or equal to 3 g above the baseline value for more than 2 s.

Ultrasound-induced defense reactions

Mice were tested for their innate fear reactions to a train of ultrasonic stimuli (US), as previously described [START_REF] Mongeau | Neural correlates of competing fear behaviors evoked by an innately aversive stimulus[END_REF]. Animals were placed one per cage for at least one week before testing. In brief, 100 ms frequency sweeps between 17-20 kHz, 85 dB, alternately ON 2 s and then OFF 2 s for 1 min were delivered into the home cage (18 x 29 cm) after a 3 min baseline period. Flight reactions triggered during the ON periods were measured as the number of running events from one side of the cage to the other followed by behavioral arrest, while the percentage time freezing to the US was quantified by sampling events of complete immobility (except respiration) every 4 s during the OFF period. These defense behaviors were measured from a video file by an observer unaware of mouse genotype.

Pargyline treatment

Mice were treated using subcutaneous osmotic minipumps (Alzet model 2004) to avoid injection handling which alters mice spontaneous defense behaviors to the US. Chronic pargyline treatment was done to achieve sustained MAOA and MAOB inhibition and optimally enhanced 5-HT neurotransmission in both control and VMAT2 sert-cre mice.

Pargyline (70 mg/kg x day; Fluka, Buchs, Zwitzerland) or vehicle (water) was administered for 3 weeks using minipumps inserted in sterile conditions on the back of the animals under pentobarbital anesthesia (55 mg/kg, i.p.).

Statistics

Data were analyzed using the Student's t-test when comparing two groups. The [ 35 S]GTP-γ-S binding data and the effects of pargyline treatment on 5-HT levels and flight behaviors in relation to the genotype were analyzed using the two-way Anova followed by Bonferonni's post-hoc test. EC 50 values were compared by a one-way Anova followed by Bonferonni's test.

Statistical significance was set at P < 0.05.

Results

Specific invalidation of the Vmat2 gene in 5-HT neurons

Conditional ablation of Vmat2 was obtained by inserting loxP sequences into the genomic sequence of the Vmat2 gene by homologous recombination, which produced the new mouse line VMAT2 lox/lox (Fig. 1). These mice were crossed to a previously described mouse line in which the 5-HT transporter (SERT, Slc6a4) promoter drives the expression of bacterial Cre recombinase [START_REF] Narboux-Neme | Serotonin transporter transgenic (SERTcre) mouse line reveals developmental targets of serotonin specific reuptake inhibitors (SSRIs)[END_REF][START_REF] Zhuang | Targeted gene expression in dopamine and serotonin neurons of the mouse brain[END_REF]. The VMAT2 lox/lox /SERT cre/+ double transgenic line (hence termed VMAT2 sert-cre ) was amplified for analysis. As with other 5-HT depleted transgenic mice [START_REF] Alenina | Growth retardation and altered autonomic control in mice lacking brain serotonin[END_REF][START_REF] Dai | Enhanced contextual fear memory in central serotonin-deficient mice[END_REF], there was an increased mortality of VMAT2 sert-cre mice between P1 and P30 compared to control mice.

We first checked the effectiveness and selectivity of the Cre-mediated excision using an antiserum that specifically stains the VMAT2 isoform. In wild type (C57BL/6J) mice and VMAT2 lox/lox mice, immunolabeling was observed in neurons of the raphe, the locus coeruleus, the substantia nigra, and the hypothalamus (Fig. 2A). In the VMAT2 sert-cre mice (Fig. 2A), VMAT2-immunostaining was abolished in the raphe neurons but was still present in noradrenergic neurons of the locus coeruleus, in dopaminergic neurons of the substantia nigra. Similarly VMAT2-immunostaining was unchanged in the hypothalamus and histamine neurons (Fig. 2A). No visible reduction in VMAT2-immunostaining was detected in heterozygote mice lacking only one allele of the VMAT2 gene (VMAT2 lox/+ /SERT cre/+ ; data not shown). 5-HT immunocytochemistry showed a major and uniform depletion of 5-HT immunostaining in all axon terminal fields (fig. 2B) with very faint residual staining in raphe neurons in the brainstem in P7 mice (Fig. 2B) and a complete lack of labeling in the adult raphe. However, the density of TPH2-positive neurons, in the rostral part of the dorsal raphe nucleus, was unchanged (Fig. 2C). To assess raphe projections, immunolabeling was performed with anti-SERT antibodies. Examination of serial coronal sections through the brain showed a comparable distribution and morphology of the SERT-labeled axons in VMAT2 sert-cre and control brains. The density of SERT-labeled axons was estimated in the cerebral cortex and hippocampus. This showed no difference in fiber density between control and mutants (Fig. 2C). Overall these results indicated that 5-HT raphe neurons developed normally in VMAT2 sert-cre mutants compared to WT mice.

Consequences on monoamines metabolism, uptake and signal transduction

Tissue monoamines' levels were measured by HPLC in various brain areas (cortex, striatum, hippocampus, brainstem), gut, and blood, as these tissues are known to produce or contain large amounts of 5-HT. Monoamines levels were not significantly different in wild-type (C57BL/6J), VMAT2 lox/lox , and VMAT2 lox/+ /SERT cre/+ mice, that lack one allele of VMAT2 (results not shown), hence, in subsequent studies the VMAT2 lox/lox and the VMAT2 lox/+ /SERT cre/+ mice were pooled as controls. Dramatic decreases in 5-HT levels were observed throughout the brain of VMAT2 sert-cre mice (from -92% to -96%), with no significant differences between brain structures (Fig. 3A, left). Remarkably, no changes in 5-HIAA levels were observed (Fig. 3A,right). This suggests (with data of Fig. 2C) that 5-HT was produced in normal amounts. Dopamine levels in the striatum and noradrenaline levels in the hippocampus and the cortex (Fig 3 ) were not or only marginally altered, although there was a 17 ± 3 % (P < 0.01) decrease of hippocampal NA in VMAT2 sert-cre mice [that could be explained by a lack of stimulatory action of 5-HT on NA terminals in that area [START_REF] Mongeau | Activation of 5-HT3 receptors enhances the electrically evoked release of [3H]noradrenaline in rat brain limbic structures[END_REF]] as compared to controls (fig. 3B). By contrast, 5-HT levels were normal in the gut (Fig. 3C), consistent with the fact that enterochromaffin cells express VMAT1 rather than VMAT2 [START_REF] Erickson | Distinct pharmacological properties and distribution in neurons and endocrine cells of two isoforms of the human vesicular monoamine transporter[END_REF]. A different situation occurred in the blood where a 80 ± 3 % (P < 0.001) decrease of 5-HT levels was noted (Fig. 3C), consistent with the notion that platelets use VMAT2 rather than VMAT1 to store 5-HT [START_REF] Holtje | The vesicular monoamine content regulates VMAT2 activity through Galphaq in mouse platelets. Evidence for autoregulation of vesicular transmitter uptake[END_REF].

The release of endogenous 5-HT was examined in synaptosomes preparations obtained from the whole brain. No potassium-induced release of 5-HT was observed in untreated synaptosomes from VMAT2 sert-cre mice (Fig. 4A). [ 3 H]5-HT uptake and MDMAand potassium-induced release were also analyzed on synaptosomes from control, SERT cre/+ and VMAT2 sert-cre mice (Fig. 4 B-D). After loading, [ 3 H]5-HT is normally distributed between two compartments, a fraction in the storage vesicles and another fraction in the axoplasm.

The MAOI pargyline (0.1 mM) was added to the medium to prevent degradation of [ 3 H]5-HT occurring in the axoplasm.

In VMAT2 sert-cre synaptosomes, uptake of [ 3 H]5-HT was reduced to a similar level as that observed in reserpine (0.1 µM) -treated synaptosomes from WT mice (Fig. 4B). In contrast, no significant alterations were noted in the SERT cre/+ synaptosomes. MDMA-induced release, occurring through the reversal of the reuptake carrier [START_REF] Hekmatpanah | 5-hydroxytryptamine uptake blockers attenuate the 5-hydroxytryptamine-releasing effect of 3,4-methylenedioxymethamphetamine and related agents[END_REF][START_REF] Mckenna | 3,4-Methylenedioxyamphetamine (MDA) analogues exhibit differential effects on synaptosomal release of 3H-dopamine and 3H-5hydroxytryptamine[END_REF][START_REF] Renoir | Differential long-term effects of MDMA on the serotoninergic system and hippocampal cell proliferation in 5-HTT knock-out vs. wild-type mice[END_REF], was analysed in synaptosomes from WT and VMAT2 sert-cre mice (Fig. 4C). The total amount of [ 3 H]-5-HT in synaptosomes was decreased in VMAT2 sert-cre or WT mice treated with reserpine compared to controls (WT = 0.13 ± 0.01; SERT cre/+ = 0.14 ± 0.02; VMAT2 sert-cre = 0.07 ± 0.01; WT + reserpine = 0.06 ± 0.01 mmol/g of protein), as expected from the reduced uptake of [ 3 H]5-HT into the storage vesicles of VMAT2 sert-cre mice compared to WT mice (Fig. 4B). There was a small but significant change in the concentration of MDMA that triggers 50% release in VMAT2 sert-cre mice compared to control mice (Fig. 4C): the dose response curve was slightly shifted to the left when using VMAT2 sert-cre synaptosomes (log EC 50 : VMAT2 sert-cre = -7.35 ± 0.11; WT = -6.86 ± 0.09 M, n=3, P < 0.01). A similar change compared to WT synaptosomes was found in WT synaptosomes treated with reserpine (log EC 50 : WT + reserpine = -7.26 ± 0.11 M, P < 0.05), but not in synaptosomes from SERT +/cre mice (log EC 50 : -6.69 M ± 0.14; Fig. 4C).

However, the concentration of KCl necessary to trigger 50% of maximal release of [ 3 H]5-HT was similar across all synaptosomes preparations (Fig. 4D; EC 50 : WT = 24.7 ± 8.5 mM; VMAT2 sert-cre = 21.8 ± 5.4 mM; SERT cre/+ = 23.9 ± 4.8 mM; WT + reserpine = 25.9 ± 8.5 mM). This release is likely to represent a predominantly axoplasmic pool of [ 3 H]5-HT since all experiments were conducted in presence of pargyline.

Potassium-induced release is only weakly dependent on the presence of calcium in the media in the above conditions (varying from 0 to 30% at 3-100 mM of KCl; not shown), suggestive of non-vesicular release. Other experimental conditions were used to test calciumdependent release. This involved the retrieval of pargyline from the media and shorter potassium pulses (30 s). In these conditions we observed a calcium dependent release of [ 3 H]5-HT (indicated in figure 4E as a decrement in the amount of radioactivity in the synaptosomes after KCl stimulation) in WT mice, whereas no significant calcium dependent vesicular release was found in synaptosomes from VMAT2 sert-cre mice.

To assess whether adaptive changes occur at 5-HT receptors as a consequence of the VMAT2 sert-cre 5-HT depletion, we measured 5-HT 1A receptor-stimulated [ 35 S]GTP-γ-S binding using 5-carboxamido-tryptamine (5-CT) as the agonist [START_REF] Fabre | Altered expression and functions of serotonin 5-HT1A and 5-HT1B receptors in knock-out mice lacking the 5-HT transporter[END_REF]. [ 35 S]GTPγ-S binding was significantly increased in the dorsal raphe nucleus in response to 5-HT 1A receptor activation by 5-CT in the VMAT2 sert-cre compared to control mice (P < 0.0001, two way Anova, Fig. 5A), suggesting an upregulation of the 5-HT 1A autoreceptors and / or enhanced coupling efficacy of these autoreceptors to G proteins. In contrast, no change in the 5-CT-induced [ 35 S]GTP-γ-S binding was found in post-synaptic sites, such as the dorsal hippocampus (Fig. 5B), the ventral hippocampus and the septum (not shown).

Behavioral consequences

We measured the consequences of VMAT2 sert-cre -induced 5-HT depletion in standard models of anxiety and depression. In the elevated plus-maze (EPM), VMAT2 sert-cre mice spent the same amount of time exploring the open arms as control mice (Fig. 6A). There was however a significant decrease (-37 ± 7 %; P<0.05) in the overall entries into both the closed and the open arms (Fig. 6A) suggesting a change in locomotor activity that could confound the interpretation of the EPM data. Indeed, specific assessment of locomotion with an actimeter also showed a 40% reduction in spontaneous locomotor activity of the VMAT2 sert-cre mice (Fig. 6B).

To further explore the anxiety-related phenotype in a model less dependent on locomotion, we applied the novelty suppressed feeding test, in which there is a conflict between the feeding drive and risk assessment behaviors. VMAT2 sert-cre mice initiated to feed much sooner (about 3 times faster; Fig. 6C) than control mice in the unfamiliar cage but not in the home cage, indicating an anxiolytic-like phenotype, rather than a change in appetite.

Furthermore, in isolated adult mice of the same weight (22 ± 1 g), food consumption measured during one week did not differ between transgenic and control mice (VMAT2 sert-cre : 3.58 ± 0.28 g; control: 3.65 ± 0.30 g; mean ± S.E.M. n=5-6). Finally, when tested in the tail suspension test (TST), VMAT2 sert-cre mice were more reactive than control mice. Indeed, immobility time was reduced (Fig. 6D; -37 ± 12 %; P<0.01) and time spent actively struggling to escape was increased (+68% ± 13%, P=0.001). These observations suggested an increase of active defense behaviors of VMAT2 sert-cre mice in the TST.

Interestingly, VMAT2 sert-cre mice also displayed a large increase of behavioral reactivity in response to stimulation with the innately aversive ultrasonic stimulus (US; P < 0.01; fig. 7A). In addition, VMAT2 sert-cre mice froze less in reaction to the US compared to control mice (control: 69 ± 7 % of time during the OFF periods; VMAT2 sert-cre : 37 ± 8 %; mean ± S.E.M.; P < 0.05, n=4-5, not shown).

To examine whether the change in behavioral reactivity to the US in VMAT2 sert-cre mice was due to the reduced 5-HT levels, we investigated the effects of a long term pargyline treatment (70 mg/kg x day, for 3 weeks). Pargyline reversed the 5-HT depletion (-94 ± 0.01%) observed in VMAT2 sert-cre mice, and even markedly increased 5-HT levels compared to vehicle treated control mice (Fig. 7D). Nevertheless, after pargyline treatment 5-HT tissue levels was still 20 ± 13 % lower in VMAT2 sert-cre mutants compared to pargyline-treated control mice (Fig. 7D). Two-way Anova indicated a significant effect (P < 0.0001) of treatment F(1,19) = 574 and of genotype F(1,19) = 46, but no significant interactions [F(1,19) = 0.3]. Pargyline also restored some 5-HT immunostaining in raphe neurons and axon terminals of VMAT2 sert-cre mice (Fig. 7B,C), although the levels were lower than those observed in controls (not shown). In response to the US, VMAT2 sert-cre mice treated with pargyline no longer displayed their increment of flight responses compared to control mice (Fig. 7A). Two-way Anova indicated significant effects (P = 0.01) of treatment F(1,12) = 9, genotype F(1,12) = 9 and a significant treatment x genotype interaction F(1,12) = 10. Furthermore, there were no changes in the percentage of time spent freezing in response to the US in pargyline treated mice (control: 87 ± 6 % of time during the OFF periods; VMAT2 sert- cre : 76 ± 24 %, not shown).

Discussion

Previous studies have shown that the majority of brain monoamines resides in the vesicular storage pools and that pharmacological inhibition of VMAT prevents vesicular release of 5-HT (Adell and Artigas 1998; [START_REF] Bunin | Release and uptake rates of 5-hydroxytryptamine in the dorsal raphe and substantia nigra reticulata of the rat brain[END_REF][START_REF] Connor | Fast cyclic voltammetry can be used to measure stimulated endogenous 5-hydroxytryptamine release in untreated rat brain slices[END_REF]. We confirm here that VMAT2 is the only vesicular transporter implicated in this effect in the CNS, since its genetic ablation in 5-HT neurons entirely reproduced the effects on 5-HT uptake of complete VMAT inhibition with reserpine. Interestingly, however, in the VMAT2 sert-cre mice, 5-HT is probably produced at normal rates, as suggested by (i) the normal levels of TPH2 gene expression, (ii) the normal amounts of 5-HIAA (the product of oxidative deamination of 5-HT), and (iii) the normal raise of 5-HT levels after MAO inhibition by pargyline. Hence, all 5-HT produced or taken up by neurons, that cannot be stored into vesicles is rapidly metabolized by mitochondrial monoamine oxidases present in raphe neurons [START_REF] Shih | Molecular biology of Monoamines oxidases A and B: their role in the degradation of serotonin[END_REF]Vitalis, et al. 2002). Because 5-HT continues to be produced in raphe neurons at a seemingly normal rate, it can still be released. While such release is likely to be marginal in standard conditions because of the low endogenous levels of 5-HT, it could still occur following administration of a MAOI which causes a major raise of 5-HT tissue levels.

Furthermore, it cannot be excluded that pargyline could enhance some form of vesicular storage [START_REF] Buu | Modification of vesicular dopamine and norepinephrine by monoamine oxidase inhibitors[END_REF]. In VMAT2 sert-cre mice MAOIs restored some functions such as the inhibitory action of 5-HT on active defense reactions (fig. 7B) as well as normal growth (Alvarez, et al. 2002;[START_REF] Narboux-Nême | Severe serotonin depletion, growth and behavioral abnormalities in conditional vesicular monoamine transporter 2 knockout mouse[END_REF]). This functional restoration in vivo fits well with the potassium-and MDMA-induced releases of [ 3 H]5-HT observed in vitro in VMAT2 sert-cre synaptosomes.

The mechanism involved in the [ 3 H]5-HT release in VMAT2 sert-cre synaptosomes is presently unclear, but two non-mutually exclusive possibilities can be discussed: 1) Release of [ 3 H]5-HT via the carrier [START_REF] Evans | The mechanism by which monoamine oxidase inhibitors give rise to a non-calcium-dependent component in the depolarization-induced release of 5-HT from rat brain synaptosomes[END_REF]. Although this was suggested to occur in VMAT2 KO mice [START_REF] Fon | Vesicular transport regulates monoamine storage and release but is not essential for amphetamine action[END_REF]), we do not presently have any data in favor of flux reversal in VMAT2 sert-cre mice. 2) Alternate vesicular storage and release pathways. It is known that 5-HT can passively cross the membrane of intracellular acidic organelles where it is protonated and remains trapped because charged monoamines cannot cross back membranes. In view of pH differences between vesicles and axoplasm, there could be at least a 100-fold increase in [ 3 H]5-HT vesicular concentration compared to the axoplasm [START_REF] Njus | Bioenergetics of secretory vesicles[END_REF], although this passively generated gradient remains very small compared to that generated by VMAT2, it may nevertheless be a way to compensate for VMAT2 loss. Future studies are clearly needed to understand how 5-HT release can occur in the VMAT2 sert-cre mice.

Beyond the cellular mechanisms involved, an important finding here is that [ 3 H]5-HT stabilized via MAO inhibition in the axoplasm can be mobilized out of the synaptosomes. The small but significant changes in the EC 50 values obtained after MDMA stimulation indicate that the process underlying 5-HT release in VMAT2 sert-cre mice is likely to be different from that of WT mice. However, the relatively low calcium dependency of our release conditions in presence of pargyline might preclude similar observation after potassium stimulation. It is also important to emphasize that in the absence of pargyline, calcium-dependent vesicular release of 5-HT was not observed in VMAT2 sert-cre mice.

The reduced 5-HT in the VMAT2 sert-cre mice caused a sensitization of the 5-HT 1A autoreceptors coupled to Gαi proteins in the dorsal raphe nucleus. In contrast, the decrease in 5-HT contents in the VMAT2 sert-cre mice caused no adaptive changes of 5-HT 1A receptors in target areas, such as the hippocampus. This is in line with the notion that raphe 5-HT 1A autoreceptors, but not hippocampal 5-HT 1A receptors, show modulation in response to changes in 5-HT levels [START_REF] Fabre | Altered expression and functions of serotonin 5-HT1A and 5-HT1B receptors in knock-out mice lacking the 5-HT transporter[END_REF][START_REF] Mannoury La Cour | Regional differences in the coupling of 5-hydroxytryptamine-1A receptors to G proteins in the rat brain[END_REF][START_REF] Mongeau | The serotonergic and noradrenergic systems of the hippocampus: their interactions and the effects of antidepressant treatments[END_REF]) and indicate that extracellular 5-HT level was much reduced at the level of serotonergic somas.

The VMAT2 sert-cre mouse line offers an interesting model of hyposerotonergia, complementary to other recently described genetic mouse models [START_REF] Trowbridge | Genetic Models of Serotonin (5-HT) Depletion: What do They Tell Us About the Developmental Role of 5-HT?[END_REF].

All recent genetic hyposerotonergic models focused on reducing 5-HT production either by targeting the central 5-HT synthesis enzyme, tryptophan hydroxylase 2 (TPH2) [START_REF] Alenina | Growth retardation and altered autonomic control in mice lacking brain serotonin[END_REF][START_REF] Beaulieu | Role of GSK3 beta in behavioral abnormalities induced by serotonin deficiency[END_REF][START_REF] Gutknecht | Deficiency of brain 5-HT synthesis but serotonergic neuron formation in Tph2 knockout mice[END_REF][START_REF] Savelieva | Genetic disruption of both tryptophan hydroxylase genes dramatically reduces serotonin and affects behavior in models sensitive to antidepressants[END_REF] or by invalidating the transcription factors controlling differentiation of raphe neurons such as pet1 and lmx1B [START_REF] Dai | Enhanced contextual fear memory in central serotonin-deficient mice[END_REF][START_REF] Hendricks | Pet-1 ETS gene plays a critical role in 5-HT neuron development and is required for normal anxiety-like and aggressive behavior[END_REF][START_REF] Kiyasova | A genetically defined morphologically and functionally unique subset of 5-HT neurons in the mouse raphe nuclei[END_REF][START_REF] Zhao | Insertion mutation at the C-terminus of the serotonin transporter disrupts brain serotonin function and emotion-related behaviors in mice[END_REF]).

The more severe 5-HT depletion observed here (-95%), compared with previous models (-70-90%), is likely due to the fact that increased 5-HT degradation, when VMAT2 is absent, affects all sources of 5-HT (e.g, produced by TPH1/TPH2). The present observations, consistent with Tph2 knockout mice [START_REF] Alenina | Growth retardation and altered autonomic control in mice lacking brain serotonin[END_REF][START_REF] Gutknecht | Deficiency of brain 5-HT synthesis but serotonergic neuron formation in Tph2 knockout mice[END_REF], showed both a normal development of the raphe neurons, and a normal density of 5-HT terminals in the hippocampus and the cerebral cortex of VMAT2 sert-cre mice. Subtle developmental defects may nonetheless exist given the large body of evidence supporting the role of 5-HT in the maturation of neural circuits [START_REF] Trowbridge | Genetic Models of Serotonin (5-HT) Depletion: What do They Tell Us About the Developmental Role of 5-HT?[END_REF].

Normal morphology of 5-HT neurons and brain specificity provides two major advantages of genetic models in general compared to pharmacological depletion methods such as, respectively, lesions of 5-HT neurons with 5,7-dihydroxytryptamine and depletion with p-chlorophenylalanine (PCPA). Compared to the Tph2 knockout which irreversibly inactivates 5-HT synthesis, the VMAT2 sert-cre mutation has also the advantage of rapid reversibility via MAO inhibition since TPH2 is not targeted or down regulated. Overall, VMAT2 selective deletion should allow more direct explorations of the function of monoamine release in otherwise normally developed raphe neurons.

Behavioral characterization of the VMAT2 sert-cre mice showed that depletion of central 5-HT stores reduces anxiety. This finding is in agreement with reports in several genetic models of 5-HT deficient mice [START_REF] Dai | Enhanced contextual fear memory in central serotonin-deficient mice[END_REF][START_REF] Kiyasova | A genetically defined morphologically and functionally unique subset of 5-HT neurons in the mouse raphe nuclei[END_REF]) and with numerous studies using pharmacological approaches to decrease brain 5-HT in rats [START_REF] Graeff | Serotonin, the periaqueductal gray and panic[END_REF][START_REF] Griebel | 5-Hydroxytryptamine-interacting drugs in animal models of anxiety disorders: more than 30 years of research[END_REF]. Furthermore, decreased 5-HT input at several receptors (including the 5-HT 2A and 5-HT 2C subtypes) clearly reduces anxiety in mice [START_REF] Heisler | Serotonin 5-HT(2C) receptors regulate anxiety-like behavior[END_REF][START_REF] Mongeau | 5-HT(2c) receptor activation prevents stress-induced enhancement of brain 5-HT turnover and extracellular levels in the mouse brain: modulation by chronic paroxetine treatment[END_REF][START_REF] Weisstaub | Cortical 5-HT2A receptor signaling modulates anxiety-like behaviors in mice[END_REF]).

VMAT2 sert-cre mice were first tested in the most standard model, the elevated plus maze (EPM), having a strong validity for anxiolysis mediated by the GABAergic system, but not so much, it seems from previous studies [START_REF] Mccreary | 5-HT systems and anxiety: multiple mechanisms in the elevated X-maze[END_REF], for the serotonergic system. Indeed, the effect of altering 5-HT on the behaviors observed in the EPM was argued to be the overall result of a balance between distinct 5-HT systems in different brain areas. For example, microinjection of a 5-HT 2C agonist generated opposite effects in the EPM depending on the brain areas targeted [START_REF] Cornelio | Anxiogenic-like effects of mCPP microinfusions into the amygdala (but not dorsal or ventral hippocampus) in mice exposed to elevated plus-maze[END_REF][START_REF] Nunes-De-Souza | 5-HT2 receptor activation in the midbrain periaqueductal grey (PAG) reduces anxiety-like behaviour in mice[END_REF]. This might explain the lack of changes in VMAT2 sert-cre mice observed here with the EPM. The observed decrease in exploratory behavior of VMAT2 sert-cre mice, as in other hyposerotonergic models [START_REF] Dringenberg | pchlorophenylalanine-induced serotonin depletion: reduction in exploratory locomotion but no obvious sensory-motor deficits[END_REF][START_REF] Hendricks | Pet-1 ETS gene plays a critical role in 5-HT neuron development and is required for normal anxiety-like and aggressive behavior[END_REF], is also a confounding factor for the interpretation of EPM data. In contrast, consistent effects of 5-HT depletion have been noted in several models involving conflicts, where reduced 5-HT tone was linked to decreased anxiety-like behaviors [START_REF] Graeff | Serotonin, the periaqueductal gray and panic[END_REF]). In the NSF, which involves a conflict between the drive to feed and the risk assessment behaviors triggered by the unfamiliar environment, VMAT2 sert-cre mice showed an anxiolytic-like behavioral profile. This agrees with the anxiolytic-like effect of PCPA-induced 5-HT depletion (Bechtholt, et al. 2007) and with observations in other 5-HT depleted mutant mice [START_REF] Dai | Enhanced contextual fear memory in central serotonin-deficient mice[END_REF][START_REF] Kiyasova | A genetically defined morphologically and functionally unique subset of 5-HT neurons in the mouse raphe nuclei[END_REF].

It is also interesting to note that VMAT2 sert-cre mice spent more time struggling to escape than control mice in the TST. This is coherent with observations in the TPH1/TPH2 KO mice which displayed increased struggling time in the forced swim test [START_REF] Savelieva | Genetic disruption of both tryptophan hydroxylase genes dramatically reduces serotonin and affects behavior in models sensitive to antidepressants[END_REF]. The increase of struggling or escape-like behavior of VMAT2 sert-cre mice in the TST might be mechanistically similar to the increase in ultrasound-induced flight also observed here with these mutants. Both of these effects can potentially be accounted for by a lack of inhibitory effect of 5-HT at the midbrain level [START_REF] Kiser | Monoaminergic mechanisms in aversive brain stimulation[END_REF]. As far as we know, the present genetic invalidation study is the first which clearly indicates a tonic inhibitory role of endogenous 5-HT on the expression of active defense behaviors in reaction to fear stimuli.

Previous pharmacological studies in rats showed that flight behaviors induced by an aversive ultrasound are decreased by the 5-HT agonist mCPP [START_REF] Beckett | Pharmacological manipulation of ultrasound induced defence behaviour in the rat[END_REF]. Furthermore, treatment with the MAOI phenelzine decreased flight behavior in the mouse defense battery test, and this was mostly apparent after chronic compared to acute treatment [START_REF] Griebel | Behavioral effects of phenelzine in an experimental model for screening anxiolytic and anti-panic drugs: correlation with changes in monoamine-oxidase activity and monoamine levels[END_REF]. In our study, we have performed a long term, rather than a short term, MAOI treatment to optimally enhance 5-HT neurotransmission via the desensitization of 5-HT 1A autoreceptors [START_REF] Mongeau | The serotonergic and noradrenergic systems of the hippocampus: their interactions and the effects of antidepressant treatments[END_REF], which were found to be hypersensitive in VMAT2 sert-cre mice (Fig 5).

In relation with depression, it is generally believed that a reduced brain 5-HT tone results in depressed-like behaviors in escape-related models of depression such as the TST or the forced swim test (FST). However, although 5-HT is necessary for the action of antidepressant drugs in these models, 5-HT depletion with PCPA generally fails to alter baseline immobility [START_REF] Cryan | The tail suspension test as a model for assessing antidepressant activity: review of pharmacological and genetic studies in mice[END_REF][START_REF] O'leary | Depletion of serotonin and catecholamines block the acute behavioral response to different classes of antidepressant drugs in the mouse tail suspension test[END_REF]. In contrast, depleting catecholamines in adult mice strongly increases baseline immobility in the TST (O' Leary, et al. 2007). Constitutive knock-downs of VMAT2 (VMAT2 +/-), which reduces the release of all monoamines, also results in an increased immobility in the FST and the TST [START_REF] Fukui | Vmat2 heterozygous mutant mice display a depressive-like phenotype[END_REF]). However, the present observation that selective deletion of VMAT2 in serotonergic neurons decreased, rather than increased, immobility in the TST, together with previous observation [START_REF] Savelieva | Genetic disruption of both tryptophan hydroxylase genes dramatically reduces serotonin and affects behavior in models sensitive to antidepressants[END_REF]) on TPH1/TPH2-KO mice subjected to the FST, indicate that a depletion of 5-HT is insufficient to induce depression-like behaviors in such escaperelated tests.

Furthermore, the antidepressant-like effect observed in the TST in VMAT2 sert-cre mice might not be paradoxical considering that antidepressant drugs exert their effect in the TST after acute rather than chronic treatment [START_REF] Cryan | The tail suspension test as a model for assessing antidepressant activity: review of pharmacological and genetic studies in mice[END_REF]. It is important to emphasize here that although chronic treatments increase 5-HT neurotransmission, acute administrations do not [START_REF] Mongeau | The serotonergic and noradrenergic systems of the hippocampus: their interactions and the effects of antidepressant treatments[END_REF]. The transient surges of extracellular 5-HT levels, associated with acute antidepressant drugs, trigger strong inhibition of both 5-HT neuronal firing and release through negative autoreceptors feedback [START_REF] Auerbach | Differential inhibition of serotonin release by 5-HT and NA reuptake blockers after systemic administration[END_REF][START_REF] Hajos | Inhibition of median and dorsal raphe neurones following administration of the selective serotonin reuptake inhibitor paroxetine[END_REF][START_REF] Hervas | Effect of fluoxetine on extracellular 5-hydroxytryptamine in rat brain. Role of 5-HT autoreceptors[END_REF][START_REF] Hjorth | Further evidence for the importance of 5-HT1A autoreceptors in the action of selective serotonin reuptake inhibitors[END_REF].

Conclusion and perspectives

Genetic invalidation of VMAT2 in 5-HT neurons provides an interesting model to test the function of endogenous 5-HT release in normally developed 5-HT raphe neurons. Using this model we show that decrease of 5-HT results in an anxiolytic profile in a conflict test, contrasting with an increase in acute stress or innate fear stimulus-induced escape-like behaviors. Chronic treatment with a MAOI prevented the behavioral abnormality of VMAT2 sert-cre mice in terms of defense to an innately aversive stimulus. Interestingly, flight reactions have often been used in validated models of panic disorders [START_REF] Griebel | Predator-elicited flight responses in Swiss-Webster mice: an experimental model of panic attacks[END_REF] and chronic MAOIs are well known to be effective anti-panic agents [START_REF] Bakish | Reversible monoamine oxidase-A inhibitors in panic disorder[END_REF].

Incidentally, these data address new questions as to whether extrasynaptic or non-vesicular 5-HT release mechanisms might be involved in the therapeutic effects of this class of antidepressant drugs. A -The release of endogenous 5-HT evoked with varying concentration of potassium in synaptosomes from WT mice was not observed in synaptosomes from VMAT2 sert-cre mice (n=3). B -[ 3 H]5-HT uptake in VMAT2 sert-cre or reserpine-treated (0.1 µM) synaptosomes compared to WT or SERT cre/+ synaptosomes. There was less total uptake in synaptosomes from VMAT2 sert-cre mice or from WT mice treated with the VMAT inhibitor reserpine (Vmax; VMAT2 sert-cre = 2.0 ± 0.1 fmol/min, reserpine = 2.6 ± 0.07, mean ± S.E.M., n = 3) compared to synaptosomes from WT or SERT cre/+ mice (Vmax: control = 6.8 ± 0.5 fmol/min; SERT cre/+ = 5.5 ± 0.3, n = 3). There were no significant changes (using one-way Anova) in the Km values between groups (WT = 18.8 ± 4.5 nM; VMAT2 sert-cre = 13.5 ± 3.2 nM; SERT cre/+ = 12.7 ± 3 nM; reserpine = 12.25 ± 1.4 nM; means ± S.E.M., n = 3. C -The concentration response curve of the MDMA-induced [ 3 H]5-HT release was slightly shifted to the left in preloaded synaptosomes from VMAT2 sert-cre mice or reserpine-treated synaptosomes. D-There were no major changes in potassium-induced [ 3 H]5-HT release between the various synaptosome preparations in conditions weakly dependent on calcium, n=3. E-In conditions in which potassium-induced release is fully dependent on the presence of calcium in the incubation buffer (brief stimulation in absence of pargyline, WT synaptosomes top panel) there was no significant release in preloaded synaptosomes from VMAT2 sert-cre mice (bottom panel). VMAT2 sert-cre mice compared to control mice, which was reversed by a chronic treatment with pargyline (70 mg/kg x day for 3 weeks). * P < 0.01 using Bonferonni's test. Error bars=S.E.M., n=3-5. B-C' -5-HT-immunoreactive neurons were observed in the raphe area (B) and positive fibers were observed in dorsolateral periaqueductal gray areas in sections from VMAT2 sert-cre treated with pargyline (C). This staining was absent in VMAT2 sert-cre treated with saline (B' and C'). D -Chronic pargyline reversed the severe 5-HT depletion occurring in VMAT2 sert-cre mice, and markedly increased 5-HT levels (in the hippocampus).

Bonferonni's test indicated a significant decrease in 5-HT levels in VMAT2 sert-cre mutants compared to control mice (### P < 0.001, # P < 0.05), and significant increases of 5-HT levels after pargyline (*** P < 0.001) compared to control mice.

Fig. 1 -

 1 Fig. 1 -Conditional ablation of the Vmat2 gene Schematic representation of the wild-type, targeted and mutated Vmat2 alleles. Two loxP sites and a neomycin-selectable marker flanked by FRT sites were inserted by homologous recombination into the introns flanking the first Vmat2 coding exon. Correct targeting event in one positive ES cell clone was evaluated by Southern blotting with a probe directed against the neomycin sequence after hydrolysis with four different restriction enzymes. The location of the restriction sites is indicated on the targeted allele (N, NsiI; Sc, ScaI; Sp, SpeI; S, SacI). Theoretical sizes of the fragments (in kb) are indicated in brackets. The neomycin cassette was subsequently excised in vivo by Flp-mediated recombination to produce the floxed allele.

Fig. 2 -

 2 Fig. 2 -Distribution of VMAT2 and serotonergic markers in VMAT2 sert-cre mice vs control mice.A -VMAT2 immunostaining shows the major sites of VMAT2 expression in control and VMAT2 sert-cre mice. Coronal sections at comparable levels in the raphe, the locus coeruleus, the substantia nigra, and the hypothalamus from P7 mice. VMAT2 immunostaining is abolished in the raphe neurons of the VMAT2 sert-cre mice compared to control mice, whereas immunostaining is unchanged in the noradrenergic, the dopaminergic or the histaminergic neurons in the other brain areas. B -A drastic depletion of 5-HT contents is visible with immunohistochemistry in both the raphe and the forebrain of VMAT2 sert-cre mice compared to control mice. A faint 5-HT immunoreactivity remains visible in the raphe neurons (arrowhead) of VMAT2 sert-cre mice. C -Tryptophan hydroxylase 2 in situ hybridization shows the distribution of neurons in the dorsal raphe of VMAT2 sert-cre mice and the histogram shows the unchanged density of positive cells compared to controls (values are mean density on a surface of 784 µm 2 , error bars=S.E.M.; n=5). 5-HT transporter immunohistochemistry shows labeled axons in the hippocampus of VMAT2 sert-cre mice with normal appearance, and there was no significant change in the density of fibers in the stratum moleculare of the dentate gyrus compared to controls (values are mean intersecting fibers on a surface of 0.462 mm 2 , error bars=S.E.M.; n=5-6).

Fig. 3 -Fig. 4 -

 34 Fig. 3 -Tissue levels of monoamines in the brain and the peripheryA -Tissue levels of 5-HT and 5-HIAA were determined in various dissected brain areas from adult control (open bars) and VMAT2 sert-cre mice (black bars). There was a highly significant decrease in 5-HT levels in all examined brain areas. ***: P<0.0001 ; Student's t-test ; error bars=S.E.M.; n=5-6. B -Tissue levels of NA in the cortex and the hippocampus and DA in the striatum. There was no change of NA in the cortex but a small reduction in the hippocampus, while striatal levels were found unchanged using the Student's t-test; n = 5-6, **; P<0.01. C -Levels of 5-HT in the gut and in the blood. There were no changes in the gut, whereas 5-HT was significantly decreased in blood ***: P<0.0001; Student's t-test; n=5-8.

Fig. 5 -

 5 Fig. 5 -[ 35 S]GTP-γ-S binding in the dorsal raphe nucleus and the hippocampus after stimulation with the 5-HT 1A agonist 5-carboxamidotryptamine (5-CT). A -There was an increased coupling efficiency as shown by more agonist-induced [ 35 S]GTP-γ-S binding in the raphe of VMAT2 sert-cre (open circles) versus control mice (filled circles). Two-way Anova indicated a significant (P < 0.001) main effect of the genotype [F(1,35) = 19], of 5-CT concentration [F(3,35) = 34], and a marginally significant (P = 0.06) genotype x 5-CT interaction [F(3,35) = 2.8]. Post-hoc Bonferroni's tests indicated significant differences at specific concentrations * P < 0.05, ** P < 0.01. B -There were no changes in the hippocampus. Error bars=S.E.M., n=6.

Fig. 6 -

 6 Fig. 6 -The VMAT2 sert-cre mutation decreased anxiety and locomotion but increased reactivity to stress. A -There was no change in the time spent in the open arms or in the number of open arm entries, but there was a significant decrease in the total number of entries in VMAT2 sert-cre mutants compared to control mice. B -There was a significant decrease of basal locomotor activity in the VMAT2 sert-cre mutants as measured over a 15 min period in an actimeter. C -The latency to feed when mice were placed in an unfamiliar cage, but not home cage, was decreased in VMAT2 sert-cre mutants. D -Tail suspension test shows an increase in the time spent struggling, and decreased immobility time in VMAT2 sert-cre mice compared to control mice. Student's t-tests, * P < 0.05, **P < = 0.01. Error bars=S.E.M., n=5-12.

Fig. 7 -

 7 Fig. 7 -Inverse relationship between brain tissue levels of 5-HT and aversive ultrasoundinduced flight behaviors in VMAT2 sert-cre mice. A -There was an enhancement of flight behaviors in reaction to the aversive ultrasound (1 min fast sweep 17-20 kHz, 85 dB) in

Acknowledgements:

We thank Xiaoxi Zhuang and René Hen for the SERT-Cre mouse line. Funding for this project was from Agence Nationale de la Recherche (ANR605-neur-046), NeRF (for NNN), and from the European Commission (FP7-health-2007-A-201714). We thank all members of the DEVANX project for stimulating discussions and suggestions. NNN was supported by Region Ile de France (NeRF). CBPM was funded by a studentship from the Ministère de l'Education Nationale et de la Recherche (France). SLD was supported by fellowhips from IBRO and from Region Ile de France DIM STEM and SD by a LeFoulon-Delalande fellowship.

Disclosure of interest:

The authors declare no conflict of interest.