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

ABSTRACT The vesicular monoamine transporter type 2 gene (_VMAT2_) has a crucial role in the storage and synaptic release of all monoamines, including serotonin (5-HT). To evaluate the

specific role of VMAT2 in 5-HT neurons, we produced a conditional ablation of _VMAT2_ under control of the serotonin transporter (_slc6a4_) promoter. VMAT2_sert−cre_ mice showed a major

(−95%) depletion of 5-HT levels in the brain with no major alterations in other monoamines. Raphe neurons contained no 5-HT immunoreactivity in VMAT2_sert−cre_ mice but developed normal

innervations, as assessed by both tryptophan hydroxylase 2 and 5-HT transporter labeling. Increased 5-HT1A autoreceptor coupling to G protein, as assessed with agonist-stimulated

[35S]GTP-_γ_-S binding, was observed in the raphe area, indicating an adaptive change to reduced 5-HT transmission. Behavioral evaluation in adult VMAT2_sert−cre_ mice showed an increase in

escape-like reactions in response to tail suspension and anxiolytic-like response in the novelty-suppressed feeding test. In an aversive ultrasound-induced defense paradigm, VMAT2_sert−cre_

mice displayed a major increase in escape-like behaviors. Wild-type-like defense phenotype could be rescued by replenishing intracellular 5-HT stores with chronic pargyline (a monoamine

oxidase inhibitor) treatment. Pargyline also allowed some form of 5-HT release, although in reduced amounts, in synaptosomes from VMAT2_sert−cre_ mouse brain. These findings are coherent

with the notion that 5-HT has an important role in anxiety, and provide new insights into the role of endogenous 5-HT in defense behaviors. SIMILAR CONTENT BEING VIEWED BY OTHERS _PTEN_ IS A

KEY INTRINSIC FACTOR REGULATING RAPHE 5-HT NEURONAL PLASTICITY AND DEPRESSIVE BEHAVIORS IN MICE Article Open access 26 March 2021 FLUOXETINE INCREASES BRAIN MECP2 IMMUNO-POSITIVE CELLS IN A

FEMALE _MECP2_ HETEROZYGOUS MOUSE MODEL OF RETT SYNDROME THROUGH ENDOGENOUS SEROTONIN Article Open access 19 July 2021 ABSENCE OF TAAR1 FUNCTION INCREASES METHAMPHETAMINE-INDUCED

EXCITABILITY OF DORSAL RAPHE SEROTONIN NEURONS AND DRIVES BINGE-LEVEL METHAMPHETAMINE INTAKE Article Open access 11 February 2025 INTRODUCTION Serotonin (5-HT) has a central importance in

the control of mood and anxiety states (Lucki, 1998). 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 (Morilak and Frazer, 2004). All these constitute entry points to pharmacological therapeutic approaches (Berton and Nestler, 2006) and

to genetic variations that impact disease predisposition. Vesicular monoamine transporters (VMATs) seem important targets with 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 (Freis, 1954). Moreover, there is evidence for a link of depression to genetic polymorphism of the _VMAT2_ gene (Christiansen et al, 2007; Gutierrez et al, 2007). 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 (Henry et al, 1998). Nevertheless, 5-HT can also function as a paracrine transmitter by acting on receptors distant from synaptic release sites (Bunin and Wightman,

1999), and non-vesicular release of neurotransmitters is known to occur under some physiological conditions (Attwell et al, 1993; Wu et al, 2007). VMATs exist as two different isoforms:

VMAT1 mainly expressed in chromaffin and enterochromaffin cells and VMAT2 essentially expressed in monoaminergic neurons (Erickson et al, 1996; Erickson and Eiden, 1993). Microdialysis

(Adell and Artigas, 1998) and voltametry (Bunin et al, 1998; O’Connor and Kruk, 1991) experiments showed that tetrabenazine, a selective VMAT2 blocker, prevents the release of 5-HT in the

raphe and in axon terminal fields, whereas _VMAT2_-KO mice showed no release of amines (Fon et al, 1997; Wang et al, 1997). _VMAT2_-KO mice do not survive beyond the first postnatal days

(Alvarez et al, 2002; Fon et al, 1997; Wang et al, 1997) 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 (Fukui et al, 2007). However, it was unclear whether

this phenotype was linked to a reduced release of 5-HT or of other amines (dopamine (DA), noradrenaline (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 sustained 5-HT depletion

was to increase the coupling of 5-HT1A 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 to anxiety (Gray and McNaughton, 2000). 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 the directives of the European

Community (council directive 86/609) and the French Agriculture and Forestry Ministry (council directive 87–848, 19 October 1987, permissions 75–977 to LL and 00782 to PG). 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 regarding the mouse line should be addressed: [email protected]). A 9-kb fragment of the _VMAT2_ gene encompassing exons 1–3 was subcloned into the targeting vector and

the two _lox_P sites and the neomycin-selectable cassette flanked by two _FRT_ sites were introduced in 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 control of the early β-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 been described

previously (Zhuang et al, 2005). 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 have shown that recombination occurs by E12 in the raphe neurons (Narboux-Neme et al, 2008) and that virtually all tryptophan hydroxylase 2 (TPH2) neurons in the raphe have

recombined in postnatal life (Zhuang et al, 2005). Male _VMAT2__lox/+_:: _SERT_ _cre/+_ or _VMAT2__lox/lox_:: _SERT_ _cre/+_ mice were mated with female VMAT2_lox/lox_ to generate the three

genotypes that were analyzed: VMAT2_lox/lox_::_SERT_ _cre/+_ (recombined), _VMAT2__lox/lox_ (control 1), and _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 h 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 (Narboux-Nême et al, 2009). 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), NA, and DA were determined using high-pressure

liquid chromatography with electrochemical detection (HPLC-ED) as described previously (Mongeau et al, 2010). Crude synaptosomes from VMAT2_sert−cre_ and control mice, prepared as described

previously (Gray and Whittaker, 1962), were used in experiments for measurements of [3H]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-HT1A receptor-mediated [35S]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 performed as described previously (Fabre et al, 2000). See Supplementary online information for further details. HISTOLOGICAL ANALYSES Mice

were perfused transcardiacally with 4% paraformaldehyde, and their 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 : 10 000, Phoenix), 5-HT (1 : 50 000, Sigma), and 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 avidin–biotin–peroxidase complex (1 : 400, Amersham). Peroxidase activity was detected with 3,3′-diaminobenzidine

peroxide. _In situ_ hybridization experiments were performed as described previously (Bally-Cuif and Wassef, 1994). The TPH2 c-DNA plasmid described previously (Cote et al, 2003) was donated

by P Ravassard (CRICM, UPMC, Paris). Images were captured using a Cool Snap FX camera fitted to a Leica DM RD microscope under consistent light conditions using 20 × /0.70 objectives.

Images were copied to 8-bit RGB digital format and analyzed using ImageJ software. The density of SERT-immunoreactive fibers was estimated by counting the number of intersecting fibers with

a grid composed of hemicycloids, as described previously (Gaspar et al, 1991). 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 250 × 250 μm2 were counted. The grid was placed over three different areas of the dorsal raphe, for each case. BEHAVIORAL TESTING

Experiments were carried out in adult (2–4 months) VMAT2_sert−cre_ mice and controls from the same litters that included both VMAT2_lox/lox_ and VMAT2_lox/+_ SERT_cre/+_ male mice. ELEVATED

PLUS MAZE The maze was made of polyvinylchloride with two lit open arms (27 × 5 cm2) and two opaque closed arms (27 × 5 × 15 cm3). The arms radiated from a central platform (5 × 5 cm2) 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 performed using a video recording using ODlog (Macropod Software) by an

observer unaware of the 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 × 150 mm2; 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 h before the experiment. NOVELTY-SUPPRESSED FEEDING Animals were placed one per cage for at least 1 week, and their bedding was changed just before food

deprivation. All mice were deprived of their regular food for 48 h and placed in the testing room for at least 1 h before the test. They were then placed into an unfamiliar arena (area: 400

× 400 mm2; height 180 mm; containing bedding) with a small plate containing food at the center of the field. 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. Latency to feed was also measured in the home cage, to assess whether 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 1 week before measuring daily food intake over 1 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_sert−cre_ mouse weights differ from

controls during postnatal life (Narboux-Nême et al, 2009), 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 (ie, excluding all the small movements), during the 6-min test period. Activity burst was defined as a force exerted on the transducer ⩾3 g above the baseline

value for >2 s. ULTRASOUND-INDUCED DEFENSE REACTIONS Mice were tested for their innate fear reactions to a train of ultrasonic stimuli (US), as described previously (Mongeau et al, 2003).

Animals were placed one per cage for at least 1 week before testing. In brief, 100 ms frequency sweeps between 17 and 20 kHz, 85 dB, alternately ON 2 s and then OFF 2 s for 1 min were

delivered into the home cage (18 × 29 cm2) after a 3-min baseline period. Flight reactions triggered during ON periods were measured as the number of running events from one side of the cage

to the other followed by behavioral arrest, whereas 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 carried out 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 × day; Fluka, Buchs, Switzerland) or vehicle (water) was administered

for 3 weeks using minipumps inserted under sterile conditions on the back of animals under pentobarbital anesthesia (55 mg/kg, i.p.). STATISTICS Data were analyzed using Student's

_t_-test when comparing two groups. The [35S]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

two-way ANOVA, followed by Bonferonni's _post hoc_ test. EC50 values were compared by 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_ (Figure 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 (Narboux-Neme et al, 2008; Zhuang et al, 2005). 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 (Alenina et al, 2009; Trowbridge et al, 2010), there was an increased mortality

of VMAT2_sert−cre_ mice between P1 and P30 compared with control mice. We first checked the effectiveness and selectivity of Cre-mediated excision using an antiserum that specifically stains

the VMAT2 isoform. In wild-type (C57BL/6J) and VMAT2_lox/lox_ mice, immunolabeling was observed in neurons of the raphe, the locus coeruleus, the substantia nigra, and the hypothalamus

(Figure 2a). In VMAT2_sert−cre_ mice (Figure 2a), VMAT2 immunostaining was abolished in 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 (Figure 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 (Figure 2b) with very faint residual staining in raphe neurons in the brainstem in P7 mice (Figure 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 (Figure 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 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 (Figure 2c). Overall,

these results indicated that 5-HT raphe neurons developed normally in VMAT2_sert−cre_ mutants compared with WT mice. CONSEQUENCES ON MONOAMINES METABOLISM, UPTAKE, AND SIGNAL TRANSDUCTION

Tissue monoamine levels were measured by HPLC in various brain areas (such as the 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, which lack one allele of _VMAT2_

(results not shown); hence, in subsequent studies, VMAT2_lox/lox_ and the VMAT2_lox/+_/SERT_cre/+_ mice were pooled as controls. Dramatic decreases in 5-HT levels were observed throughout

the brains of VMAT2_sert−cre_ mice (from −92 to −96%), with no significant differences between brain structures (Figure 3a, left). Remarkably, no changes in 5-HIAA levels were observed

(Figure 3a, right). This suggests (with data of Figure 2c) that 5-HT was produced in normal amounts. DA levels in the striatum and NA levels in the hippocampus and the cortex (Figure 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 (Mongeau et al, 1994)) as compared with controls (Figure 3b). By contrast, 5-HT levels were normal in the gut (Figure 3c), consistent with the fact that

enterochromaffin cells express VMAT1 rather than VMAT2 (Erickson et al, 1996). A different situation occurred in the blood in which a 80±3% (_p_<0.001) decrease in 5-HT levels was noted

(Figure 3c), consistent with the notion that platelets use VMAT2 rather than VMAT1 to store 5-HT (Holtje et al, 2003). The release of endogenous 5-HT was examined in synaptosome preparations

obtained from the whole brain. No potassium-induced release of 5-HT was observed in untreated synaptosomes from VMAT2_sert−cre_ mice (Figure 4a). [3H]5-HT uptake and MDMA- and

potassium-induced release were also analyzed on synaptosomes from control, SERT_cre/+_, and VMAT2_sert−cre_ mice (Figure 4b–d). After loading, [3H]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 [3H]5-HT occurring in the

axoplasm. In VMAT2_sert−cre_ synaptosomes, uptake of [3H]5-HT was reduced to a similar level as that observed in reserpine (0.1 μM)-treated synaptosomes from WT mice (Figure 4b). In

contrast, no significant alterations were noted in SERT_cre/+_ synaptosomes. MDMA-induced release, occurring through the reversal of the reuptake carrier (Hekmatpanah and Peroutka, 1990;

McKenna et al, 1991; Renoir et al, 2008), was analyzed in synaptosomes from WT and VMAT2_sert−cre_ mice (Figure 4c). The total amount of [3H]-5-HT in synaptosomes was decreased in

VMAT2_sert−cre_ or WT mice treated with reserpine compared with 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 [3H]5-HT into the storage vesicles of VMAT2_sert−cre_ mice compared with WT mice (Figure 4b). There was a small but significant change in the

concentration of MDMA that triggers 50% release in VMAT2_sert−cre_ mice compared with control mice (Figure 4c): the dose-response curve was slightly shifted to the left when using

VMAT2_sert−cre_ synaptosomes (−log EC50: VMAT2_sert−cre_=7.35±0.11; WT=6.86±0.09, _n_=3, _p_<0.01). A similar change compared with WT synaptosomes was found in WT synaptosomes treated

with reserpine (−log EC50: WT+reserpine=7.26±0.11, _p_<0.05), but not in synaptosomes from SERT+/cre mice (−log EC50: 6.69±0.14; Figure 4c). However, the concentration of KCl necessary to

trigger 50% of maximal release of [3H]5-HT was similar across all synaptosomes preparations (Figure 4d; EC50: 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 [3H]5-HT as all experiments were conducted in the presence of pargyline. Potassium-induced

release is only weakly dependent on the presence of calcium in the media under the above-described 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 calcium-dependent release. This involved the retrieval of pargyline from the media and shorter potassium pulses (30 s).

Under these conditions, we observed a calcium-dependent release of [3H]5-HT (indicated in Figure 4e as a decrease in the amount of radioactivity in 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 VMAT2_sert−cre_ 5-HT depletion, we measured 5-HT1A receptor-stimulated [35S]GTP-_γ_-S binding using 5-CT as the agonist (Fabre et al, 2000). [35S]GTP-_γ_-S binding was

significantly increased in the dorsal raphe nucleus in response to 5-HT1A receptor activation by 5-CT in VMAT2_sert−cre_ compared with control mice (_p_<0.0001, two-way ANOVA, Figure 5a),

suggesting an upregulation of 5-HT1A autoreceptors and/or enhanced coupling efficacy of these autoreceptors to G proteins. In contrast, no change in 5-CT-induced [35S]GTP-_γ_-S binding was

found in post-synaptic sites, such as the dorsal hippocampus (Figure 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 did control mice (Figure 6a). However, there was a significant decrease (−37±7%; _p_<0.05) in the overall entries into both the closed and the open arms (Figure 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 VMAT2_sert−cre_ mice (Figure 6b). To further explore the anxiety-related phenotype in a model less dependent on locomotion, we applied the novelty-suppressed feeding

(NSF) 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 three times faster; Figure 6c)

than did 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 1 week did not differ between transgenic and control mice (VMAT2_sert−cre_: 3.58±0.28 g; control: 3.65±0.30 g; mean±SEM. _n_=5–6).

Finally, when tested in the tail suspension test (TST), VMAT2_sert−cre_ mice were more reactive than were control mice. Indeed, immobility time was reduced (Figure 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 in active defense behaviors of VMAT2_sert−cre_ mice in the TST.

Interestingly, VMAT2_sert−cre_ mice also displayed a large increase in behavioral reactivity in response to stimulation with the innately aversive US (_p_<0.01; Figure 7a). In addition,

VMAT2_sert−cre_ mice froze less in reaction to the US than did control mice (control: 69±7% of time during the OFF periods; VMAT2_sert−cre_: 37±8%; mean±SEM.; _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 reduced 5-HT levels, we investigated the effects of a long-term pargyline

treatment (70 mg/kg × 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 with

vehicle-treated control mice (Figure 7d). Nevertheless, after pargyline treatment, 5-HT tissue levels were still 20±13% lower in VMAT2_sert−cre_ mutants than in pargyline-treated control

mice (Figure 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 (Figures 7b and 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 with control mice (Figure 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 × 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; Bunin et al, 1998; O’Connor and Kruk, 1991). We confirm here that VMAT2 is the only vesicular transporter implicated in this effect in the CNS, as its genetic

ablation in 5-HT neurons entirely reproduced the effects on 5-HT uptake of complete VMAT inhibition with reserpine. Interestingly, however, in VMAT2_sert−cre_ mice, 5-HT is probably produced

at normal rates, as suggested by (1) the normal levels of _TPH2_ gene expression, (2) the normal amounts of 5-HIAA (the product of oxidative deamination of 5-HT), and (3) the normal

increase in 5-HT levels after MAO inhibition by pargyline. Hence, all 5-HT produced or taken up by neurons, which cannot be stored into vesicles are rapidly metabolized by mitochondrial

monoamine oxidases present in raphe neurons (Shih et al, 1997; Vitalis et al, 2002). As 5-HT continues to be produced in raphe neurons at a seemingly normal rate, it can still be released.

Although such release is likely to be marginal under standard conditions because of the low endogenous levels of 5-HT, it could still occur after administration of a MAOI which causes a

major increase in 5-HT tissue levels. Furthermore, it cannot be excluded that pargyline could enhance some form of vesicular storage (Buu, 1989). In both _VMAT2_ KO and VMAT2_sert−cre_ mice,

MAOIs restored some functions such as the inhibitory action of 5-HT on active defense reactions (Figure 7b) and normal growth (Alvarez et al, 2002; Narboux-Nême et al, 2009). This

functional restoration _in vivo_ fits well with the potassium- and MDMA-induced releases of [3H]5-HT observed _in vitro_ in VMAT2_sert−cre_ synaptosomes. The mechanism involved in the

[3H]5-HT release in VMAT2_sert−cre_ synaptosomes is presently unclear, but three non-mutually exclusive possibilities can be discussed: (1) Release of [3H]5-HT through the carrier (Evans and

Collard, 1988). Although this was suggested to occur in VMAT2 KO mice (Fon et al, 1997), 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 [3H]5-HT vesicular concentration compared with

the axoplasm (Njus et al, 1986), although this passively generated gradient remains very small compared with that generated by VMAT2, it may nevertheless be a way to compensate for VMAT2

loss. (3) Finally, there is evidence for uptake of 5-HT in catecholaminergic neurons (Cases et al, 1998; Zhou et al, 2005). Storage of [3H]5-HT in any non-serotonergic neurons, still

normally expressing VMAT2 in VMAT2_sert−cre_ mice, could account for some of the [3H]5-HT release observed here. Future studies are clearly required to understand how 5-HT release can occur

in VMAT2_sert−cre_ mice. Beyond the cellular mechanisms involved, an important finding here is that [3H]5-HT stabilized through MAO inhibition in the axoplasm can be mobilized out of the

synaptosomes. The small but significant changes in EC50 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 the 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. Reduced 5-HT levels in

VMAT2_sert−cre_ mice caused a sensitization of the 5-HT1A autoreceptors coupled to G_α_i proteins in the dorsal raphe nucleus. In contrast, the decrease in 5-HT contents in VMAT2_sert−cre_

mice caused no adaptive changes of 5-HT1A receptors in target areas, such as the hippocampus. This is in line with the notion that raphe 5-HT1A autoreceptors, but not hippocampal 5-HT1A

receptors, show modulation in response to changes in 5-HT levels (Fabre et al, 2000; Mannoury La Cour et al, 2006; Mongeau et al, 1997) 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

(Trowbridge et al, 2010). All recent genetic hyposerotonergic models focused on reducing 5-HT production either by targeting the central 5-HT synthesis enzyme, TPH2 (Alenina et al, 2009;

Beaulieu et al, 2008; Gutknecht et al, 2008; Savelieva et al, 2008) or by invalidating the transcription factors controlling differentiation of raphe neurons such as _pet1_ and _lmx1B_ (Dai

et al, 2008; Hendricks et al, 2003; Kiyasova et al, 2011; Zhao et al, 2006). The more severe 5-HT depletion observed here (−95%), compared with previous models (−70 to 90%), is likely due to

the fact that increased 5-HT degradation, when VMAT2 is absent, affects all sources of 5-HT (eg, produced by TPH1/TPH2). The present observations, consistent with Tph2 knockout mice

(Alenina et al, 2009; Gutknecht et al, 2008), 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 (Trowbridge et al,

2010). Normal morphology of 5-HT neurons and brain specificity provides two major advantages of genetic models in general compared with pharmacological depletion methods such as,

respectively, lesions of 5-HT neurons with 5,7-dihydroxytryptamine and depletion with _p_-chlorophenylalanine (PCPA). Compared with the Tph2 knockout which irreversibly inactivates 5-HT

synthesis, the VMAT2_sert−cre_ mutation has also the advantage of rapid reversibility through MAO inhibition as TPH2 is not targeted or downregulated. Overall, VMAT2 selective deletion

should allow more direct explorations of the function of monoamine release in otherwise normally developed raphe neurons. Behavioral characterization of 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 (Dai et al, 2008; Kiyasova et al, 2011) and with

numerous studies using pharmacological approaches to decrease brain 5-HT in rats (Graeff, 2004; Griebel, 1995). Furthermore, decreased 5-HT input at several receptors (including the 5-HT2A

and 5-HT2C subtypes) clearly reduces anxiety in mice (Heisler et al, 2007; Mongeau et al, 2010; Weisstaub et al, 2006). VMAT2_sert−cre_ mice were first tested in the most standard model, the

EPM, having a strong validity for anxiolysis mediated by the GABAergic system, but not so much, it seems from previous studies (McCreary et al, 1996), for the serotonergic system. Indeed,

the effect of altering 5-HT on 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-HT2C agonist generated opposite effects in the EPM depending on the brain areas targeted (Cornelio and Nunes-de-Souza, 2007; Nunes-de-Souza et al, 2008). This might

explain the lack of changes in VMAT2_sert−cre_ mice observed here with the EPM. The observed decrease in the exploratory behavior of VMAT2_sert−cre_ mice, as in other hyposerotonergic models

(Dringenberg et al, 1995; Hendricks et al, 2003), 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, in which reduced 5-HT tone was linked to decreased anxiety-like behaviors (Graeff, 2004). In the NSF, which involves a conflict between the drive to feed

and 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 (Dai et al, 2008; Kiyasova et al, 2011). It is also interesting to note that

VMAT2_sert−cre_ mice spent more time struggling to escape than did control mice in the TST. This is coherent with observations in Tph1::Tph2 DKO mice which displayed increased struggling

time in the forced swim test (FST) (Savelieva et al, 2008). 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 (Kiser and Lebovitz, 1975). As far as we know, the present genetic invalidation study is the first that 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 (Beckett et al, 1996). 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 (Griebel et al, 1998). In our study, we have performed a long-term, rather than a short-term, MAOI treatment to optimally enhance 5-HT neurotransmission by

desensitization of 5-HT1A autoreceptors (Mongeau et al, 1997), which were found to be hypersensitive in VMAT2_sert−cre_ mice (Figure 5). In relation to 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 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 (Cryan et al, 2005; O’Leary et al, 2007). In contrast, depleting catecholamines in

adult mice strongly increases baseline immobility in the TST (O’Leary et al, 2007). Constitutive knockdowns of _VMAT2_ (_VMAT2_+/−), which reduces the release of all monoamines, also

results in an increased immobility in the FST and the TST (Fukui et al, 2007). However, the present observation that selective deletion of VMAT2 in serotonergic neurons decreased, rather

than increased, immobility in the TST, together with previous observation (Savelieva et al, 2008) on Tph1::Tph2 DKO mice subjected to the FST, indicate that a depletion of 5-HT is

insufficient to induce depression-like behaviors in such escape-related 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 (Cryan et al, 2005). It is important to emphasize here that although

chronic treatments increase 5-HT neurotransmission, acute administrations do not (Mongeau et al, 1997). 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 (Auerbach et al, 1995; Hajos et al, 1995; Hervas and Artigas,

1998; Hjorth and Auerbach, 1994). 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 in 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 (Griebel et al, 1996) and chronic MAOIs are well known to be effective anti-panic agents

(Bakish et al, 1993). 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. REFERENCES * Adell A, Artigas F (1998). A microdialysis study of the _in vivo_ release of 5-HT in the median raphe nucleus of the rat. _Br J Pharmacol_

125: 1361–1367. Article  CAS  PubMed  PubMed Central  Google Scholar  * Alenina N, Kikic D, Todiras M, Mosienko V, Qadri F, Plehm R _et al_ (2009). Growth retardation and altered autonomic

control in mice lacking brain serotonin. _Proc Natl Acad Sci USA_ 106: 10332–10337. Article  CAS  PubMed  PubMed Central  Google Scholar  * Alvarez C, Vitalis T, Fon EA, Hanoun N, Hamon M,

Seif I _et al_ (2002). Effects of genetic depletion of monoamines on somatosensory cortical development. _Neuroscience_ 115: 753–764. Article  CAS  PubMed  Google Scholar  * Attwell D,

Barbour B, Szatkowski M (1993). Nonvesicular release of neurotransmitter. _Neuron_ 11: 401–407. Article  CAS  PubMed  Google Scholar  * Auerbach SB, Lundberg JF, Hjorth S (1995).

Differential inhibition of serotonin release by 5-HT and NA reuptake blockers after systemic administration. _Neuropharmacology_ 34: 89–96. Article  CAS  PubMed  Google Scholar  * Bakish D,

Saxena BM, Bowen R, D’Souza J (1993). Reversible monoamine oxidase-A inhibitors in panic disorder. _Clin Neuropharmacol_ 16 (Suppl 2): S77–S82. PubMed  Google Scholar  * Bally-Cuif L, Wassef

M (1994). Ectopic induction and reorganization of Wnt-1 expression in quail/chick chimeras. _Development_ 120: 3379–3394. CAS  PubMed  Google Scholar  * Beaulieu JM, Zhang X, Rodriguiz RM,

Sotnikova TD, Cools MJ, Wetsel WC _et al_ (2008). Role of GSK3 beta in behavioral abnormalities induced by serotonin deficiency. _Proc Natl Acad Sci USA_ 105: 1333–1338. Article  CAS  PubMed

  PubMed Central  Google Scholar  * Bechtholt AJ, Hill TE, Lucki I (2007). Anxiolytic effect of serotonin depletion in the novelty-induced hypophagia test. _Psychopharmacology (Berl)_ 190:

531–540. CAS  Google Scholar  * Beckett SR, Aspley S, Graham M, Marsden CA (1996). Pharmacological manipulation of ultrasound induced defence behaviour in the rat. _Psychopharmacology

(Berl)_ 127: 384–390. Article  CAS  Google Scholar  * Berton O, Nestler EJ (2006). New approaches to antidepressant drug discovery: beyond monoamines. _Nat Rev Neurosci_ 7: 137–151. Article

  CAS  PubMed  Google Scholar  * Bunin MA, Prioleau C, Mailman RB, Wightman RM (1998). Release and uptake rates of 5-hydroxytryptamine in the dorsal raphe and substantia nigra reticulata of

the rat brain. _J Neurochem_ 70: 1077–1087. Article  CAS  PubMed  Google Scholar  * Bunin MA, Wightman RM (1999). Paracrine neurotransmission in the CNS: involvement of 5-HT. _Trends

Neurosci_ 22: 377–382. Article  CAS  PubMed  Google Scholar  * Buu NT (1989). Modification of vesicular dopamine and norepinephrine by monoamine oxidase inhibitors. _Biochem Pharmacol_ 38:

1685–1692. Article  CAS  PubMed  Google Scholar  * Cases O, Lebrand C, Giros B, Vitalis T, De Maeyer E, Caron MG _et al_ (1998). Plasma membrane transporters of serotonin, dopamine, and

norepinephrine mediate serotonin accumulation in atypical locations in the developing brain of monoamine oxidase A knock-outs. _J Neurosci_ 18: 6914–6927. Article  CAS  PubMed  PubMed

Central  Google Scholar  * Christiansen L, Tan Q, Iachina M, Bathum L, Kruse TA, McGue M _et al_ (2007). Candidate gene polymorphisms in the serotonergic pathway: influence on depression

symptomatology in an elderly population. _Biol Psychiatry_ 61: 223–230. Article  CAS  PubMed  Google Scholar  * Cornelio AM, Nunes-de-Souza RL (2007). Anxiogenic-like effects of mCPP

microinfusions into the amygdala (but not dorsal or ventral hippocampus) in mice exposed to elevated plus-maze. _Behav Brain Res_ 178: 82–89. Article  CAS  PubMed  Google Scholar  * Cote F,

Thevenot E, Fligny C, Fromes Y, Darmon M, Ripoche MA _et al_ (2003). Disruption of the nonneuronal tph1 gene demonstrates the importance of peripheral serotonin in cardiac function. _Proc

Natl Acad Sci USA_ 100: 13525–13530. Article  CAS  PubMed  PubMed Central  Google Scholar  * Cryan JF, Mombereau C, Vassout A (2005). The tail suspension test as a model for assessing

antidepressant activity: review of pharmacological and genetic studies in mice. _Neurosci Biobehav Rev_ 29: 571–625. Article  CAS  PubMed  Google Scholar  * Dai JX, Han HL, Tian M, Cao J,

Xiu JB, Song NN _et al_ (2008). Enhanced contextual fear memory in central serotonin-deficient mice. _Proc Natl Acad Sci USA_ 105: 11981–11986. Article  CAS  PubMed  PubMed Central  Google

Scholar  * Dringenberg HC, Hargreaves EL, Baker GB, Cooley RK, Vanderwolf CH (1995). _p_-Chlorophenylalanine-induced serotonin depletion: reduction in exploratory locomotion but no obvious

sensory-motor deficits. _Behav Brain Res_ 68: 229–237. Article  CAS  PubMed  Google Scholar  * Erickson JD, Eiden LE (1993). Functional identification and molecular cloning of a human brain

vesicle monoamine transporter. _J Neurochem_ 61: 2314–2317. Article  CAS  PubMed  Google Scholar  * Erickson JD, Schafer MK, Bonner TI, Eiden LE, Weihe E (1996). Distinct pharmacological

properties and distribution in neurons and endocrine cells of two isoforms of the human vesicular monoamine transporter. _Proc Natl Acad Sci USA_ 93: 5166–5171. Article  CAS  PubMed  PubMed

Central  Google Scholar  * Evans SM, Collard KJ (1988). 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. _Br J Pharmacol_ 95: 950–956. Article  CAS  PubMed  PubMed Central  Google Scholar  * Fabre V, Beaufour C, Evrard A, Rioux A, Hanoun N, Lesch KP

_et al_ (2000). Altered expression and functions of serotonin 5-HT1A and 5-HT1B receptors in knock-out mice lacking the 5-HT transporter. _Eur J Neurosci_ 12: 2299–2310. Article  CAS  PubMed

  Google Scholar  * Fon EA, Pothos EN, Sun BC, Killeen N, Sulzer D, Edwards RH (1997). Vesicular transport regulates monoamine storage and release but is not essential for amphetamine

action. _Neuron_ 19: 1271–1283. Article  CAS  PubMed  Google Scholar  * Freis ED (1954). Mental depression in hypertensive patients treated for long periods with large doses of reserpine. _N

Engl J Med_ 251: 1006–1008. Article  CAS  PubMed  Google Scholar  * Fukui M, Rodriguiz RM, Zhou J, Jiang SX, Phillips LE, Caron MG _et al_ (2007). Vmat2 heterozygous mutant mice display a

depressive-like phenotype. _J Neurosci_ 27: 10520–10529. Article  CAS  PubMed  PubMed Central  Google Scholar  * Gaspar P, Duyckaerts C, Alvarez C, Javoy-Agid F, Berger B (1991). Alterations

of dopaminergic and noradrenergic innervations in motor cortex in Parkinson's disease. _Ann Neurol_ 30: 365–374. Article  CAS  PubMed  Google Scholar  * Graeff FG (2004). Serotonin,

the periaqueductal gray and panic. _Neurosci Biobehav Rev_ 28: 239–259. Article  CAS  PubMed  Google Scholar  * Gray EG, Whittaker VP (1962). The isolation of nerve endings from brain: an

electron-microscopic study of cell fragments derived by homogenization and centrifugation. _J Anat_ 96: 79–88. CAS  PubMed  PubMed Central  Google Scholar  * Gray JA, McNaughton N (2000).

_The Neuropsychology of Anxiety_. Oxford Medical Publications: Oxford. Google Scholar  * Griebel G (1995). 5-Hydroxytryptamine-interacting drugs in animal models of anxiety disorders: more

than 30 years of research. _Pharmacol Ther_ 65: 319–395. Article  CAS  PubMed  Google Scholar  * Griebel G, Blanchard DC, Blanchard RJ (1996). Predator-elicited flight responses in

Swiss-Webster mice: an experimental model of panic attacks. _Prog Neuropsychopharmacol Biol Psychiatry_ 20: 185–205. Article  CAS  PubMed  Google Scholar  * Griebel G, Curet O, Perrault G,

Sanger DJ (1998). 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. _Neuropharmacology_ 37: 927–935. Article  CAS  PubMed  Google Scholar  * Gutierrez B, Rosa A, Papiol S, Arrufat FJ, Catalan R, Salgado P _et al_ (2007). Identification of

two risk haplotypes for schizophrenia and bipolar disorder in the synaptic vesicle monoamine transporter gene (SVMT). _Am J Med Genet B Neuropsychiatr Genet_ 144B: 502–507. Article  CAS 

PubMed  Google Scholar  * Gutknecht L, Waider J, Kraft S, Kriegebaum C, Holtmann B, Reif A _et al_ (2008). Deficiency of brain 5-HT synthesis but serotonergic neuron formation in Tph2

knockout mice. _J Neural Transm_ 115: 1127–1132. Article  CAS  PubMed  Google Scholar  * Hajos M, Gartside SE, Sharp T (1995). Inhibition of median and dorsal raphe neurones following

administration of the selective serotonin reuptake inhibitor paroxetine. _Naunyn Schmiedebergs Arch Pharmacol_ 351: 624–629. Article  CAS  PubMed  Google Scholar  * Heisler LK, Zhou L, Bajwa

P, Hsu J, Tecott LH (2007). Serotonin 5-HT(2C) receptors regulate anxiety-like behavior. _Genes Brain Behav_ 6: 491–496. Article  CAS  PubMed  Google Scholar  * Hekmatpanah CR, Peroutka SJ

(1990). 5-Hydroxytryptamine uptake blockers attenuate the 5-hydroxytryptamine-releasing effect of 3,4-methylenedioxymethamphetamine and related agents. _Eur J Pharmacol_ 177: 95–98. Article

  CAS  PubMed  Google Scholar  * Hendricks TJ, Fyodorov DV, Wegman LJ, Lelutiu NB, Pehek EA, Yamamoto B _et al_ (2003). Pet-1 ETS gene plays a critical role in 5-HT neuron development and is

required for normal anxiety-like and aggressive behavior. _Neuron_ 37: 233–247. Article  CAS  PubMed  Google Scholar  * Henry JP, Sagne C, Bedet C, Gasnier B (1998). The vesicular monoamine

transporter: from chromaffin granule to brain. _Neurochem Int_ 32: 227–246. Article  CAS  PubMed  Google Scholar  * Hervas I, Artigas F (1998). Effect of fluoxetine on extracellular

5-hydroxytryptamine in rat brain. Role of 5-HT autoreceptors. _Eur J Pharmacol_ 358: 9–18. Article  CAS  PubMed  Google Scholar  * Hjorth S, Auerbach SB (1994). Further evidence for the

importance of 5-HT1A autoreceptors in the action of selective serotonin reuptake inhibitors. _Eur J Pharmacol_ 260: 251–255. Article  CAS  PubMed  Google Scholar  * Holtje M, Winter S,

Walther D, Pahner I, Hortnagl H, Ottersen OP _et al_ (2003). The vesicular monoamine content regulates VMAT2 activity through Galphaq in mouse platelets. Evidence for autoregulation of

vesicular transmitter uptake. _J Biol Chem_ 278: 15850–15858. Article  PubMed  Google Scholar  * Kiser RS, Lebovitz RM (1975). Monoaminergic mechanisms in aversive brain stimulation.

_Physiol Behav_ 15: 47–53. Article  CAS  PubMed  Google Scholar  * Kiyasova V, Fernandez SP, Laine J, Stankovski L, Muzerelle A, Doly S _et al_ (2011). A genetically defined morphologically

and functionally unique subset of 5-HT neurons in the mouse raphe nuclei. _J Neurosci_ 31: 2756–2768. Article  CAS  PubMed  PubMed Central  Google Scholar  * Lucki I (1998). The spectrum of

behaviors influenced by serotonin. _Biol Psychiatry_ 44: 151–162. Article  CAS  PubMed  Google Scholar  * Mannoury La Cour C, El Mestikawy S, Hanoun N, Hamon M, Lanfumey L (2006). Regional

differences in the coupling of 5-hydroxytryptamine-1A receptors to G proteins in the rat brain. _Mol Pharmacol_ 70: 1013–1021. Article  PubMed  Google Scholar  * McCreary AC, McBlane JW,

Spooner HA, Handley SL (1996). 5-HT systems and anxiety: multiple mechanisms in the elevated X-maze. _Pol J Pharmacol_ 48: 1–12. CAS  PubMed  Google Scholar  * McKenna DJ, Guan XM, Shulgin

AT (1991). 3,4-Methylenedioxyamphetamine (MDA) analogues exhibit differential effects on synaptosomal release of 3H-dopamine and 3H-5-hydroxytryptamine. _Pharmacol Biochem Behav_ 38:

505–512. Article  CAS  PubMed  Google Scholar  * Mongeau R, Blier P, de Montigny C (1997). The serotonergic and noradrenergic systems of the hippocampus: their interactions and the effects

of antidepressant treatments. _Brain Res Brain Res Rev_ 23: 145–195. Article  CAS  PubMed  Google Scholar  * Mongeau R, de Montigny C, Blier P (1994). Activation of 5-HT3 receptors enhances

the electrically evoked release of [3H]noradrenaline in rat brain limbic structures. _Eur J Pharmacol_ 256: 269–279. Article  CAS  PubMed  Google Scholar  * Mongeau R, Martin CB, Chevarin C,

Maldonado R, Hamon M, Robledo P _et al_ (2010). 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. _J Neurochem_ 115: 438–449. Article  CAS  PubMed  Google Scholar  * Mongeau R, Miller GA, Chiang E, Anderson DJ (2003). Neural correlates of

competing fear behaviors evoked by an innately aversive stimulus. _J Neurosci_ 23: 3855–3868. Article  CAS  PubMed  PubMed Central  Google Scholar  * Morilak DA, Frazer A (2004).

Antidepressants and brain monoaminergic systems: a dimensional approach to understanding their behavioural effects in depression and anxiety disorders. _Int J Neuropsychopharmacol_ 7:

193–218. CAS  PubMed  Google Scholar  * Narboux-Nême N, Mongeau R, Sagne C, Angenard G, Hamon M, Martres MP _et al_ (2009). Severe serotonin depletion, growth and behavioral abnormalities in

conditional vesicular monoamine transporter 2 knockout mouse. _Soc Neurosci Abstract_ 90.9. * Narboux-Neme N, Pavone LM, Avallone L, Zhuang X, Gaspar P (2008). Serotonin transporter

transgenic (SERTcre) mouse line reveals developmental targets of serotonin specific reuptake inhibitors (SSRIs). _Neuropharmacology_ 55: 994–1005. Article  CAS  PubMed  Google Scholar  *

Njus D, Kelley PM, Harnadek GJ (1986). Bioenergetics of secretory vesicles. _Biochim Biophys Acta_ 853: 237–265. Article  CAS  PubMed  Google Scholar  * Nunes-de-Souza V, Nunes-de-Souza RL,

Rodgers RJ, Canto-de-Souza A (2008). 5-HT2 receptor activation in the midbrain periaqueductal grey (PAG) reduces anxiety-like behaviour in mice. _Behav Brain Res_ 187: 72–79. Article  CAS 

PubMed  Google Scholar  * O’Connor JJ, Kruk ZL (1991). Fast cyclic voltammetry can be used to measure stimulated endogenous 5-hydroxytryptamine release in untreated rat brain slices. _J

Neurosci Methods_ 38: 25–33. Article  PubMed  Google Scholar  * O’Leary OF, Bechtholt AJ, Crowley JJ, Hill TE, Page ME, Lucki I (2007). Depletion of serotonin and catecholamines block the

acute behavioral response to different classes of antidepressant drugs in the mouse tail suspension test. _Psychopharmacology (Berl)_ 192: 357–371. Article  Google Scholar  * Renoir T,

Paizanis E, El Yacoubi M, Saurini F, Hanoun N, Melfort M _et al_ (2008). Differential long-term effects of MDMA on the serotoninergic system and hippocampal cell proliferation in 5-HTT

knock-out _vs_ wild-type mice. _Int J Neuropsychopharmacol_ 11: 1149–1162. Article  CAS  PubMed  Google Scholar  * Savelieva KV, Zhao S, Pogorelov VM, Rajan I, Yang Q, Cullinan E _et al_

(2008). Genetic disruption of both tryptophan hydroxylase genes dramatically reduces serotonin and affects behavior in models sensitive to antidepressants. _PLoS One_ 3: e3301. Article 

PubMed  PubMed Central  Google Scholar  * Shih JC, Grimsby J, Chen K (1997). Molecular biology of monoamines oxidases A and B: their role in the degradation of serotonin. In: _Handbook of

Experimental Pharmacology_. Springer-Verlag: Berlin. pp 655–670. Google Scholar  * Trowbridge S, Narboux-Neme N, Gaspar P (2010). Genetic models of serotonin (5-HT) depletion: what do they

tell us about the developmental role of 5-HT? _Anat Rec (Hoboken)_ (ahead of print). * Vitalis T, Fouquet C, Alvarez C, Seif I, Price D, Gaspar P _et al_ (2002). Developmental expression of

monoamine oxidases A and B in the central and peripheral nervous systems of the mouse. _J Comp Neurol_ 442: 331–347. Article  CAS  PubMed  Google Scholar  * Wang YM, Gainetdinov RR,

Fumagalli F, Xu F, Jones SR, Bock CB _et al_ (1997). Knockout of the vesicular monoamine transporter 2 gene results in neonatal death and supersensitivity to cocaine and amphetamine.

_Neuron_ 19: 1285–1296. Article  CAS  PubMed  Google Scholar  * Weisstaub NV, Zhou M, Lira A, Lambe E, Gonzalez-Maeso J, Hornung JP _et al_ (2006). Cortical 5-HT2A receptor signaling

modulates anxiety-like behaviors in mice. _Science_ 313: 536–540. Article  CAS  PubMed  Google Scholar  * Wu Y, Wang W, Diez-Sampedro A, Richerson GB (2007). Nonvesicular inhibitory

neurotransmission via reversal of the GABA transporter GAT-1. _Neuron_ 56: 851–865. Article  CAS  PubMed  PubMed Central  Google Scholar  * Zhao S, Edwards J, Carroll J, Wiedholz L,

Millstein RA, Jaing C _et al_ (2006). Insertion mutation at the C-terminus of the serotonin transporter disrupts brain serotonin function and emotion-related behaviors in mice.

_Neuroscience_ 140: 321–334. Article  CAS  PubMed  Google Scholar  * Zhou FM, Liang Y, Salas R, Zhang L, De Biasi M, Dani JA (2005). Corelease of dopamine and serotonin from striatal

dopamine terminals. _Neuron_ 46: 65–74. Article  CAS  PubMed  Google Scholar  * Zhuang X, Masson J, Gingrich JA, Rayport S, Hen R (2005). Targeted gene expression in dopamine and serotonin

neurons of the mouse brain. _J Neurosci Methods_ 143: 27–32. Article  CAS  PubMed  Google Scholar  Download references 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 (ANR-08-MNPS-032), the Région I?le de France, DIM 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. NN-N 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 fellowships from IBRO and from Region Ile de France DIM STEM and SD by a

LeFoulon-Delalande fellowship. AUTHOR INFORMATION AUTHORS AND AFFILIATIONS * INSERM, UMR-S 839, Institut du Fer à Moulin, Paris, France Nicolas Narboux-Nême, Stephane Doly, Silvina L Diaz, 

Gaelle Angenard & Patricia Gaspar * Université Pierre and Marie Curie, Paris, France Nicolas Narboux-Nême, Stephane Doly, Silvina L Diaz, Cédric B P Martin, Gaelle Angenard, 

Marie-Pascale Martres, Bruno Giros, Michel Hamon, Laurence Lanfumey, Patricia Gaspar & Raymond Mongeau * CNRS UMR8192-Université Paris Descartes, Paris, France Corinne Sagné * INSERM,

U894, Paris, France Cédric B P Martin, Michel Hamon, Laurence Lanfumey & Raymond Mongeau * INSERM, U952, Paris, France Marie-Pascale Martres & Bruno Giros * CNRS UMR7224, Paris,

France Marie-Pascale Martres & Bruno Giros * Department of Psychiatry, Douglas Hospital, McGill University, Montreal, Canada Bruno Giros Authors * Nicolas Narboux-Nême View author

publications You can also search for this author inPubMed Google Scholar * Corinne Sagné View author publications You can also search for this author inPubMed Google Scholar * Stephane Doly

View author publications You can also search for this author inPubMed Google Scholar * Silvina L Diaz View author publications You can also search for this author inPubMed Google Scholar *

Cédric B P Martin View author publications You can also search for this author inPubMed Google Scholar * Gaelle Angenard View author publications You can also search for this author inPubMed

 Google Scholar * Marie-Pascale Martres View author publications You can also search for this author inPubMed Google Scholar * Bruno Giros View author publications You can also search for

this author inPubMed Google Scholar * Michel Hamon View author publications You can also search for this author inPubMed Google Scholar * Laurence Lanfumey View author publications You can

also search for this author inPubMed Google Scholar * Patricia Gaspar View author publications You can also search for this author inPubMed Google Scholar * Raymond Mongeau View author

publications You can also search for this author inPubMed Google Scholar CORRESPONDING AUTHOR Correspondence to Patricia Gaspar. ETHICS DECLARATIONS COMPETING INTERESTS The authors declare

no conflict of interest. ADDITIONAL INFORMATION Supplementary Information accompanies the paper on the Neuropsychopharmacology website SUPPLEMENTARY INFORMATION SUPPLEMENTARY INFORMATION

(DOC 77 KB) POWERPOINT SLIDES POWERPOINT SLIDE FOR FIG. 1 POWERPOINT SLIDE FOR FIG. 2 POWERPOINT SLIDE FOR FIG. 3 POWERPOINT SLIDE FOR FIG. 4 POWERPOINT SLIDE FOR FIG. 5 POWERPOINT SLIDE FOR

FIG. 6 POWERPOINT SLIDE FOR FIG. 7 RIGHTS AND PERMISSIONS Reprints and permissions ABOUT THIS ARTICLE CITE THIS ARTICLE Narboux-Nême, N., Sagné, C., Doly, S. _et al._ Severe Serotonin

Depletion after Conditional Deletion of the Vesicular Monoamine Transporter 2 Gene in Serotonin Neurons: Neural and Behavioral Consequences. _Neuropsychopharmacol_ 36, 2538–2550 (2011).

https://doi.org/10.1038/npp.2011.142 Download citation * Received: 16 May 2011 * Revised: 09 June 2011 * Accepted: 15 June 2011 * Published: 03 August 2011 * Issue Date: November 2011 * DOI:

https://doi.org/10.1038/npp.2011.142 SHARE THIS ARTICLE Anyone you share the following link with will be able to read this content: Get shareable link Sorry, a shareable link is not

currently available for this article. Copy to clipboard Provided by the Springer Nature SharedIt content-sharing initiative KEYWORDS * conditional knockout mice * serotonin transporter *

depression * anxiety * raphe * defense behaviors