Alain M. Gardier

Neurogenesis-Dependent and -Independent Effects of Fluoxetine in an Animal Model of Anxiety/Depression

Understanding the physiopathology of affective disorders and their treatment relies on the availability of experimental models that accurately mimic aspects of the disease. Here we describe a mouse model of an anxiety/depressive-like state induced by chronic corticosterone treatment. Furthermore, chronic antidepressant treatment reversed the behavioral dysfunctions and the inhibition of hippocampal neurogenesis induced by corticosterone treatment. In corticosterone-treated mice where hippocampal neurogenesis is abolished by X-irradiation, the efficacy of fluoxetine is blocked in some, but not all, behavioral paradigms, suggesting both neurogenesis-dependent and -independent mechanisms of antidepressant action. Finally, we identified a number of candidate genes, the expression of which is decreased by chronic corticosterone and normalized by chronic fluoxetine treatment selectively in the hypothalamus. Importantly, mice deficient in one of these genes, beta-arrestin 2, displayed a reduced response to fluoxetine in multiple tasks, suggesting that beta-arrestin signaling is necessary for the antidepressant effects of fluoxetine.

Nicotine reinforcement and cognition restored by targeted expression of nicotinic receptors

Worldwide, 100 million people are expected to die this century from the consequences of nicotine addiction, but nicotine is also known to enhance cognitive performance. Identifying the molecular mechanisms involved in nicotine reinforcement and cognition is a priority and requires the development of new in vivo experimental paradigms. The ventral tegmental area (VTA) of the midbrain is thought to mediate the reinforcement properties of many drugs of abuse. Here we specifically re-expressed the β2-subunit of the nicotinic acetylcholine receptor (nAChR) by stereotaxically injecting a lentiviral vector into the VTA of mice carrying β2-subunit deletions. We demonstrate the efficient re-expression of electrophysiologically responsive, ligand-binding nicotinic acetylcholine receptors in dopamine-containing neurons of the VTA, together with the recovery of nicotine-elicited dopamine release and nicotine self-administration. We also quantified exploratory behaviours of the mice, and showed that β2-subunit re-expression restored slow exploratory behaviour (a measure of cognitive function) to wild-type levels, but did not affect fast navigation behaviour. We thus demonstrate the sufficient role of the VTA in both nicotine reinforcement and endogenous cholinergic regulation of cognitive functions.

Subunit Composition of Functional Nicotinic Receptors in Dopaminergic Neurons Investigated with Knock-Out Mice

Nicotinic acetylcholine receptors (nAChRs) expressed by dopaminergic (DA) neurons have long been considered as potential therapeutic targets for the treatment of several neuropsychiatric diseases, including nicotine and cocaine addiction or Parkinsons disease. However, DA neurons express mRNAs coding for most, if not all, neuronal nAChR subunits, and the subunit composition of functional nAChRs has been difficult to establish. Immunoprecipitation experiments performed on mouse striatal extracts allowed us to identify three main types of heteromeric nAChRs (α4β2*, α6β2*, and α4α6β2*) in DA terminal fields. The functional relevance of these subtypes was then examined by studying nicotine-induced DA release in striatal synaptosomes and recording ACh-elicited currents in DA neurons fromα4, α6, α4α6, and β2 knock-out mice. Our results establish that α6β2* nAChRs are functional and sensitive to α-conotoxin MII inhibition. These receptors are mainly located on DA terminals and consistently do not contribute to DA release induced by systemic nicotine administration, as evidenced by in vivo microdialysis. In contrast, (nonα6)α4β2* nAChRs represent the majority of functional heteromeric nAChRs on DA neuronal soma. Thus, whereas a combination of α6β2* and α4β2* nAChRs may mediate the endogenous cholinergic modulation of DA release at the terminal level, somato-dendritic (nonα6)α4β2* nAChRs most likely contribute to nicotine reinforcement.

Effects of nicotine in the dopaminergic system of mice lacking the alpha4 subunit of neuronal nicotinic acetylcholine receptors.

The mesostriatal dopaminergic system influences locomotor activity and the reinforcing properties of many drugs of abuse including nicotine. Here we investigate the role of the α4 nicotinic acetylcholine receptor (nAChR) subunit in mediating the effects of nicotine in the mesolimbic dopamine system in mice lacking the α4 subunit. We show that there are two distinct populations of receptors in the substantia nigra and striatum by using autoradiographic labelling with 125I α‐conotoxin MII. These receptors are comprised of the α4, β2 and α6 nAChR subunits and non‐α4, β2, and α6 nAChR subunits. Non‐α4 subunit‐containing nAChRs are located on dopaminergic neurons, are functional and respond to nicotine as demonstrated by patch clamp recordings. In vivo microdialysis performed in awake, freely moving mice reveal that mutant mice have basal striatal dopamine levels which are twice as high as those observed in wild‐type mice. Despite the fact that both wild‐type and α4 null mutant mice show a similar increase in dopamine release in response to intrastriatal KCl perfusion, a nicotine‐elicited increase in dopamine levels is not observed in mutant mice. Locomotor activity experiments show that there is no difference between wild‐type and mutant mice in basal activity in both habituated and non‐habituated environments. Interestingly, mutant mice sustain an increase in cocaine‐elicited locomotor activity longer than wild‐type mice. In addition, mutant mice recover from depressant locomotor activity in response to nicotine at a faster rate. Our results indicate that α4‐containing nAChRs exert a tonic control on striatal basal dopamine release, which is mediated by a heterogeneous population of nAChRs.

Role of 5-HT1A autoreceptors in the mechanism of action of serotoninergic antidepressant drugs : recent findings from in vivo microdialysis studies

Summary— Although a new generation of selective serotonin reuptake inhibitors (SSRIs) has been introduced in therapeutics as antidepressant drugs, a two to four week lag period still occurs between starting treatment with SSRIs and the onset of therapeutic effects in man. In vivo cerebral microdialysis can be used to measure extracellular concentrations of serotonin (5‐hydroxytryptamine, 5‐HT), which reflect intrasynaptic events. With the coupling of this new experimental method to very sensitive analytical assays such as liquid chromatography with electrochemical detection, it has recently been possible to obtain two major arguments supporting the hypothesis that somatodendritic 5‐HT1A autoreceptors situated in the raphe nuclei play an important role in the mechanism of action of SSRIs. First, in the rat, single administration of SSRIs at low doses comparable to those used therapeutically increases extracellular 5‐HT concentrations in the vicinity of the cell body and the dendrites of serotoninergic neurones of the raphe nuclei. This effect is more marked than that observed in regions rich in nerve endings (frontal cortex). The magnitude of the activation of the serotoninergic neurotransmission depends on the brain area studied and the dose of the SSRIs administered to rats. This could be explained by simultaneous activation of somatodendritic 5‐HT1A autoreceptors by endogenous 5‐HT in the raphe nuclei, thereby limiting the corticofrontal effects of the antidepressant. Second, SSRIs cause a larger increase in extracellular 5‐HT concentrations in the nerve endings when administered chronically: 5‐HT autoreceptors may have gradually desensitized during the 2–4 weeks of treatment with SSRIs. Preliminary studies of patients with depression appear to confirm these experimental results, as co‐administration of a 5‐HT1A autoreceptor antagonist and a SSRI accelerated the onset of the antidepressant effect (< 1 week).

Molecular evidence for BDNF- and GABA-related dysfunctions in the amygdala of female subjects with Major Depression

Women are twice as likely as men to develop major depressive disorder (MDD) and are more prone to recurring episodes. Hence, we tested the hypothesis that the illness may associate with robust molecular changes in female subjects, and investigated large-scale gene expression in the post-mortem brain of MDD subjects paired with matched controls (n=21 pairs). We focused on the lateral/basolateral/basomedian complex of the amygdala as a neural hub of mood regulation affected in MDD. Among the most robust findings were downregulated transcripts for genes coding for γ-aminobutyric acid (GABA) interneuron-related peptides, including somatostatin (SST), tachykinin, neuropeptide Y (NPY) and cortistatin, in a pattern reminiscent to that previously reported in mice with low brain-derived neurotrophic factor (BDNF). Changes were confirmed by quantitative PCR and not explained by demographic, technical or known clinical parameters. BDNF itself was significantly downregulated at the RNA and protein levels in MDD subjects. Investigating putative mechanisms, we show that this core MDD-related gene profile (including SST, NPY, TAC1, RGS4 and CORT) is recapitulated by complementary patterns in mice with constitutive (BDNF-heterozygous) or activity-dependent (exon IV knockout) decreases in BDNF function, with a common effect on SST and NPY. Together, these results provide both direct (low RNA/protein) and indirect (low BDNF-dependent gene pattern) evidence for reduced BDNF function in the amygdala of female subjects with MDD. Supporting studies in mutant mice models suggest a complex mechanism of low constitutive and activity-dependent BDNF function in MDD, particularly affecting SST/NPY-related GABA neurons, thus linking the neurotrophic and GABA hypotheses of depression.

Effects of acute fluoxetine on extracellular serotonin levels in the raphe: an in vivo microdialysis study.

Acute administration of fluoxetine (1, 10 and 20 mg/kg i.p.) increased extracellular levels of serotonin (5-hydroxytryptamine, 5-HT) in the frontal cortex, ventral hippocampus and raphe nuclei as measured by in vivo microdialysis in anaesthetized rats. In the frontal cortex, fluoxetine showed a marked dose-response effect whereas in the ventral hippocampus and raphe nuclei the fluoxetine-induced effect was maximum at 10 mg/kg. However, the maximal increase in 5-HT was observed in the cell body-containing area, the raphe nuclei. The order of changes in extracellular 5-HT was raphe nuclei > ventral hippocampus > frontal cortex. Our results add further arguments in favour of the key role played by raphe nuclei in the mechanism of action of serotoninergic antidepressant drugs.

Serotonin-1A Autoreceptors Are Necessary and Sufficient for the Normal Formation of Circuits Underlying Innate Anxiety

Identifying the factors contributing to the etiology of anxiety and depression is critical for the development of more efficacious therapies. Serotonin (5-HT) is intimately linked to both disorders. The inhibitory serotonin-1A (5-HT1A) receptor exists in two separate populations with distinct effects on serotonergic signaling: (1) an autoreceptor that limits 5-HT release throughout the brain and (2) a heteroreceptor that mediates inhibitory responses to released 5-HT. Traditional pharmacologic and transgenic strategies have not addressed the distinct roles of these two receptor populations. Here we use a recently developed genetic mouse system to independently manipulate 5-HT1A autoreceptor and heteroreceptor populations. We show that 5-HT1A autoreceptors act to affect anxiety-like behavior. In contrast, 5-HT1A heteroreceptors affect responses to forced swim stress, without effects on anxiety-like behavior. Together with our previously reported work, these results establish distinct roles for the two receptor populations, providing evidence that signaling through endogenous 5-HT1A autoreceptors is necessary and sufficient for the establishment of normal anxiety-like behavior.

Behavioral and serotonergic consequences of decreasing or increasing hippocampus brain-derived neurotrophic factor protein levels in mice.

Antidepressants such as Selective Serotonin Reuptake Inhibitors (SSRI) act as indirect agonists of serotonin (5-HT) receptors. Although these drugs produce a rapid blockade of serotonin transporters (SERTs) in vitro, several weeks of treatment are necessary to observe clinical benefits. This paradox has not been solved yet. Recent studies have identified modifications of intracellular signaling proteins and target genes that could contribute to antidepressant-like activity of SSRI (e.g., increases in neurogenesis and BDNF protein levels), and may explain, at least in part, their long delay of action. Although these data suggest a positive regulation of 5-HT on the expression of the gene coding for BDNF, the reciprocal effects of BDNF on brain 5-HT neurotransmission remains poorly documented. To study the impact of BDNF on serotonergic activity, a dual experimental strategy was used to analyze neurochemical and behavioral consequences of its decrease (strategy 1) or increase (strategy 2) in the brain of adult male mice. (1) In heterozygous BDNF+/- mice in which brain BDNF protein levels were decreased by half, an enhancement of basal extracellular 5-HT levels (5-HText) that induced a down-regulation of SERT, i.e., a decrease in its capacity to reuptake 5-HT, was found in the hippocampus. In addition, the SSRI, paroxetine, failed to increase hippocampal 5-HText in BDNF+/- mice, while it produces robust effects in wild-type littermates. Thus, BDNF+/- mice can be viewed as an animal model of genetic resistance to serotonergic antidepressant drugs. (2) In wild-type BDNF+/+ mice, the effects of intra-hippocampal (vHi) injection of BDNF (100 ng) in combination with a SSRI was examined by using intracerebral microdialysis and behavioral paradigms that predict an antidepressant- and anxiolytic-like activity of a molecule [the forced swim test (FST) and the open field paradigm (OF) respectively]. BDNF induced a rapid and transient increase in paroxetine response on 5-HText in the adult hippocampus, which was correlated with a potentiation of its antidepressant-like activity in the FST. The effects of BDNF were selectively blocked by K252a, an antagonist of its high-affinity TrkB receptor. Such a correlation between neurochemical and behavioral effects of [BDNF+SSRI] co-administration suggests that its antidepressant-like activity is linked to the activation of 5-HT neurotransmission in the adult hippocampus. BDNF also had a facilitatory effect on anxiety-like behavior in the OF test, and paroxetine prevented this anxiogenesis. What was the mechanism by which BDNF exerted these latter effects? Surprisingly, by using zero net flux method of quantitative microdialysis in vivo, we found that an intra-hippocampal BDNF injection in wild-type mice decreased the functional activity of SERT as observed in BDNF+/- mice. However, the decreased capacity of SERT to reuptake 5-HT was not associated to an increase in basal 5-HText in the hippocampus of WT mice. Interestingly, using in situ hybridization experiments indicated that TrkB receptor mRNA was expressed in the hippocampus and dorsal raphe nucleus in adult mice suggesting that the neurochemical and behavioral effects of intra-hippocampal BDNF injection can mobilize both pre- and post-synaptic elements of the brain 5-HT neurotransmission. Taken together, these set of experiments unveiled a relative opposition of neurochemical and behavioral responses following either a decrease (in BDNF+/- mutant mice) or an increase in brain BDNF levels (bilateral intra-hippocampal injection) in adult mice. In view of developing new antidepressant drug strategy, a poly-therapy combining BDNF with a chronic SSRI treatment could thus improve the efficacy of current medications.

VGLUT3 (Vesicular Glutamate Transporter Type 3) Contribution to the Regulation of Serotonergic Transmission and Anxiety

Three different subtypes of H+-dependent carriers (named VGLUT1–3) concentrate glutamate into synaptic vesicles before its exocytotic release. Neurons using other neurotransmitter than glutamate (such as cholinergic striatal interneurons and 5-HT neurons) express VGLUT3. It was recently reported that VGLUT3 increases acetylcholine vesicular filling, thereby, stimulating cholinergic transmission. This new regulatory mechanism is herein designated as vesicular-filling synergy (or vesicular synergy). In the present report, we found that deletion of VGLUT3 increased several anxiety-related behaviors in adult and in newborn mice as early as 8 d after birth. This precocious involvement of a vesicular glutamate transporter in anxiety led us to examine the underlying functional implications of VGLUT3 in 5-HT neurons. On one hand, VGLUT3 deletion caused a significant decrease of 5-HT1A-mediated neurotransmission in raphe nuclei. On the other hand, VGLUT3 positively modulated 5-HT transmission of a specific subset of 5-HT terminals from the hippocampus and the cerebral cortex. VGLUT3- and VMAT2-positive serotonergic fibers show little or no 5-HT reuptake transporter. These results unravel the existence of a novel subset of 5-HT terminals in limbic areas that might play a crucial role in anxiety-like behaviors. In summary, VGLUT3 accelerates 5-HT transmission at the level of specific 5-HT terminals and can exert an inhibitory control at the raphe level. Furthermore, our results suggest that the loss of VGLUT3 expression leads to anxiety-associated behaviors and should be considered as a potential new target for the treatment of this disorder.