Bruno P. Guiard

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.

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.

Cross-Talk between Dopaminergic and Noradrenergic Systems in the Rat Ventral Tegmental Area, Locus Ceruleus, and Dorsal Hippocampus

A decreased central dopaminergic and/or noradrenergic transmission is believed to be involved in the pathophysiology of depression. It is known that dopamine (DA) neurons in the ventral tegmental area (VTA) and norepinephrine (NE) neurons in the locus ceruleus (LC) are autoregulated by somatodendritic D2-like and α2-adrenoceptors, respectively. Complementing these autoreceptor-mediated inhibitory feedbacks, anatomical and functional studies have established a role for noradrenergic inputs in regulating dopaminergic activity, and reciprocally. In the present study, a microiontophoretic approach was used to characterize the postsynaptic catecholamine heteroreceptors involved in such regulations. In the VTA, the application of DA and NE significantly reduced the firing activity of DA neurons. In addition to a role for D2-like receptors in the inhibitory effects of both catecholamines, it was demonstrated that the α2-adrenoceptor antagonist idazoxan dampened the DA- and NE-induced attenuations of DA neuronal activity, indicating that both of these receptors are involved in the responsiveness of VTA DA neurons to catecholamines. In the LC, the effectiveness of iontophoretically applied NE and DA to suppress NE neuronal firing was blocked by idazoxan but not by the D2-like receptor antagonist raclopride, which suggested that only α2-adrenoceptors were involved. In the dorsal hippocampus, a forebrain region having a sparse dopaminergic innervation but receiving a dense noradrenergic input, the suppressant effects of DA and NE on pyramidal neurons were attenuated by idazoxan but not by raclopride. The suppressant effect of DA was prolonged by administration of the selective NE reuptake inhibitor desipramine and, to lesser extent, of the selective DA reuptake inhibitor 1-(2-[bis(4-fluorophenyl)methoxy]ethyl)-4-(3-phenylpropyl)-piperazine (GBR12909), suggesting that both the NE and DA transporters were involved in DA uptake in the hippocampus. These findings might help in designing new antidepressant strategies aimed at enhancing DA and NE neurotransmission.

BDNF overexpression in mouse hippocampal astrocytes promotes local neurogenesis and elicits anxiolytic-like activities

The therapeutic activity of selective serotonin (5-HT) reuptake inhibitors (SSRIs) relies on long-term adaptation at pre- and post-synaptic levels. The sustained administration of SSRIs increases the serotonergic neurotransmission in response to a functional desensitization of the inhibitory 5-HT1A autoreceptor in the dorsal raphe. At nerve terminal such as the hippocampus, the enhancement of 5-HT availability increases brain-derived neurotrophic factor (BDNF) synthesis and signaling, a major event in the stimulation of adult neurogenesis. In physiological conditions, BDNF would be expressed at functionally relevant levels in neurons. However, the recent observation that SSRIs upregulate BDNF mRNA in primary cultures of astrocytes strongly suggest that the therapeutic activity of antidepressant drugs might result from an increase in BDNF synthesis in this cell type. In this study, by overexpressing BDNF in astrocytes, we balanced the ratio between astrocytic and neuronal BDNF raising the possibility that such manipulation could positively reverberate on anxiolytic-/antidepressant-like activities in transfected mice. Our results indicate that BDNF overexpression in hippocampal astrocytes produced anxiolytic-/antidepressant-like activity in the novelty suppressed feeding in relation with the stimulation of hippocampal neurogenesis whereas it did not potentiate the effects of the SSRI fluoxetine on these parameters. Moreover, overexpressing BDNF revealed the anxiolytic-like activity of fluoxetine in the elevated plus maze while attenuating 5-HT neurotransmission in response to a blunted downregulation of the 5-HT1A autoreceptor. These results emphasize an original role of hippocampal astrocytes in the synthesis of BDNF, which can act through neurogenesis-dependent and -independent mechanisms to regulate different facets of anxiolytic-like responses.

Prospect of a Dopamine Contribution in the Next Generation of Antidepressant Drugs: The Triple Reuptake Inhibitors

Biogenic amine transporters for serotonin and norepinephrine (5-HT and NE respectively), are major targets for currently available antidepressant drugs, particularly those inhibiting the reuptake of 5-HT and/or NE. Compelling evidence suggest that dopamine (DA) is also involved in the pathophysiology and treatment of depression, leading to the development of a new class of antidepressants: the triple reuptake inhibitors that simultaneously inhibit 5-HT, NE and DA reuptake thereby prolonging their duration of action at postsynaptic levels. Although preclinical studies strongly suggest that triple reuptake inhibitors display antidepressant-like activity in various behavioural paradigms, including the forced swimming and the tail suspension tests, it has yet to be demonstrated that the addition of the dopaminergic component produces more robust effects than single- or dual-acting compounds. Several arguments favour this hypothesis and particularly the observation that DA may promote neurotrophic processes in the adult hippocampus, as 5-HT and NE do. It is thus possible that the stimulation of multiple signalling pathways resulting from the elevation of all three monoamines may account, in part, for an accelerated and/or greater antidepressant response. To predict the efficacy of triple reuptake inhibitors, it is important to take into consideration the existence of dense connections between monoaminergic neurons. Indeed, it is well established that the increase in central dopaminergic transmission regulates the neuronal activity of 5-HT and NE in the dorsal raphe (DR) and locus coeruleus (LC), respectively, while in turn, the ventral tegmental area (VTA), is sensitive to changes in 5-HT and NE release. This review synthetizes the pertinent litterature, focusing on the contribution of DA, to illustrate the rationale for designing improved antidepressants.

Antidepressant and anxiolytic potential of the multimodal antidepressant vortioxetine (Lu AA21004) assessed by behavioural and neurogenesis outcomes in mice

Vortioxetine (Lu AA21004) is an investigational novel antidepressant with multimodal activity that functions as a 5-HT3, 5-HT7 and 5-HT(1D) receptor antagonist, 5-HT(1B) receptor partial agonist, 5-HT(1A) receptor agonist and inhibitor of the 5-HT transporter in vitro. Here we explore its anxiolytic and antidepressant potential in adult mice. Vortioxetine was assessed in BalB/cJ@RJ mice using the open-field and forced-swim tests (acute: p.o. 1 h, repeated: daily p.o. 21 days), and in 129S6/SvEvTac mice using the novelty suppressed feeding paradigm (acute: p.o. 1 h, sustained: daily p.o. 14 or 21 days). Fluoxetine and diazepam were controls. Acute and repeated dosing of vortioxetine produced more pronounced anxiolytic- and antidepressant-like activities than fluoxetine. Vortioxetine significantly increased cell proliferation and cell survival and stimulated maturation of immature granule cells in the subgranular zone of the dentate gyrus of the hippocampus after 21 days of treatment. After 14 days, a high dose of vortioxetine increased dendritic length and the number of dendrite intersections, suggesting that vortioxetine accelerates the maturation of immature neurons. Vortioxetine displays an antidepressant and anxiolytic profile following repeated administration associated with increased neurogenesis at several stages. Vortioxetine effects were observed at low levels of 5-HT transporter occupancy, suggesting an alternative mechanism of action to 5-HT reuptake inhibition.

Organic cation transporter 2 controls brain norepinephrine and serotonin clearance and antidepressant response

High-affinity transporters for norepinephrine (NE) and serotonin (5-HT), which ensure neurotransmitter clearance at the synapse, are the principal targets of widely used antidepressant drugs. Antidepressants targeting these high-affinity transporters, however, do not provide positive treatment outcomes for all patients. Other monoamine transport systems, with lower affinity, have been detected in the brain, but their role is largely unknown. Here we report that OCT2, a member of the polyspecific organic cation transporter (OCT) family, is expressed notably in the limbic system and implicated in anxiety and depression-related behaviors in the mouse. Genetic deletion of OCT2 in mice produced a significant reduction in brain tissue concentrations of NE and 5-HT and in ex vivo uptake of both these neurotransmitters in the presence of the dual 5-HT–NE transport blocker, venlafaxine. In vivo clearance of NE and 5-HT evaluated using microiontophoretic electrophysiology was diminished in the hippocampus of OCT2−/− mice in the presence of venlafaxine, thereby affecting postsynaptic neuronal activity. OCT2−/− mice displayed an altered sensitivity to acute treatments with NE- and/or 5-HT-selective transport blockers in the forced-swim test. Moreover, the mutant mice were insensitive to long-term venlafaxine treatment in a more realistic, corticosterone-induced, chronic depression model. Our findings identify OCT2 as an important postsynaptic determinant of aminergic tonus and mood-related behaviors and a potential pharmacological target for mood disorders therapy.

Blockade of substance P (neurokinin 1) receptors enhances extracellular serotonin when combined with a selective serotonin reuptake inhibitor: an in vivo microdialysis study in mice

Substance P antagonists of the neurokinin‐1 receptor type (NK1) are gaining growing interest as new antidepressant therapies. It has been postulated that these drugs exert this putative therapeutic effect without direct interactions with serotonin (5‐HT) neurones. Our recent microdialysis experiment performed in NK1 receptor knockout mice suggested evidence of changes in 5‐HT neuronal function ( Froger et al. 2001 ). The aim of the present study was to evaluate the effects of coadministration of the selective 5‐HT reuptake inhibitor (SSRI) paroxetine with a NK1 receptor antagonist (GR205171 or L733060), given either intraperitoneally (i.p.) or locally into the dorsal raphe nucleus, on extracellular levels of 5‐HT ([5‐HT]ext) in the frontal cortex and the dorsal raphe nucleus using in vivo microdialysis in awake, freely moving mice. The systemic or intraraphe administration of a NK1 receptor antagonist did not change basal cortical [5‐HT]ext in mice. A single systemic dose of paroxetine (4 mg/kg; i.p.) resulted in a statistically significant increase in [5‐HT]ext with a larger extent in the dorsal raphe nucleus (+ 138% over basal AUC values), than in the frontal cortex (+ 52% over basal AUC values). Co‐administration of paroxetine (4 mg/kg; i.p.) with the NK1 receptor antagonists, GR205171 (30 mg/kg; i.p.) or L733060 (40 mg/kg; i.p.), potentiated the effects of paroxetine on cortical [5‐HT]ext in wild‐type mice, whereas GR205171 (30 mg/kg; i.p.) had no effect on paroxetine‐induced increase in cortical [5‐HT]ext in NK1 receptor knock‐out mice. When GR205171 (300 µmol/L) was perfused by ‘reverse microdialysis’ into the dorsal raphe nucleus, it potentiated the effects of paroxetine on cortical [5‐HT]ext, and inhibited paroxetine‐induced increase in [5‐HT]ext in the dorsal raphe nucleus. Finally, in mice whose 5‐HT transporters were first blocked by a local perfusion of 1 µmol/L of citalopram into the frontal cortex, a single dose of paroxetine (4 mg/kg i.p.) decreased cortical 5‐HT release, and GR205171 (30 mg/kg i.p.) reversed this effect. The present findings suggest that NK1 receptor antagonists, when combined with a SSRI, augment 5‐HT release by modulating substance P/5–HT interactions in the dorsal raphe nucleus.