Naoya Nishitani

Control of intermale aggression by medial prefrontal cortex activation in the mouse.

Aggressive behavior is widely observed throughout the animal kingdom because of its adaptiveness for social animals. However, when aggressive behavior exceeds the species-typical level, it is no longer adaptive, so there should be a mechanism to control excessive aggression to keep it within the adaptive range. Using optogenetics, we demonstrate that activation of excitatory neurons in the medial prefrontal cortex (mPFC), but not the orbitofrontal cortex (OFC), inhibits inter-male aggression in mice. At the same time, optogenetic silencing of mPFC neurons causes an escalation of aggressive behavior both quantitatively and qualitatively. Activation of the mPFC suppresses aggressive bursts and reduces the intensity of aggressive behavior, but does not change the duration of the aggressive bursts. Our findings suggest that mPFC activity has an inhibitory role in the initiation and execution, but not the termination, of aggressive behavior, and maintains such behavior within the adaptive range.

Role of the 5-HT4 receptor in chronic fluoxetine treatment-induced neurogenic activity and granule cell dematuration in the dentate gyrus

BackgroundChronic treatment with selective serotonin (5-HT) reuptake inhibitors (SSRIs) facilitates adult neurogenesis and reverses the state of maturation in mature granule cells (GCs) in the dentate gyrus (DG) of the hippocampus. Recent studies have suggested that the 5-HT4 receptor is involved in both effects. However, it is largely unknown how the 5-HT4 receptor mediates neurogenic effects in the DG and, how the neurogenic and dematuration effects of SSRIs interact with each other.ResultsWe addressed these issues using 5-HT4 receptor knockout (5-HT4R KO) mice. Expression of the 5-HT4 receptor was detected in mature GCs but not in neuronal progenitors of the DG. We found that chronic treatment with the SSRI fluoxetine significantly increased cell proliferation and the number of doublecortin-positive cells in the DG of wild-type mice, but not in 5-HT4R KO mice. We then examined the correlation between the increased neurogenesis and the dematuration of GCs. As reported previously, reduced expression of calbindin in the DG, as an index of dematuration, by chronic fluoxetine treatment was observed in wild-type mice but not in 5-HT4R KO mice. The proliferative effect of fluoxetine was inversely correlated with the expression level of calbindin in the DG. The expression of neurogenic factors in the DG, such as brain derived neurotrophic factor (Bdnf), was also associated with the progression of dematuration. These results indicate that the neurogenic effects of fluoxetine in the DG are closely associated with the progression of dematuration of GCs. In contrast, the DG in which neurogenesis was impaired by irradiation still showed significant reduction of calbindin expression by chronic fluoxetine treatment, suggesting that dematuration of GCs by fluoxetine does not require adult neurogenesis in the DG.ConclusionsWe demonstrated that the 5-HT4 receptor plays an important role in fluoxetine-induced adult neurogenesis in the DG in addition to GC dematuration, and that these phenomena are closely associated. Our results suggest that 5-HT4 receptor-mediated phenotypic changes, including dematuration in mature GCs, underlie the neurogenic effect of SSRIs in the DG, providing new insight into the cellular mechanisms of the neurogenic actions of SSRIs in the hippocampus.

Olanzapine augments the effect of selective serotonin reuptake inhibitors by suppressing GABAergic inhibition via antagonism of 5-HT6 receptors in the dorsal raphe nucleus

The combination of the selective serotonin reuptake inhibitors (SSRIs) and atypical antipsychotic drugs shows better therapeutic efficacy than SSRI monotherapy in the treatment of depression. However, the underlying mechanisms responsible for the augmenting effects of olanzapine are not fully understood. Here, we report that olanzapine enhances the SSRI-induced increase in extracellular serotonin (5-HT) levels and antidepressant-like effects by inhibiting GABAergic neurons through 5-HT6 receptor antagonism in the dorsal raphe nucleus (DRN). In organotypic raphe slice cultures, treatment with olanzapine (1-100 μM) enhanced the increase in extracellular 5-HT levels in the presence of fluoxetine (10 μM) or citalopram (1 μM). The enhancing effect of olanzapine was not further augmented by the GABAA receptor antagonist bicuculline. Electrophysiological analysis revealed that olanzapine (50 μM) decreased the firing frequency of GABAergic neurons in acute DRN slices. Among many serotonergic agents, the 5-HT6 receptor antagonist SB399885 (1-100 μM) mimicked the effects of olanzapine by enhancing the SSRI-induced increase in extracellular 5-HT levels, which was not further augmented by bicuculline or olanzapine. SB399885 (50 μM) also decreased the firing frequency of GABAergic neurons in the DRN. In addition, an intraperitoneal administration of SB399885 (10 mg/kg) to mice significantly enhanced the antidepressant-like effect of a subeffective dose of citalopram (3 mg/kg) in the tail-suspension test. These results suggest that olanzapine decreases local inhibitory GABAergic tone in the DRN through antagonism of 5-HT6 receptors, thereby increasing the activity of at least part of serotonergic neurons, which may contribute to the augmentation of the efficacy of SSRIs.

Chronic effects of antidepressants on serotonin release in rat raphe slice cultures: high potency of milnacipran in the augmentation of serotonin release.

Most clinically-used antidepressants acutely increase monoamine levels in synaptic clefts, while their therapeutic effects often require several weeks of administration. Slow neuroadaptive changes in serotonergic neurons are considered to underlie this delayed onset of beneficial actions. Recently, we reported that sustained exposure of rat organotypic raphe slice cultures containing abundant serotonergic neurons to selective serotonin (5-HT) reuptake inhibitors (citalopram, fluoxetine and paroxetine) caused the augmentation of exocytotic serotonin release. However, the ability of other classes of antidepressants to evoke a similar outcome has not been clarified. In this study, we investigated the sustained actions of two tricyclic antidepressants (imipramine and desipramine), one tetracyclic antidepressant (mianserin), three 5-HT and noradrenaline reuptake inhibitors (milnacipran, duloxetine and venlafaxine) and one noradrenergic and specific serotonergic antidepressant (mirtazapine) on serotonin release in the slice cultures. For seven of nine antidepressants, sustained exposure to the agents at concentrations of 0.1-100 μ m augmented the level of increase in extracellular serotonin. The rank order of their potency was as follows: milnacipran>duloxetine>citalopram>venlafaxine>imipramine>fluoxetine>desipramine. Neither mirtazapine nor mianserin caused any augmentation. The highest augmentation by sustained exposure to milnacipran was partially attenuated by an α 1-adrenoceptor antagonist, benoxathian, while the duloxetine-, venlafaxine- and citalopram-mediated increases were not affected. These results suggest that inhibition of the 5-HT transporter is required for the enhancement of serotonin release. Furthermore, the potent augmentation by milnacipran is apparently due to the accompanied activation of the α 1-adrenoceptor.

Chronic antidepressant potentiates spontaneous activity of dorsal raphe serotonergic neurons by decreasing GABA B receptor-mediated inhibition of L-type calcium channels

Spontaneous activity of serotonergic neurons of the dorsal raphe nucleus (DRN) regulates mood and motivational state. Potentiation of serotonergic function is one of the therapeutic strategies for treatment of various psychiatric disorders, such as major depression, panic disorder and obsessive-compulsive disorder. However, the control mechanisms of the serotonergic firing activity are still unknown. In this study, we examined the control mechanisms for serotonergic spontaneous activity and effects of chronic antidepressant administration on these mechanisms by using modified ex vivo electrophysiological recording methods. Serotonergic neurons remained firing even in the absence of glutamatergic and GABAergic ionotropic inputs, while blockade of L-type voltage dependent Ca2+ channels (VDCCs) in serotonergic neurons decreased spontaneous firing activity. L-type VDCCs in serotonergic neurons received gamma-aminobutyric acid B (GABAB) receptor-mediated inhibition, which maintained serotonergic slow spontaneous firing activity. Chronic administration of an antidepressant, citalopram, disinhibited the serotonergic spontaneous firing activity by weakening the GABAB receptor-mediated inhibition of L-type VDCCs in serotonergic neurons. Our results provide a new mechanism underlying the spontaneous serotonergic activity and new insights into the mechanism of action of antidepressants.

Inhibition of histone deacetylases enhances the function of serotoninergic neurons in organotypic raphe slice cultures.

Inhibition of histone deacetylases (HDACs) is a promising approach for the treatment of mood disorders. However, the effects of HDAC inhibition on the serotonin (5-HT) system, a common target for psychiatric disorders, are poorly understood. Here, we show that a broad-spectrum HDAC inhibitor, trichostatin A (TSA), enhances the function of 5-HT neurons in organotypic raphe slice cultures. Sustained treatment with TSA (1μM) for 2 or 4 days significantly increased the 5-HT tissue content and tryptophan hydroxylase 2 (TPH2) expression, which were accompanied by hyper-acetylation of histone H3 in the promoter region of the TPH2 gene. TSA treatment for 4 days increased the extracellular 5-HT level, which was significantly suppressed in the presence of the selective AMPA receptor (AMPAR) antagonist NBQX. Moreover, the expression of both the AMPAR subunit GluA2 and Ca(2+)/calmodulin-dependent kinase II α (CaMKIIα) mRNAs were significantly increased by TSA treatment. Co-treatment with the CaMKII inhibitors KN-62 and KN-93 prevented the TSA-induced increase in 5-HT release, but had no effect on the increases in 5-HT tissue content. These results suggest that inhibition of HDACs increases 5-HT synthesis and release by epigenetic mechanisms, and that 5-HT release is mediated by the enhancement of AMPAR-mediated excitatory inputs and CaMKII signaling.

TRPM2 confers susceptibility to social stress but is essential for behavioral flexibility

Transient receptor potential melastatin 2 (TRPM2) is a Ca2+-permeable, nonselective cation channel and a member of the TRP channel superfamily that acts as a sensor of intracellular redox states. TRPM2 is widely distributed in many tissues and highly expressed in the brain, but the physiological roles of TRPM2 in the central nervous system remain unclear. In this study, TRPM2-deficient mice were examined in a series of behavioral tests. TRPM2-deficient mice did not significantly differ from wild-type littermates in muscle strength, light/dark transition test, rotarod, elevated plus maze, social interaction, prepulse inhibition, Y-maze, forced swim test, cued and contextual fear conditioning, and tail suspension test. In the Barnes circular maze, TRPM2-deficient mice learned the fixed escape box position at similar extent to wild-type littermates, suggesting normal reference memory. However, performance of the first reversal trial and probe test were significantly impaired in TRPM2-deficient mice. In the T-maze delayed alternation task, TRPM2 deficiency significantly reduced choice accuracy. These results indicate that TRPM2-deficient mice shows behavioral inflexibility. Meanwhile, social avoidance induced by repeated social defeat stress was significantly attenuated in TRPM2-deficient mice, suggesting that TRPM2 deficiency confers stress resiliency. Our findings indicate that TRPM2 plays an essential role in maintaining behavioral flexibility but it increases susceptibility to stress.

Ketamine-Induced Prefrontal Serotonin Release Is Mediated by Cholinergic Neurons in the Pedunculopontine Tegmental Nucleus

Abstract Background Ketamine rapidly elicits antidepressive effects in humans and mice in which serotonergic activity is involved. Although α4β2 nicotinic acetylcholine receptor (α4β2 nAChR) in the dorsal raphe nucleus plays a key role in the ketamine-induced prefrontal serotonin release, the source of cholinergic afferents, and its role is unclear. Methods Prefrontal serotonin levels after ketamine injection were measured by microdialysis in rats. Electrolytic lesion of pedunculopontine tegmental nucleus and laterodorsal tegmental nucleus was made with constant direct current. Results Bilateral lesion of the pedunculopontine tegmental nucleus, but not laterodorsal tegmental nucleus, attenuated prefrontal serotonin release induced by systemic ketamine. Intra-pedunculopontine tegmental nucleus, but not intra-laterodorsal tegmental nucleus ketamine perfusion, increased prefrontal serotonin release. This increase was attenuated by intra-dorsal raphe nucleus injection of dihydro-β-erythroidine, an α4β2 nAChR antagonist, or NBQX, an AMPA receptor antagonist. Conclusions These results suggest the ketamine-induced serotonin release in medial prefrontal cortex is mediated by cholinergic neurons projecting from pedunculopontine tegmental nucleus to dorsal raphe nucleus via α4β2 nAChRs.

Activation of GABAergic Neurons in the Nucleus Accumbens Mediates the Expression of Cocaine-Associated Memory

Cocaine-associated environmental cues elicit craving and relapse to cocaine use by recalling the rewarding memory of cocaine. However, the neuronal mechanisms underlying the expression of cocaine-associated memory are not fully understood. Here, we investigated the possible contribution of γ-aminobutyrate (GABA)ergic neurons in the nucleus accumbens (NAc), a key brain region associated with the rewarding and reinforcing effects of cocaine, to the expression of cocaine-associated memory using the conditioned place preference (CPP) paradigm combined with designer receptors exclusively activated by designer drugs (DREADD) technology. The inhibitory DREADD hM4Di was selectively expressed in NAc GABAergic neurons of vesicular GABA transporter-Cre (vGAT-Cre) mice by infusing adeno-associated virus (AAV) vectors. Ex vivo electrophysiological recordings revealed that bath application of clozapine-N-oxide (CNO) significantly hyperpolarized membrane potentials and reduced the number of spikes induced by depolarizing current injections in hM4Di-positive NAc neurons. Additionally, systemic CNO injections into cocaine-conditioned mice 30 min before posttest session significantly reduced CPP scores compared to saline-injected mice. These results indicate that chemogenetic inhibition of NAc GABAergic neurons attenuated the expression of cocaine CPP, suggesting that NAc GABAergic neuronal activation is required for the environmental context-induced expression of cocaine-associated memory.