Search

Major depressive disorder (MDD) is among the most common mental illnesses. Serotonergic (5-HT) neurons are central to the pathophysiology and treatment of MDD. Repeatedly recalling positive episodes is effective for MDD. Stimulating 5-HT neurons of the dorsal raphe nucleus (DRN) or neuronal ensembles in the dorsal dentate gyrus (dDG) associated with positive memories reverses the stress-induced behavioral abnormalities. Despite this phenotypic similarity, their causal relationship is unclear. This study revealed that the DRN 5-HT neurons activate dDG neurons; surprisingly, this activation was specifically observed in positive memory ensembles rather than neutral or negative ensembles. Furthermore, we revealed that dopaminergic signaling induced by activation of DRN 5-HT neurons projecting to the ventral tegmental area mediates an increase in active coping behavior and positive dDG ensemble reactivation. Our study identifies a role of DRN 5-HT neurons as specific reactivators of positive memories and provides insights into how serotonin elicits antidepressive effects., セロトニン神経による海馬神経アンサンブル制御 --快経験で活性化する神経アンサンブルの選択的再活性化--. 京都大学プレスリリース. 2023-02-28.

Previous findings have proposed that drugs targeting 5-HT

Serotonin (5-HT) in the central nervous system regulates a variety of biological functions, from the basic homeostatic control to higher brain functions, by acting on fourteen known receptor subtypes. However, it is still usually unclear which receptor subtype is responsible for a specific function due to the lack of highly selective ligands for most of these receptors. Although 5-HT receptor knockout mice are useful, the brain-wide distribution of various receptors makes it difficult to dissect receptor functions in specific and brain regions and cell types. Recent advances in CRISPR/Cas9-mediated in vivo genome editing technology may overcome this problem. In this study, we constructed a viral vector expressing a single guide (sg)RNA targeting Htr1a (sgHtr1a) and Cre recombinase under the control of a neuron-specific promoter. Injection of the viral vector into the dorsal raphe nucleus (DRN) of Cre-dependent Cas9 knock-in mice induced Cre-dependent Cas9 expression mainly in DRN serotonin and GABA neurons. Mismatch cleavage assay and Sanger sequencing showed insertion or deletion formation at the target site. 5-HT1A receptor agonist-induced hypothermia was attenuated and antidepressant effect of a selective serotonin reuptake inhibitor (SSRI) was enhanced by microinjection of the viral vector expressing sgHtr1a into the DRN of Cre-dependent Cas9 knock-in mice. These results suggest that this in vivo CRISPR/Cas9-mediated 5-HT receptor gene knockout strategy provides a reliable and low-cost method for elucidating 5-HT receptor functions in specific cell types and brain regions. Further, we demonstrate that the neuronal 5-HT1A receptor in the DRN regulates body temperature and antidepressant effect of SSRI.

Stress enhances cocaine craving. We recently reported that acute restraint stress increases cocaine conditioned place preference (CPP) in mice; however, the underlying mechanisms remain unclear. This study aimed to examine the role of serotonergic transmission in the medial prefrontal cortex (mPFC) in cocaine CPP enhancement by acute restraint stress, which increases extracellular serotonin (5-HT) levels in the mPFC. Intra-mPFC infusion of the selective serotonin reuptake inhibitor (S)-citalopram prior to the test session significantly increased the cocaine CPP score under non-stressed conditions. This is indicative of the substantial role of increased mPFC 5-HT levels in cocaine CPP enhancement. Moreover, intra-mPFC and systemic administration of the 5-HT

Background: Reward processing is fundamental for animals to survive and reproduce. Many studies have shown the importance of dorsal raphe nucleus (DRN) serotonin (5-HT) neurons in this process, but the strongly correlative link between the activity of DRN 5-HT neurons and rewarding/aversive potency is under debate. Our primary objective was to reveal this link using two different strategies to transduce DRN 5-HT neurons. Methods: For transduction of 5-HT neurons in wildtype mice, adeno-associated virus (AAV) bearing the mouse tryptophan hydroxylase 2 (TPH2) gene promoter was used. For transduction in Tph2-tTA transgenic mice, AAVs bearing the tTA-dependent TetO enhancer were used. To manipulate the activity of 5-HT neurons, optogenetic actuators (CheRiff, eArchT) were expressed by AAVs. For measurement of rewarding/aversive potency, we performed a nose-poke self-stimulation test and conditioned place preference (CPP) test. Results: We found that stimulation of DRN 5-HT neurons and their projections to the ventral tegmental area (VTA) increased the number of nose-pokes in self-stimulation test and CPP scores in both targeting methods. Concomitantly, CPP scores were decreased by inhibition of DRN 5-HT neurons and their projections to VTA. Conclusion: Our findings indicate that the activity of DRN 5-HT neurons projecting to the VTA is a key modulator of balance between reward and aversion.

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.

Adapting to changing environmental conditions requires a prospective inference of future actions and their consequences, a strategy also known as model-based decision making.1-3 In stable environments, extensive experience of actions and their consequences leads to a shift from a model-based to a model-free strategy, whereby behavioral selection is primarily governed by retrospective experiences of positive and negative outcomes. Human and animal studies, where subjects are required to speculate about implicit information and adjust behavioral responses over multiple sessions, point to a role for the central serotonergic system in model-based decision making.4-8 However, to directly test a causal relationship between serotonergic activity and model-based decision making, phase-specific manipulation of serotonergic activity is needed in a one-shot test, where learning by trial and error is neutralized. Moreover, the serotonergic origin responsible for this effect is yet to be determined. Herein, we demonstrate that optogenetic silencing of serotonin neurons in the dorsal raphe nucleus, but not in the median raphe nucleus, disrupts model-based decision making in lithium-induced outcome devaluation tasks.9-11 Our data indicate that the serotonergic behavioral effects are not due to increased locomotor activity, anxiolytic effects, or working memory deficits. Our findings provide insights into the neural mechanisms underlying neural weighting between model-free and model-based strategies.

Serotonin (5-HT) in the central nervous system regulates a variety of biological functions, from the basic homeostatic control to higher brain functions, by acting on fourteen known receptor subtypes. However, it is still usually unclear which receptor subtype is responsible for a specific function due to the lack of highly selective ligands for most of these receptors. Although 5-HT receptor knockout mice are useful, the brain-wide distribution of various receptors makes it difficult to dissect receptor functions in specific and brain regions and cell types. Recent advances in CRISPR/Cas9-mediated in vivo genome editing technology may overcome this problem. In this study, we constructed a viral vector expressing a single guide (sg)RNA targeting Htr1a (sgHtr1a) and Cre recombinase under the control of a neuron-specific promoter. Injection of the viral vector into the dorsal raphe nucleus (DRN) of Cre-dependent Cas9 knock-in mice induced Cre-dependent Cas9 expression mainly in DRN serotonin and GABA neurons. Mismatch cleavage assay and Sanger sequencing showed insertion or deletion formation at the target site. 5-HT

Stress enhances cocaine craving. We recently reported that acute restraint stress increases cocaine conditioned place preference (CPP) in mice; however, the underlying mechanisms remain unclear. This study aimed to examine the role of serotonergic transmission in the medial prefrontal cortex (mPFC) in cocaine CPP enhancement by acute restraint stress, which increases extracellular serotonin (5-HT) levels in the mPFC. Intra-mPFC infusion of the selective serotonin reuptake inhibitor (S)-citalopram prior to the test session significantly increased the cocaine CPP score under non-stressed conditions. This is indicative of the substantial role of increased mPFC 5-HT levels in cocaine CPP enhancement. Moreover, intra-mPFC and systemic administration of the 5-HT1A receptor antagonist WAY100635 immediately before restraint stress exposure significantly attenuated stress-induced cocaine CPP enhancement. Our findings suggest that enhanced serotonergic transmission via 5-HT1A receptors in the mPFC is involved in acute stress-induced augmentation of rewarding memory of cocaine; moreover, the 5-HT1A receptor could be a therapeutic target for stress-induced cocaine craving.

Serotonergic agents have been widely used for treatment of psychiatric disorders, but the therapeutic effects are insufficient and these drugs often induce severe side effects. We need to specify the distinct serotonergic pathways underlying each mental function to overcome these problems. Preclinical studies have demonstrated that the central serotonergic system is involved in several emotional/cognitive functions including anxiety, depression, and impulse control, but it remains unclear whether each function is regulated by a different serotonergic system. We used optogenetic strategy to increase central serotonergic activity in mice and evaluated the behavioral consequences. Pharmacological and genetic tools were used to determine the subtype of 5-HT receptors responsible for the observed effects. We demonstrated that the serotonergic activation in the median raphe nucleus enhanced anxiety-like behavior, the serotonergic activation in the dorsal raphe nucleus exerted antidepressant-like effects, and the serotonergic activation in the median or dorsal raphe nucleus suppressed impulsive action. We also found that different serotonergic terminals, ventral hippocampus, ventral tegmental area/substantia nigra, and subthalamic/parasubthalamic nucleus, are involved in regulating anxiety-like behavior, antidepressant-like, and anti-impulsive effects, respectively. Furthermore, we found, using triple-transgenic mice, that the stimulation of the 5-HT2C receptor is required to evoke anxiety-like behavior, but not to exert anti-impulsive effects. These results suggest the need for pathway-specific treatments and provide important insights that will help the development of more effective and safer therapeutics. This article is part of the special issue entitled 'Serotonin Research: Crossing Scales and Boundaries'.

Pharmacological studies have suggested that the serotonin 5-HT2C receptor is involved in locomotor activity, anxiety, and fear memory. However, the results of locomotor activity and anxiety in 5-HT2C receptor knockout mice have been mixed, and the effects of 5-HT2C receptor knockout on contextual fear memory have not yet been addressed. In the present study, we reconcile these inconsistent results by analyzing behavioral data in detail and by examining the effects of 5-HT2C receptor knockout on contextual fear memory. We demonstrated that the higher locomotor activity in 5-HT2C receptor knockout mice was observed only in the late phase of the test, indicating that the analyses in the previous study using the total locomotor activity would lead to variable results. Moreover, by analyzing mouse behavior in detail, we found that 5-HT2C receptor knockout mice displayed a hesitating attitude by staying in the central area as well as risk assessment behavior in the elevated plus-maze test. However, the time spent in the open arms was longer in 5-HT2C receptor knockout mice than in wild-type littermates when a zero-maze test lacking the central area was used. In the contextual fear conditioning test, 5-HT2C receptor knockout mice showed rapid within-session extinction of fear, but not between-session extinction, compared with wild-type littermates. However, this remains inconclusive because the facilitation of extinction might be confounded with higher locomotor activity in 5-HT2C receptor knockout mice. Taken together, the present results provide reasonable explanations about previous inconsistent findings and partially filled the gaps between pharmacological and genetic findings.

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.

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.

BACKGROUND:Chronic 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. RESULTS:We 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. CONCLUSIONS:We 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.

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 µm 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 α1-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 α1-adrenoceptor.