Differential Impact of Serotonin Signaling Methylphenidate on Young versus Adult: Insights from Behavioral and Dorsal Raphe Nucleus Neuronal Recordings from Freely Behaving Rats.

Methylphenidate (MPD) remains a cornerstone pharmacological intervention for managing ADHD, yet its increasing usage among ordinary youth and adults outside clinical contexts necessitates a thorough investigation into its developmental effects. This study seeks to simultaneously investigate the behavioral and neuronal changes within the dorsal raphe (DR) nucleus, a center of serotonergic neurons in the mammalian brain, before and after the administration of varying doses of acute and chronic MPD in freely behaving young and adult rats implanted with DR recording electrodes. Wireless neuronal and behavioral recording systems were used over 10 consecutive experimental days. Eight groups were examined: saline, 0.6, 2.5, and 10.0 mg/kg MPD for both young and adult rats. Six daily MPD injections were administered on experimental days 1 to 6, followed by a three-day washout period and MPD re-administration on experimental day 10 (ED10). The analysis of neuronal activity recorded from 504 DR neurons (DRNs) in young rats and 356 DRNs in adult rats reveals significant age-dependent differences in acute and chronic MPD responses. This study emphasizes the importance of aligning electrophysiological evaluations with behavioral outcomes following extended MPD exposure, elucidating the critical role of DRNs and serotonin signaling in modulating MPD responses and delineating age-specific variations in young versus adult rat models.

Behavioural changes in young ovariectomized mice via GPR30-dependent serotonergic nervous system.

Fluctuations in estradiol levels at each stage of life in women are considered one of the causes of mental diseases through their effects on the central nervous system. During menopause, a decrease in estradiol levels has been reported to affect the serotonin nervous system and induce depression-like and anxiety symptoms. However, the regulation of brain and behaviour during childhood and adolescence is poorly understood. Moreover, the role of oestrogen receptors α and ß in the regulation of the serotonergic nervous system has been reported, but little is known about the involvement of G protein-coupled receptor 30. Therefore, in this study, we used an ovariectomized childhood mouse model to analyse behaviour and investigate the effects on the serotonin nervous system. We showed that ovariectomy surgery at 4 weeks of age, which is the weaning period, induced a decrease in spontaneous locomotor activity during the active period and a preference for novel mice over familiar mice in the three-chamber social test at 10 weeks of age. In addition, the administration of G-1, a protein-coupled receptor 30 agonist, to ovariectomized mice suppressed spontaneous locomotor activity and the preference for novel mice. Furthermore, we demonstrated that childhood ovariectomy induces increased tryptophan hydroxylase gene expression in the raphe nucleus and increased serotonin release in the amygdaloid nucleus, and administration of G-1 ameliorated these effects. Our study suggests that G protein-coupled receptor 30-mediated regulation of serotonin synthesis is involved in changes in activity and social-cognitive behaviour due to decreased estradiol levels during childhood.

The dorsal raphe-to-ventral hippocampal projection modulates reactive aggression through 5-HT1B receptors.

Maladaptive reactive aggression is a core symptom of neuropsychiatric disorders such as schizophrenia. While uncontrolled aggression dampens societal safety, there is a limited understanding of the neural regulation involved in reactive aggression and its treatment. High levels of aggression have been linked to low serotonin (5-HT) levels. Additionally, post-weaning socially isolated (SI) mice exhibit outbursts of aggression following encountering acute stress, and hyperactivated ventral hippocampus (vHip) involves this stress-provoked escalated aggression. Here, we investigated the potential role of the raphe nucleus projecting to the vHip in modulating aggressive behavior. Chemogenetically activating the dorsal raphe nucleus (DRN) soma projecting the vHip or DRN nerve terminals in the vHip reduced reactive aggression. The reduction of attack behavior was abolished by the pretreatment of 5-HT1B receptor antagonist SB-224289. However, activating the median raphe nucleus (MRN)-to-vHip pathway ameliorated depression-like behavior but did not affect reactive aggression. DRN→vHip activation suppressed the vHip downstream area, the ventromedial hypothalamus (VMH), which is a core aggression area. Intra-vHip infusion of 5-HT1B receptor agonists (anpirtoline, CP-93129) suppressed reactive aggression and decreased c-Fos levels in the vHip neurons projecting to the VMH, suggesting an inhibition mechanism. Our findings indicate that activating the DRN projecting to the vHip is sufficient to inhibit reactive aggression in a 5-HT1B receptor-dependent manner. Thus, targeting 5-HT1B receptor could serve as a promising therapeutic approach to ameliorate symptoms of reactive aggression.

Revealing a role of brainstem monoaminergic nuclei on the pronociceptive effect of sleep restriction.

Sleep disturbances and persistent pain conditions are public health challenges worldwide. Although it is well-known that sleep deficit increases pain sensitivity, the underlying mechanisms remain elusive. We have recently demonstrated the involvement of nucleus accumbens (NAc) and anterior cingulate cortex (ACC) in the pronociceptive effect of sleep restriction. In this study, we found that sleep restriction increases c-Fos expression in NAc and ACC, suggesting hyperactivation of these regions during prolonged wakefulness in male Wistar rats. Blocking adenosine A2A receptors in the NAc or GABAA receptors in the ventral tegmental area (VTA), dorsal raphe nucleus (DRN), or locus coeruleus (LC) effectively mitigated the pronociceptive effect of sleep restriction. In contrast, the blockade of GABAA receptors in each of these nuclei only transiently reduced carrageenan-induced hyperalgesia. Pharmacological activation of dopamine D2, serotonin 5-HT1A and noradrenaline alpha-2 receptors within the ACC also prevented the pronociceptive effect of sleep restriction. While pharmacological inhibition of these same monoaminergic receptors in the ACC restored the pronociceptive effect which had been prevented by the GABAergic disinhibition of the of the VTA, DRN or LC. Overall, these findings suggest that the pronociceptive effect of sleep restriction relies on increased adenosinergic activity on NAc, heightened GABAergic activity in VTA, DRN, and LC, and reduced inhibitory monoaminergic activity on ACC. These findings advance our understanding of the interplay between sleep and pain, shedding light on potential NAc-brainstem-ACC mechanisms that could mediate increased pain sensitivity under conditions of sleep impairment.

Differential Roles of Key Brain Regions: Ventral Tegmental Area, Locus Coeruleus, Dorsal Raphe, Nucleus Accumbens, Caudate Nucleus, and Prefrontal Cortex in Regulating Response to Methylphenidate: Insights from Neuronal and Behavioral Studies in Freely Behaving Rats.

A total of 3102 neurons were recorded before and following acute and chronic methylphenidate (MPD) administration. Acute MPD exposure elicits mainly increases in neuronal and behavioral activity in dose-response characteristics. The response to chronic MPD exposure, as compared to acute 0.6, 2.5, or 10.0 mg/kg MPD administration, elicits electrophysiological and behavioral sensitization in some animals and electrophysiological and behavioral tolerance in others when the neuronal recording evaluations were performed based on the animals' behavioral responses, or amount of locomotor activity, to chronic MPD exposure. The majority of neurons recorded from those expressing behavioral sensitization responded to chronic MPD with further increases in firing rate as compared to the initial MPD responses. The majority of neurons recorded from animals expressing behavioral tolerance responded to chronic MPD with decreases in their firing rate as compared to the initial MPD exposures. Each of the six brain areas studied-the ventral tegmental area, locus coeruleus, dorsal raphe, nucleus accumbens, prefrontal cortex, and caudate nucleus (VTA, LC, DR, NAc, PFC, and CN)-responds significantly (p < 0.001) differently to MPD, suggesting that each one of the above brain areas exhibits different roles in the response to MPD. Moreover, this study demonstrates that it is essential to evaluate neuronal activity responses to psychostimulants based on the animals' behavioral responses to acute and chronic effects of the drug from several brain areas simultaneously to obtain accurate information on each area's role in response to the drug.

Dorsal raphe to basolateral amygdala corticotropin-releasing factor circuit regulates cocaine-memory reconsolidation.

Environmental stimuli elicit drug craving and relapse in cocaine users by triggering the retrieval of strong cocaine-related contextual memories. Retrieval can also destabilize drug memories, requiring reconsolidation, a protein synthesis-dependent storage process, to maintain memory strength. Corticotropin-releasing factor (CRF) signaling in the basolateral amygdala (BLA) is necessary for cocaine-memory reconsolidation. We have hypothesized that a critical source of CRF in the BLA is the dorsal raphe nucleus (DR) based on its neurochemistry, anatomical connectivity, and requisite involvement in cocaine-memory reconsolidation. To test this hypothesis, male and female Sprague-Dawley rats received adeno-associated viruses to express Gi-coupled designer receptors exclusively activated by designer drugs (DREADDs) selectively in CRF neurons of the DR and injection cannulae directed at the BLA. The rats were trained to self-administer cocaine in a distinct environmental context then received extinction training in a different context. Next, they were briefly re-exposed to the cocaine-predictive context to destabilize (reactivate) cocaine memories. Intra-BLA infusions of the DREADD agonist deschloroclozapine (DCZ; 0.1 mM, 0.5 µL/hemisphere) immediately after memory reactivation attenuated cocaine-memory strength, relative to vehicle infusion. This was indicated by a selective, DCZ-induced and memory reactivation-dependent decrease in drug-seeking behavior in the cocaine-predictive context in DREADD-expressing males and females at test compared to respective controls. Notably, BLA-projecting DR CRF neurons that exhibited increased c-Fos expression during memory reconsolidation co-expressed the glutamatergic neuronal marker, vesicular glutamate transporter 3. Together, these findings suggest that the DRCRF → BLA circuit is engaged to maintain cocaine-memory strength after memory destabilization, and this phenomenon may be mediated by DR CRF and/or glutamate release in the BLA.

Raphe glucose-sensing serotonergic neurons stimulate KNDy neurons to enhance LH pulses via 5HT2CR: rat and goat studies.

Dysfunction of central serotonergic neurons is known to cause depressive disorders in humans, who often show reproductive and/or glucose metabolism disorders. This study examined whether dorsal raphe (DR) serotonergic neurons sense high glucose availability to upregulate reproductive function via activating hypothalamic arcuate (ARC) kisspeptin neurons (= KNDy neurons), a dominant stimulator of gonadotropin-releasing hormone (GnRH)/gonadotropin pulses, using female rats and goats. RNA-seq and histological analysis revealed that stimulatory serotonin-2C receptor (5HT2CR) was mainly expressed in the KNDy neurons in female rats. The serotonergic reuptake inhibitor administration into the mediobasal hypothalamus (MBH), including the ARC, significantly blocked glucoprivic suppression of luteinizing hormone (LH) pulses and hyperglycemia induced by intravenous 2-deoxy-D-glucose (2DG) administration in female rats. A local infusion of glucose into the DR significantly increased in vivo serotonin release in the MBH and partly restored LH pulses and hyperglycemia in the 2DG-treated female rats. Furthermore, central administration of serotonin or a 5HT2CR agonist immediately evoked GnRH pulse generator activity, and central 5HT2CR antagonism blocked the serotonin-induced facilitation of GnRH pulse generator activity in ovariectomized goats. These results suggest that DR serotonergic neurons sense high glucose availability to reduce gluconeogenesis and upregulate reproductive function by activating GnRH/LH pulse generator activity in mammals.

Computational modeling of light processing in the habenula and dorsal raphe based on laser ablation of functionally-defined cells.

BACKGROUND: The habenula is a major regulator of serotonergic neurons in the dorsal raphe, and thus of brain state. The functional connectivity between these regions is incompletely characterized. Here, we use the ability of changes in irradiance to trigger reproducible changes in activity in the habenula and dorsal raphe of zebrafish larvae, combined with two-photon laser ablation of specific neurons, to establish causal relationships. RESULTS: Neurons in the habenula can show an excitatory response to the onset or offset of light, while neurons in the anterior dorsal raphe display an inhibitory response to light, as assessed by calcium imaging. The raphe response changed in a complex way following ablations in the dorsal habenula (dHb) and ventral habenula (vHb). After ablation of the ON cells in the vHb (V-ON), the raphe displayed no response to light. After ablation of the OFF cells in the vHb (V-OFF), the raphe displayed an excitatory response to darkness. After ablation of the ON cells in the dHb (D-ON), the raphe displayed an excitatory response to light. We sought to develop in silico models that could recapitulate the response of raphe neurons as a function of the ON and OFF cells of the habenula. Early attempts at mechanistic modeling using ordinary differential equation (ODE) failed to capture observed raphe responses accurately. However, a simple two-layer fully connected neural network (NN) model was successful at recapitulating the diversity of observed phenotypes with root-mean-squared error values ranging from 0.012 to 0.043. The NN model also estimated the raphe response to ablation of D-off cells, which can be verified via future experiments. CONCLUSION: Lesioning specific cells in different regions of habenula led to qualitatively different responses to light in the dorsal raphe. A simple neural network is capable of mimicking experimental observations. This work illustrates the ability of computational modeling to integrate complex observations into a simple compact formalism for generating testable hypotheses, and for guiding the design of biological experiments.

Transient Adenosine Modulates Serotonin Release Indirectly in the Dorsal Raphe Nuclei.

Rapid adenosine transiently regulates dopamine and glutamate via A1 receptors, but other neurotransmitters, such as serotonin, have not been studied. In this study, we examined the rapid modulatory effect of adenosine on serotonin release in the dorsal raphe nuclei (DRN) of mouse brain slices by using fast-scan cyclic voltammetry. To mimic adenosine release during damage, a rapid microinjection of adenosine at 50 pmol was applied before electrical stimulation of serotonin release. Transient adenosine significantly reduced electrically evoked serotonin release in the first 20 s after application, but serotonin release recovered to baseline as adenosine was cleared from the slice. The continuous perfusion of adenosine did not change the evoked serotonin release. Surprisingly, the modulatory effects of adenosine were not regulated by A1 receptors as adenosine still inhibited serotonin release in A1KO mice and also after perfusion of an A1 antagonist (8-cyclopentyl-1,3-dipropyl xanthine). The inhibition was also not regulated by A3 receptors as perfusion of the A3 antagonist (MRS 1220) in A1KO brain slices did not eliminate the inhibitory effects of transient adenosine. In addition, adenosine also inhibited serotonin release in A2AKO mice, showing that A2A did not modulate serotonin. However, perfusion of a selective 5HT1A autoreceptor antagonist drug [(S)-WAY 100135 dihydrochloride] abolished the inhibitory effect of transient adenosine on serotonin release. Thus, the transient neuromodulatory effect of adenosine on DRN serotonin release is regulated by serotonin autoreceptors and not by adenosine receptors. Rapid, transient adenosine modulation of neurotransmitters such as serotonin may have important implications for diseases such as depression and brain injury.

Estrogen signaling in the dorsal raphe regulates binge-like drinking in mice.

Estrogens promote binge alcohol drinking and contribute to sex differences in alcohol use disorder. However, the mechanisms are largely unknown. This study aims to test if estrogens act on 5-hydroxytryptamine neurons in the dorsal raphe nucleus (5-HTDRN) to promote binge drinking. We found that female mice drank more alcohol than male mice in chronic drinking in the dark (DID) tests. This sex difference was associated with distinct alterations in mRNA expression of estrogen receptor α (ERα) and 5-HT-related genes in the DRN, suggesting a potential role of estrogen/ERs/5-HT signaling. In supporting this view, 5-HTDRN neurons from naïve male mice had lower baseline firing activity but higher sensitivity to alcohol-induced excitation compared to 5-HTDRN neurons from naïve female mice. Notably, this higher sensitivity was blunted by 17ß-estradiol treatment in males, indicating an estrogen-dependent mechanism. We further showed that both ERα and ERß are expressed in 5-HTDRN neurons, whereas ERα agonist depolarizes and ERß agonist hyperpolarizes 5-HTDRN neurons. Notably, both treatments blocked the stimulatory effects of alcohol on 5-HTDRN neurons in males, even though they have antagonistic effects on the activity dynamics. These results suggest that ERs' inhibitory effects on ethanol-induced burst firing of 5-HTDRN neurons may contribute to higher levels of binge drinking in females. Consistently, chemogenetic activation of ERα- or ERß-expressing neurons in the DRN reduced binge alcohol drinking. These results support a model in which estrogens act on ERα/ß to prevent alcohol-induced activation of 5-HTDRN neurons, which in return leads to higher binge alcohol drinking.

Effects of pre-gestational exposure to the stressors and perinatal bupropion administration on the firing activity of serotonergic neurons and anxiety-like behavior in rats.

Exposure by women to stressors before pregnancy increases their risk of contracting prenatal depression, a condition which typically may require antidepressant treatment. And even though such perinatal antidepressant treatment is generally considered to be safe. For the mother, its effects on the development and functioning of the offspring`s brain remain unknown. In this study, we aimed to investigate the effects of pregestational chronic unpredictable stress (CUS) and perinatal bupropion on the anxiety behavior and firing activity of the dorsal raphe nucleus (DRN) serotonin (5-HT) neurons. Female rats underwent CUS for three weeks before mating. Bupropion was administered to them from gestation day ten until their offspring were weaned. Behavioral (elevated plus maze or EPM test) and neurophysiological (single-unit in vivo electrophysiology) assessments were performed on offspring who reached the age of 48-56 days. We found that maternal CUS and perinatal bupropion, as separate factors on their own, did not change offspring behavior. There was, however, an interaction between their effects on the number of entries to the open arms and time spent in the intersection: maternal CUS tended to decrease these values, and perinatal bupropion tended to diminish CUS effect. Maternal CUS increased the firing activity of 5-HT neurons in males, but not females. Perinatal bupropion did not alter the firing activity of 5-HT neurons but tended to potentiate the maternal CUS-induced increase in 5-HT neuronal firing activity. The CUS-induced increase in firing activity of 5-HT neurons might be a compensatory mechanism that diminishes the negative effects of maternal stress. Perinatal bupropion does not alter the offspring`s anxiety and firing activity of 5-HT, but it does intervene in the effects of maternal stress.

Dopamine, Norepinephrine and Serotonin Participate Differently in Methylphenidate Action in Concomitant Behavioral and Ventral Tegmental Area, Locus Coeruleus and Dorsal Raphe Neuronal Study in Young Rats.

Methylphenidate (MPD), known as Ritalin, is a psychostimulant used to treat children, adults, and the elderly. MPD exerts its effects through increasing concentrations of dopamine (DA), norepinephrine (NE), and serotonin (5-HT) in the synaptic cleft. Concomitant behavioral and neuronal recording from the ventral tegmental area (VTA), locus coeruleus (LC), and from the dorsal raphe (DR) nucleus, which are the sources of DA, NE, and 5-HT to the mesocorticolimbic circuit, were investigated following acute and repetitive (chronic) saline, 0.6, 2.5, or 10.0 mg/kg MPD. Animals received daily saline or MPD administration on experimental days 1 to 6 (ED1-6), followed by a 3-day washout period and MPD rechallenge on ED10. Each chronic MPD dose elicits behavioral sensitization in some animals while inducing behavioral tolerance in others. The uniqueness of this study is in the evaluation of neuronal activity based on the behavioral response to chronic MPD. Neuronal excitation was observed mainly in brain areas of animals exhibiting behavioral sensitization, while neuronal attenuation following chronic MPD was observed in animals expressing behavioral tolerance. Different ratios of excitatory/inhibitory neuronal responses were obtained from the VTA, LC, or DR following chronic MPD. Thus, each brain area responds differently to each MPD dose used, suggesting that DA, NE, and 5-HT in the VTA, LC, and DR exert different effects.

Mesenchymal stromal cells alleviate depressive and anxiety-like behaviors via a lung vagal-to-brain axis in male mice.

Major depressive disorder (MDD) is one of the most common and disabling mental disorders, and current strategies remain inadequate. Although mesenchymal stromal cells (MSCs) have shown beneficial effects in experimental models of depression, underlying mechanisms remain elusive. Here, using murine depression models, we demonstrated that MSCs could alleviate depressive and anxiety-like behaviors not due to a reduction in proinflammatory cytokines, but rather activation of dorsal raphe nucleus (DRN) 5-hydroxytryptamine (5-HT) neurons. Mechanistically, peripheral delivery of MSCs activated pulmonary innervating vagal sensory neurons, which projected to the nucleus tractus solitarius, inducing the release of 5-HT in DRN. Furthermore, MSC-secreted brain-derived neurotrophic factor activated lung sensory neurons through tropomyosin receptor kinase B (TrkB), and inhalation of a TrkB agonist also achieved significant therapeutic effects in male mice. This study reveals a role of peripheral MSCs in regulating central nervous system function and demonstrates a potential "lung vagal-to-brain axis" strategy for MDD.

Methylphenidate induces a different response in the dorsal raphe as compared to ventral tegmental area and locus coeruleus: behavioral and concomitant neuronal recordings in adult rats.

Methylphenidate (MPD) is a psychostimulant used to treat attention deficit hyperactivity disorder. MPD exerts its neurocognitive effects through increasing concentrations of dopamine (DA), norepinephrine (NE), and serotonin (5-HT) in the neuronal synapse. This study recorded from adult freely behaving rats a total of 1170 neurons, 403 from the ventral tegmental area (VTA), 409 from locus coeruleus (LC), and 356 from dorsal raphe (DR) nucleus, which are the main sources of DA, NE, and 5-HT to the mesocorticolimbic circuitry, respectively. Electrophysiological and behavioral activities were recorded simultaneously following acute and repetitive (chronic) saline or 0.6, 2.5, or 10.0 mg/kg MPD. The uniqueness of this study is the evaluation of neuronal activity based on the behavioral response to chronic MPD. Animals received daily saline or MPD administration on experimental days 1-6 (ED1-6), followed by a 3-day wash-out period, and then MPD rechallenge on ED10. Each chronic MPD dose elicits behavioral sensitization in some animals, while in others, behavioral tolerance. Neuronal excitation following chronic MPD was observed in brains areas of animals exhibiting behavioral sensitization, while neuronal attenuation following chronic MPD was observed in those animals expressing behavioral tolerance. DR neuronal activity was most affected in response to acute and chronic MPD administration and responded differently compared to the neurons recorded from VTA and LC neurons at all doses. This suggests that although not directly related, DR and 5-HT are involved in the acute and chronic effects of MPD in adult rats, but exhibit a different role in response to MPD.

Preoperative Pain Facilitates Postoperative Cognitive Dysfunction via Periaqueductal Gray Matter-Dorsal Raphe Circuit.

Postoperative cognitive dysfunction (POCD) is a medically induced, rapidly occurring postoperative disease, which is hard to recover and seriously threatens the quality of life, especially for elderly patients, so it is important to identify the risk factors for POCD and apply early intervention to prevent POCD. As we have known, pain can impair cognition, and many surgery patients experience different preoperative pain, but it is still unknown whether these patients are vulnerable for POCD. Here we found that chronic pain (7 days, but not 1 day acute pain) induced by Complete Freund's Adjuvant (CFA) injected in the hind paw of rats could easily induce spatial cognition and memory impairment after being exposed to sevoflurane anesthesia. Next, for the mechanisms, we focused on the Periaqueductal Gray Matter (PAG), a well-known pivotal nucleus in pain process. It was detected the existence of neural projection from ventrolateral PAG (vlPAG) to adjacent nucleus Dorsal Raphe (DR), the origin of serotonergic projection for the whole cerebrum, through virus tracing and patch clamp recordings. The Immunofluorescence staining and western blot results showed that Tryptophan Hydroxylase 2 (TPH2) for serotonin synthesis in the DR was increased significantly in the rats treated with CFA for 7 days and sevoflurane for 3 hours, while chemo-genetic inhibition of the vlPAG-DR projection induced obvious spatial learning and memory impairment. Our study suggests that preoperative chronic pain may facilitate cognitive function impairment after receiving anesthesia through the PAG-DR neural circuit, and preventative analgesia should be a considerable measure to reduce the incidence of POCD.

Temporal derivative computation in the dorsal raphe network revealed by an experimentally driven augmented integrate-and-fire modeling framework.

By means of an expansive innervation, the serotonin (5-HT) neurons of the dorsal raphe nucleus (DRN) are positioned to enact coordinated modulation of circuits distributed across the entire brain in order to adaptively regulate behavior. Yet the network computations that emerge from the excitability and connectivity features of the DRN are still poorly understood. To gain insight into these computations, we began by carrying out a detailed electrophysiological characterization of genetically identified mouse 5-HT and somatostatin (SOM) neurons. We next developed a single-neuron modeling framework that combines the realism of Hodgkin-Huxley models with the simplicity and predictive power of generalized integrate-and-fire models. We found that feedforward inhibition of 5-HT neurons by heterogeneous SOM neurons implemented divisive inhibition, while endocannabinoid-mediated modulation of excitatory drive to the DRN increased the gain of 5-HT output. Our most striking finding was that the output of the DRN encodes a mixture of the intensity and temporal derivative of its input, and that the temporal derivative component dominates this mixture precisely when the input is increasing rapidly. This network computation primarily emerged from prominent adaptation mechanisms found in 5-HT neurons, including a previously undescribed dynamic threshold. By applying a bottom-up neural network modeling approach, our results suggest that the DRN is particularly apt to encode input changes over short timescales, reflecting one of the salient emerging computations that dominate its output to regulate behavior.

Shared and Distinct Brain Regions Targeted for Immediate Early Gene Expression by Ketamine and Psilocybin.

Psilocybin is a psychedelic with therapeutic potential. While there is growing evidence that psilocybin exerts its beneficial effects through enhancing neural plasticity, the exact brain regions involved are not completely understood. Determining the impact of psilocybin on plasticity-related gene expression throughout the brain can broaden our understanding of the neural circuits involved in psychedelic-evoked neural plasticity. In this study, whole-brain serial two-photon microscopy and light sheet microscopy were employed to map the expression of the immediate early gene, c-Fos, in male and female mice. The drug-induced c-Fos expression following psilocybin administration was compared to that of subanesthetic ketamine and saline control. Psilocybin and ketamine produced acutely comparable elevations in c-Fos expression in numerous brain regions, including anterior cingulate cortex, locus coeruleus, primary visual cortex, central and basolateral amygdala, medial and lateral habenula, and claustrum. Select regions exhibited drug-preferential differences, such as dorsal raphe and insular cortex for psilocybin and the CA1 subfield of hippocampus for ketamine. To gain insights into the contributions of receptors and cell types, the c-Fos expression maps were related to brain-wide in situ hybridization data. The transcript analyses showed that the endogenous levels of Grin2a and Grin2b predict whether a cortical region is sensitive to drug-evoked neural plasticity for both ketamine and psilocybin. Collectively, the systematic mapping approach produced an unbiased list of brain regions impacted by psilocybin and ketamine. The data are a resource that highlights previously underappreciated regions for future investigations. Furthermore, the robust relationships between drug-evoked c-Fos expression and endogenous transcript distributions suggest glutamatergic receptors as a potential convergent target for how psilocybin and ketamine produce their rapid-acting and long-lasting therapeutic effects.

Sexually dimorphic effects of estrogen receptor 2 deletion in the dorsal raphe nucleus on emotional behaviors.

Sex differences in emotional behaviors and affective disorders have been widely noted, of which sexually dimorphic secretion of gonadal steroid hormones such as estrogen is suspected to play a role. However, the underlying neural mechanisms remain poorly understood. We noted that the expression of estrogen receptor 2 (Esr2, or ERß), a key mediator of estrogen signaling in the brain, was enriched in the dorsal raphe nucleus (DRN), a region involved in emotion regulation. To investigate whether DRN Esr2 expression confers sex-specific susceptibility or vulnerability in emotional behaviors, we generated a conditional allele of Esr2 that allowed for site-specific deletion of Esr2 in the DRN via local injection of Cre-expressing viruses. DRN-specific Esr2 deletion mildly increased anxiety behaviors in females, as shown by decreased time spent in the center zone of an open field in knockout females. By contrast, DRN Esr2 deletion had no effects on anxiety levels in males, as demonstrated by knockout males spending comparable time in the center zone of an open field and open arms of an elevated-plus maze. Furthermore, in the tail suspension test, DRN Esr2 deletion reduced immobility, a depression-like behavior, in a male-biased manner. Together, these results reveal sex-specific functions of DRN Esr2 in regulating emotional behaviors and suggest targeted manipulation of DRN Esr2 signaling as a potential therapeutic strategy to treat sex-biased affective disorders.

Histochemical Characterization of the Dorsal Raphe-Periaqueductal Grey Dopamine Transporter Neurons Projecting to the Extended Amygdala.

The dorsal raphe (DR) nucleus contains many tyrosine hydroxylase (TH)-positive neurons which are regarded as dopaminergic (DA) neurons. These DA neurons in the DR and periaqueductal gray (PAG) region (DADR-PAG neurons) are a subgroup of the A10 cluster, which is known to be heterogeneous. This DA population projects to the central nucleus of the amygdala (CeA) and the bed nucleus of the stria terminalis (BNST) and has been reported to modulate various affective behaviors. To characterize, the histochemical features of DADR-PAG neurons projecting to the CeA and BNST in mice, the current study combined retrograde labeling with Fluoro-Gold (FG) and histological techniques, focusing on TH, dopamine transporter (DAT), vasoactive intestinal peptide (VIP), and vesicular glutamate transporter 2 (VGlut2). To identify putative DA neurons, DAT-Cre::Ai14 mice were used. It was observed that DATDR-PAG neurons consisted of the following two subpopulations: TH+/VIP- and TH-/VIP+ neurons. The DAT+/TH-/VIP+ subpopulation would be non-DA noncanonical DAT neurons. Anterograde labeling of DATDR-PAG neurons with AAV in DAT-Cre mice revealed that the fibers exclusively innervated the lateral part of the CeA and the oval nucleus of the BNST. Retrograde labeling with FG injections into the CeA or BNST revealed that the two subpopulations similarly innervated these regions. Furthermore, using VGlut2-Cre::Ai14 mice, it was turned out that the TH-/VIP+ subpopulations innervating both CeA and BNST were VGlut2-positive neurons. These two subpopulations of DATDR-PAG neurons, TH+/VIP- and TH-/VIP+, might differentially interfere with the extended amygdala, thereby modulating affective behaviors.

The ATP Level in the Medial Prefrontal Cortex Regulates Depressive-like Behavior via the Medial Prefrontal Cortex-Lateral Habenula Pathway.

BACKGROUND: Depression is the most common mental illness. Mounting evidence suggests that dysregulation of extracellular ATP (adenosine triphosphate) is involved in the pathophysiology of depression. However, the cellular and neural circuit mechanisms through which ATP modulates depressive-like behavior remain elusive. METHODS: By use of ex vivo slice electrophysiology, chemogenetic manipulations, RNA interference, gene knockout, behavioral testing, and two depression mouse models, one induced by chronic social defeat stress and one caused by a IP3R2-null mutation, we systematically investigated the cellular and neural circuit mechanisms underlying ATP deficiency-induced depressive-like behavior. RESULTS: Deficiency of extracellular ATP in both defeated susceptible mice and IP3R2-null mutation mice led to reduced GABAergic (gamma-aminobutyric acidergic) inhibition and elevated excitability in lateral habenula-projecting, but not dorsal raphe-projecting, medial prefrontal cortex (mPFC) neurons. Furthermore, the P2X2 receptor in GABAergic interneurons mediated ATP modulation of lateral habenula-projecting mPFC neurons and depressive-like behavior. Remarkably, chemogenetic activation of the mPFC-lateral habenula pathway induced depressive-like behavior in C57BL/6J mice, while inhibition of this pathway was sufficient to alleviate the behavioral impairment in both defeated susceptible and IP3R2-null mutant mice. CONCLUSIONS: Overall, our study provides compelling evidence that ATP level in the mPFC is critically involved in regulating depressive-like behavior in a pathway-specific manner. These results shed new light on the mechanisms underlying depression and the antidepressant effect of ATP.