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Astrocytes and microglia play important roles in organizing the structure and function of neuronal networks in the central nervous system (CNS). The dorsal diencephalic connection system (DDCS) is a phylogenetically ancient regulatory system by which the forebrain influences the activity of cholinergic and ascending monoaminergic pathways in the midbrain. The DDCS is probably important in inducing aspects of mental disorders, such as depression and addiction. The habenula is the small but highly complex connecting center of the DDCS in the epithalamus that consists of a medial (MHb) and lateral (LHb) division. MHb and LHb are built differently and connect different brain structures. Studies in animal models and human biomarker research provide good evidence that astroglia and microglia also affect the symptoms of mental disorders (such as depression). The significance of these neuroglia in habenular neurotransmission has not been extensively studied. This review article provides arguments for doing so more thoroughly.

The lateral habenula (LHb) is a critical brain structure involved in the aversive response to drug abuse. It has been determined that the gamma-aminobutyric acid (GABA)-ergic system plays the main role in morphine dependency. The role of GABA type A receptors (GABAARs) in LHb on morphine-induced conditioned place preference (CPP) remains unknown. In this study, the effect of bilateral intra-LHb microinjection of GABAAR agonist and antagonist on the acquisition and expression phases of CPP, utilizing a 5-day CPP paradigm in male rats, was evaluated. Subcutaneous administration of different doses of morphine caused a dose-dependent CPP. Intra-LHb microinjection of the GABAAR agonist, muscimol, in combination with morphine (5 mg/kg; subcutaneously) enhanced CPP scores in the acquisition phase of morphine CPP, whereas the GABAAR antagonist, bicuculline, significantly reduced the conditioning scores in the acquisition phase. Furthermore, pretreatment with a high dose of bicuculline reversed the additive effect of muscimol during the acquisition phase, yet the low dose of antagonist had no significant effect on agonist-induced CPP scores. On the other hand, muscimol (3 µg/rat) significantly increased CPP scores in the expression phase but bicuculline did not induce a significant effect on CPP scores. Bicuculline and muscimol microinjections did not affect locomotor activity in the testing sessions. Our results confirm that GABAARs in LHb play an active role in morphine reward. In addition, microinjections of bicuculline/muscimol may alter the morphine response through the GABAergic system.

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.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.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.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.

The lateral habenula (LHb) is an epithalamic nuclei that has been shown to signal the aversive properties of ethanol. The present study tested the hypothesis that activity of the LHb is required for the acquisition and/or expression of dependence-induced escalation of ethanol drinking and somatic withdrawal symptoms. Male Sprague-Dawley rats completed 4 weeks of baseline drinking under a standard intermittent access two-bottle choice (2BC) paradigm before undergoing 2 weeks of daily chronic intermittent ethanol (CIE) via vapor inhalation. Following this CIE exposure period, rats resumed 2BC drinking to assess dependence-induced changes in voluntary ethanol consumption. CIE exposed rats exhibited a significant increase in ethanol drinking that was associated with high levels of blood alcohol and a reduction in somatic symptoms of ethanol withdrawal. However, despite robust cFos activation in the LHb during ethanol withdrawal, chemogenetic inhibition of the LHb did not alter either ethanol consumption or somatic signs of ethanol withdrawal. Consistent with this observation, ablating LHb outputs via electrolytic lesions of the fasciculus retroflexus (FR) did not alter the acquisition of somatic withdrawal symptoms or escalation of ethanol drinking in CIE-exposed rats. The LHb controls activity of the rostromedial tegmental nucleus (RMTg), a midbrain nucleus activated by aversive experiences including ethanol withdrawal. During ethanol withdrawal, both FR lesioned and sham control rats exhibited similar cFos activation in the RMTg, suggesting that RMTg activation during ethanol withdrawal does not require LHb input. These data suggest that, at least in male rats, the LHb is not necessary for the acquisition or expression of escalation of ethanol consumption or expression of somatic symptoms of ethanol withdrawal. Overall, our findings provide evidence that the LHb is dispensable for some of the negative consequences of ethanol withdrawal.

Chronic stress contributes to the risk of developing depression; the habenula, a nucleus in epithalamus, is associated with many neuropsychiatric disorders. Using genome-wide gene expression analysis, we analyzed the transcriptome of the habenula in rats exposed to chronic restraint stress for 14 days. We identified 379 differentially expressed genes (DEGs) that were affected by chronic stress. These genes were enriched in neuroactive ligand-receptor interaction, the cAMP (cyclic adenosine monophosphate) signaling pathway, circadian entrainment, and synaptic signaling from the Kyoto Encyclopedia of Genes and Genomes pathway analysis and responded to corticosteroids, positive regulation of lipid transport, anterograde trans-synaptic signaling, and chemical synapse transmission from the Gene Ontology analysis. Based on protein-protein interaction network analysis of the DEGs, we identified neuroactive ligand-receptor interactions, circadian entrainment, and cholinergic synapse-related subclusters. Additionally, cell type and habenular regional expression of DEGs, evaluated using a recently published single-cell RNA sequencing study (GSE137478), strongly suggest that DEGs related to neuroactive ligand-receptor interaction and trans-synaptic signaling are highly enriched in medial habenular neurons. Taken together, our findings provide a valuable set of molecular targets that may play important roles in mediating the habenular response to stress and the onset of chronic stress-induced depressive behaviors.

The lateral habenula (LHb), which is a critical neuroanatomical hub and a regulator of midbrain monoaminergic centers, is activated by events resulting in negative valence and contributes to the expression of both appetitive and aversive behaviors. However, whole-brain cell-type-specific monosynaptic inputs to the LHb in both sexes remain incompletely elucidated. In this study, we used viral tracing combined with in situ hybridization targeting vesicular glutamate transporter 2 (vGlut2) and glutamic acid decarboxylase 2 (Gad2) to generate a comprehensive whole-brain atlas of inputs to glutamatergic and γ-aminobutyric acid (GABA)ergic neurons in the LHb. We found30 ipsilateral and contralateral brain regions that projected to the LHb. Of these, there were significantly more monosynaptic LHb-projecting neurons from the lateral septum, anterior hypothalamus, dorsomedial hypothalamus, and ventromedial hypothalamus in females than in males. More interestingly, we found a stronger GABAergic projection from the medial septum to the LHb in males than in females. Our results reveal a comprehensive connectivity atlas of glutamatergic and GABAergic inputs to the LHb in both sexes, which may facilitate a better understanding of sexual dimorphism in physiological and pathological brain functions.

The lateral habenula (LHb) is a tiny structure that acts as a hub, relaying signals from the limbic forebrain structures and basal ganglia to the brainstem modulatory area. Facilitated by updated knowledge and more precise manipulation of circuits, the progress in figuring out the neural circuits and functions of the LHb has increased dramatically over the past decade. Importantly, LHb is found to play an integrative role and has profound effects on a variety of behaviors associated with pain, including depression-like and anxiety-like behaviors, antireward or aversion, aggression, defensive behavior, and substance use disorder. Thus, LHb is a potential target for improving pain management and related disorders. In this review, we focused on the functions, related circuits, and neurotransmissions of the LHb in pain processing and related behaviors. A comprehensive understanding of the relationship between the LHb and pain will help to find new pain treatments.

GABABreceptors in habenula cholinergic neurons mediate strong presynaptic excitation and control aversive memory expression. K+channel tetramerization domain (KCTD) proteins are key interacting partners of GABABreceptors; it remains unclear whether and how KCTDs contribute to GABABexcitatory signaling. Here, we show that KCTD8 and KCTD12 in these neurons facilitate the GABABreceptors expression in axonal terminals and contribute to presynaptic excitation by GABABreceptors. Genetically knocking outKCTD8/12/16orKCTD8/12, but not other combinations of the threeKCTDisoforms, substantially reduced GABABreceptors–mediated potentiation of glutamate release and presynaptic Ca2+entry in response to axonal stimulation, whereas they had no effect on GABAB-mediated inhibition in the somata of cholinergic neurons within the habenulo–interpeduncular pathway in mice of either sex. The physiological phenotypes were associated with a significant decrease in the GABABexpression within the axonal terminals but not the somata. Overexpressing either KCTD8 or KCTD12 in theKCTD8/12/16triple knock-out mice reversed the changes in axonal GABABexpression and presynaptic excitation. In mice lacking the KCTDs, aversion-predicting cues produced stronger neuronal activation in the interpeduncular nucleus, and the infusion of GABABagonist in this nucleus produced a weaker effect on fear extinction. Collectively, our results reveal isoform-specific roles of KCTD proteins in enriching the axonal expression of GABABreceptors, facilitating their presynaptic signaling, and modulating aversion-related memory processes.SIGNIFICANCE STATEMENTGABABreceptors represent the principal inhibitory neurotransmitter receptor, but they mediate strong presynaptic excitation in the habenulo–interpeduncular pathway and modulate aversion memory expression. KCTD proteins are integral constituents of GABABreceptors. By analyzing the physiological, neuroanatomical, and behavioral phenotypes of multiple KCTD knock-out mouse lines, we show that KCTD8 and KCTD12 facilitate the axonal expression and hence presynaptic excitation of GABABreceptors in habenula cholinergic neurons and control cued-aversion memory formation and expression in the habenulo–interpeduncular pathway. These results expand the physiological and behavioral functions of KCTDs in modulating the brain neural circuits.

Recently, an animal model for depression has shown that ketamine, an N-methyl- d-aspartate receptor (NMDAR) antagonist, elicits a rapid-acting antidepressant effect by blocking NMDAR-dependent bursting in the lateral habenula (Hb). However, evidence from human studies remains scarce.This study explored the changes of resting-state functional connectivity (FC) of the Hb in responders and nonresponders who was diagnosed with unipolar or bipolar depression before and after ketamine treatment. The response was defined as a ≥50% reduction in the total MADRS score at Day 13 (24 h following the sixth infusion) in comparison with the baseline score. Correlation analyses were performed to identify an association between symptom improvement and the signals of the significantly different brain regions detected in the above imaging analysis.In the post-hoc region-of-interest analysis, an enhanced baseline FC between Hb and several hubs of the default mode network (including angulate cortex, precuneus, medial prefrontal cortex, and middle temporal cortex) was observed in responders (≥50% decrease in the Montgomery-Asberg Scale at 2 weeks) compared with nonresponders.These pilot findings may suggest a potential neural mechanism by which ketamine exerts its robust antidepressant efficacy via downregulation of aberrant habenular FC with parts of the default mode network.

Environmental cues and internal states such as mood, reward, or aversion directly influence feeding behaviors beyond homeostatic necessity. The hypothalamus has been extensively investigated for its role in homeostatic feeding. However, many of the neural circuits that drive more complex, non-homeostatic feeding that integrate valence and sensory cues (such as taste and smell) remain unknown. Here, we describe a basal forebrain (BF)-to-lateral habenula (LHb) circuit that directly modulates non-homeostatic feeding behavior. Using viral-mediated circuit mapping, we identified a population of glutamatergic neurons within the BF that project to the LHb, which responds to diverse sensory cues, including aversive and food-related odors. Optogenetic activation of BF-to-LHb circuitry drives robust, reflexive-like aversion. Furthermore, activation of this circuitry suppresses the drive to eat in a fasted state. Together, these data reveal a role of basal forebrain glutamatergic neurons in modulating LHb-associated aversion and feeding behaviors by sensing environmental cues.

As a critical node connecting the forebrain with the midbrain, the lateral habenula (LHb) processes negative feedback in response to aversive events and plays an essential role in value-based decision-making. Compulsive drug use, a hallmark of substance use disorder, is attributed to maladaptive decision-making regarding aversive drug-use-related events and has been associated with dysregulation of various frontal–midbrain circuits. To understand the contributions of frontal–habenula–midbrain circuits in the development of drug dependence, we employed a rat model of methamphetamine self-administration (SA) in the presence of concomitant footshock, which has been proposed to model compulsive drug-taking in humans. In this longitudinal study, functional MRI data were collected at pretraining baseline, after 20 d of long-access SA phase, and after 5 d of concomitant footshock coupled with SA (punishment phase). Individual differences in response to punishment were quantified by a “compulsivity index (CI),” defined as drug infusions at the end of punishment phase, normalized by those at the end of SA phase. Functional connectivity of LHb with the frontal cortices and substantia nigra (SN) after the punishment phase was positively correlated with the CI in rats that maintained drug SA despite receiving increasing-intensity footshock. In contrast, functional connectivity of the same circuits was negatively correlated with CI in rats that significantly reduced SA. These findings suggest that individual differences in compulsive drug-taking are reflected by alterations within frontal–LHb–SN circuits after experiencing the negative consequences from SA, suggesting these circuits may serve as unique biomarkers and potential therapeutic targets for individualized treatment of addiction.

Circadian rhythms are those variations in behavioral and molecular processes of organisms that follow roughly 24 h cycles in the absence of any external cue. The hypothalamic suprachiasmatic nucleus (SCN) harbors the principal brain pacemaker driving circadian rhythms. The epithalamic habenula (Hb) contains a self-sustained circadian clock functionally coupled to the SCN. Anatomically, the Hb projects to the midbrain dopamine (DA) and serotonin (5-HT) systems, and it receives inputs from the forebrain, midbrain, and brainstem. The SCN is set by internal signals such as 5-HT or melatonin from the raphe nuclei and pineal gland, respectively. However, how the Hb clock is set by internal cues is not well characterized. Hence, in the present study, we determined whether DA, noradrenaline (NA), 5-HT, and the neuropeptides orexin (ORX) and vasopressin influence the Hb circadian clock. Using PER2::Luciferase transgenic mice, we found that the amplitude of the PER2 protein circadian oscillations from Hb explants was strongly affected by DA and NA. Importantly, these effects were dose-and region (rostral vs. caudal) dependent for NA, with a main effect in the caudal part of the Hb. Furthermore, ORX also induced a significant change in the amplitude of PER2 protein oscillations in the caudal Hb. In conclusion, catecholaminergic (DA, NA) and ORXergic transmission impacts the clock properties of the Hb clock likely contributing to the circadian regulation of motivated behaviors. Accordingly, pathological conditions that lead in alterations of catecholamine or ORX activity (drug intake, compulsive feeding) might affect the Hb clock and conduct to circadian disturbances.

The identification of proteins that are altered following nicotine/tobacco exposure can facilitate and positively impact the investigation of related diseases. In this report, we investigated the effects of chronic (–)-menthol exposure in fourteen murine brain regions for changes in total β2 subunit protein levels and changes in epibatidine binding levels using immunoblotting and radioligand binding assays. We identified the habenula as a region of interest due to the region’s marked decreases in β2 subunit and nAChR levels in response to chronic (–)-menthol alone. Thus, we further examined the habenula, a brain region associated with both the reward and withdrawal components of addiction, for additional protein level alterations using mass spectrometry. A total of 552 proteins with altered levels were identified after chronic (–)-menthol exposure. Enriched in the proteins with altered levels after (–)-menthol exposure were proteins associated with signaling, immune systems, RNA regulation, and protein transport. The continuation and expansion of the brain region-specific protein profiling in response to (–)-menthol will provide a better understanding of how this common flavorant in tobacco and e-liquid products may affect addiction and general health.

Background Major depressive disorder (MDD) is a common cause of disability and morbidity, affecting about 10% of the population worldwide. Subclinical depression (SD) can be understood as a precursor of MDD, and therefore provides an MDD risk indicator. The pathogenesis of MDD and SD in humans is still unclear, and the current diagnosis lacks accurate biomarkers and gold standards. Methods A total of 40 MDD, 34 SD, and 40 healthy control (HC) participants matched by age, gender, and education were included in this study. Resting-state functional magnetic resonance images (rs-fMRI) were used to analyze the functional connectivity (FC) of the posterior parietal thalamus (PPtha), which includes the lateral habenula, as the region of interest. Analysis of variance with the post hoc t-test test was performed to find significant differences in FC and clarify the variations in FC among the HC, SD, and MDD groups. Results Increased FC was observed between PPtha and the left inferior temporal gyrus (ITG) for MDD versus SD, and between PPtha and the right ITG for SD versus HC. Conversely, decreased FC was observed between PPtha and the right middle temporal gyrus (MTG) for MDD versus SD and MDD versus HC. The FC between PPtha and the middle frontal gyrus (MFG) in SD was higher than that in MDD and HC. Compared with the HC group, the FC of PPtha-ITG (left and right) increased in both the SD and MDD groups, PPtha-MTG (right) decreased in both the SD and MDD groups and PPtha-MFG (right) increased in the SD group and decreased in the MDD group. Conclusion Through analysis of FC measured by rs-fMRI, the altered FC between PPtha and several brain regions (right and left ITG, right MTG, and right MFG) has been identified in participants with SD and MDD. Different alterations in FC between PPtha and these regions were identified for patients with depression. These findings might provide insights into the potential pathophysiological mechanisms of SD and MDD, especially related to PPtha and the lateral habenula.

Background: The auditory system processes how we hear and understand sounds within the environment. It comprises both peripheral and central structures. Sympathetic nervous system projections are present throughout the auditory system. The function of sympathetic fibers in the cochlea has not been studied extensively due to the limited number of direct projections in the auditory system. Nevertheless, research on adrenergic and noradrenergic regulation of the cochlea and central auditory system is growing. With the rapid development of neuroscience, auditory central regulation is an extant topic of focus in research on hearing. Summary: As such, understanding sympathetic nervous system regulation of auditory function is a growing topic of interest. Herein, we review the distribution and putative physiological and pathological roles of sympathetic nervous system projections in hearing. Key Messages: In the peripheral auditory system, the sympathetic nervous system regulates cochlear blood flow, modulates cochlear efferent fibers, affects hair cells, and influences the habenula region. In central auditory pathways, norepinephrine is essential for plasticity in the auditory cortex and affects auditory cortex activity. In pathological states, the sympathetic nervous system is associated with many hearing disorders. The mechanisms and pathways of sympathetic nervous system modulation of auditory function is still valuable for us to research and discuss.

The orphan G-protein-coupled receptor GPR139 is highly expressed in the habenula, a small brain nucleus that has been linked to depression, schizophrenia (SCZ), and substance-use disorder. High-throughput screening and a medicinal chemistry structure-activity relationship strategy identified a novel series of potent and selective benzotriazinone-based GPR139 agonists. Herein, we describe the chemistry optimization that led to the discovery and validation of multiple potent and selective in vivo GPR139 agonist tool compounds, including our clinical candidate TAK-041, also known as NBI-1065846 (compound 56). The pharmacological characterization of these GPR139 agonists in vivo demonstrated GPR139-agonist-dependent modulation of habenula cell activity and revealed consistent in vivo efficacy to rescue social interaction deficits in the BALB/c mouse strain. The clinical GPR139 agonist TAK-041 is being explored as a novel drug to treat negative symptoms in SCZ.

The forebrain plays a critical role in a broad range of neural processes encompassing sensory integration and initiation/selection of behaviour. The forebrain functions through an interaction between different cortical areas, the thalamus, the basal ganglia with the dopamine system, and the habenulae. The ambition here is to compare the mammalian forebrain with that of the lamprey representing the oldest now living group of vertebrates, by a review of earlier studies. We show that the lamprey dorsal pallium has a motor, a somatosensory, and a visual area with retinotopic representation. The lamprey pallium was previously thought to be largely olfactory. There is also a detailed similarity between the lamprey and mammals with regard to other forebrain structures like the basal ganglia in which the general organisation, connectivity, transmitters and their receptors, neuropeptides, and expression of ion channels are virtually identical. These initially unexpected results allow for the possibility that many aspects of the basic design of the vertebrate forebrain had evolved before the lamprey diverged from the evolutionary line leading to mammals. Based on a detailed comparison between the mammalian forebrain and that of the lamprey and with due consideration of data from other vertebrate groups, we propose a compelling account of a pan-vertebrate schema for basic forebrain structures, suggesting a common ancestry of over half a billion years of vertebrate evolution.

The mu opioid receptor (MOR) and the orphan GPR151 receptor are inhibitory G protein coupled receptors that are enriched in the habenula, a small brain region involved in aversion processing, addiction and mood disorders. While MOR expression in the brain is widespread, GPR151 expression is restricted to the habenula. In a previous report, we created conditional ChrnB4-Cre × Oprm1fl/fl (so-called B4MOR) mice, where MORs are deleted specifically in Chrnb4-positive neurons restricted to the habenula, and shown a role for these receptors in naloxone aversion. Here we characterized the implication of habenular MORs in social behaviors. B4MOR−/− mice and B4MOR+/+ mice were compared in several social behavior measures, including the chronic social stress defeat (CSDS) paradigm, the social preference (SP) test and social conditioned place preference (sCPP). In the CSDS, B4MOR−/− mice showed lower preference for the social target (unfamiliar mouse of a different strain) at baseline, providing a first indication of deficient social interactions in mice lacking habenular MORs. In the SP test, B4MOR−/− mice further showed reduced sociability for an unfamiliar conspecific mouse. In the sCPP, B4MOR−/− mice also showed impaired place preference for their previous familiar littermates after social isolation. We next created and tested Gpr151−/− mice in the SP test, and also found reduced social preference compared to Gpr151+/+ mice. Altogether our results support the underexplored notion that the habenula regulates social behaviors. Also, our data suggest that the inhibitory habenular MOR and GPR151 receptors normally promote social reward, possibly by dampening the aversive habenula activity.

The Stria Medullaris (SM) is a white-matter tract that contains afferent fibres that connect the cognitive-emotional areas in the forebrain to the Habenula (Hb). The Hb plays an important role in behavioral responses to reward, stress, anxiety, pain, and sleep through its action on neuromodulator systems. The Fasciculus Retroflexus (FR) forms the primary output of the Hb to the midbrain. The SM, Hb, and FR are part of a special pathway between the forebrain and the midbrain known as the Dorsal Diencephalic Conduction system (DDC). Hb dysfunction is accompanied by different types of neuropsychiatric disorders, such as schizophrenia, depression, and Treatment-Resistant Depression (TRD). Due to difficulties in the imaging assessment of the SM and HB in vivo, they had not been a focus of clinical studies until the invention of Diffusion Tensor Imaging (DTI), which has revolutionized the imaging and investigation of the SM and Hb. DTI has facilitated the imaging of the SM and Hb and has provided insights into their properties through the investigation of their monoamine dysregulation. DTI is a well-established technique for mapping brain microstructure and white matter tracts; it provides indirect information about the microstructural architecture and integrity of white matter in vivo, based on water diffusion properties in the intra- and extracellular space, such as Axial Diffusivity (AD), Radial Diffusivity (RD), mean diffusivity, and Fractional Anisotropy (FA). Neurosurgeons have recognized the potential value of DTI in the direct anatomical targeting of the SM and Hb prior to Deep Brain Stimulation (DBS) surgery for the treatment of certain neuropsychiatric conditions, such as TRD. DTI is the only non-invasive method that offers the possibility of visualization in vivo of the white-matter tracts and nuclei in the human brain. This review study summarizes the use of DTI as a promising new imaging method for accurate identification of the SM and Hb, with special emphasis on direct anatomical targeting of the SM and Hb prior to DBS surgery.

The lateral habenula (LHb) is a brain structure which is known to be pathologically hyperactive in depression, whereby it shuts down the brains' reward systems. Interestingly, inhibition of the LHb has been shown to have an antidepressant effect, hence making the LHb a fascinating subject of study for developing novel antidepressant therapies. Despite this however, the exact mechanisms by which inhibitory signalling is processed within the LHb remain incompletely understood. Some studies have proposed the existence of locally‐targeting inhibitory interneuron populations within the LHb. One such population is believed to be akin to neocortical neurogliaform cells, yet specific molecular markers for studying these neurons are sparse and hence their function remains elusive. Recently, neuron‐derived neurotrophic factor (NDNF) has been proposed as one such marker for neocortical neurogliaform cells. Using a combination of histological, physiological and optogenetic tools, we hence sought to first validate if NDNF was selectively expressed by such inhibitory neurons within the neocortex, and then if it was confined to a similar population within the LHb. While we report this to be true for the neocortex, we find no such evidence within the LHb; rather that NDNF is expressed without restriction to a particular neuronal sub‐population. These results hence indicate that molecular markers can represent broadly diverse populations of neurons on a region‐to‐region basis, and that therefore each population as defined by molecular marker expression should be validated in each brain structure.

The conserved bilateral habenular nuclei (HA) in vertebrate diencephalon develop into compartmentalized structures containing neurons derived from different cell lineages. Despite extensive studies demonstrated that zebrafish larval HA display distinct left-right (L-R) asymmetry in gene expression and connectivity, the spatial gene expression domains were mainly obtained from two-dimensional (2D) snapshots of colorimetric RNA in situ hybridization staining which could not properly reflect different HA neuronal lineages constructed in three-dimension (3D). Combing the tyramide-based fluorescent mRNA in situ hybridization, confocal microscopy and customized imaging processing procedures, we have created spatial distribution maps of four genes for 4-day-old zebrafish and in sibling fish whose L-R asymmetry was spontaneously reversed. 3D volumetric analyses showed that ratios of cpd2, lov, ron, and nrp1a expression in L-R reversed HA were reversed according to the parapineal positions. However, the quantitative changes of gene expression in reversed larval brains do not mirror the gene expression level in the obverse larval brains. There were a total 87.78% increase in lov+ nrp1a+ and a total 12.45% decrease in lov+ ron+ double-positive neurons when the L-R asymmetry of HA was reversed. Thus, our volumetric analyses of the 3D maps indicate that changes of HA neuronal cell fates are associated with the reversal of HA laterality. These changes likely account for the behavior changes associated with HA laterality alterations.

Nodal signaling is essential for mesoderm and endoderm formation, as well as neural plate induction and establishment of left-right asymmetry. However, the mechanisms controlling expression of Nodal pathway genes in these contexts are not fully known. Previously, we showed that Cdx1b induces expression of downstream Nodal signaling factors during early endoderm formation. In this study, we show that Cdx1b also regulates epithalamic asymmetry in zebrafish embryos by modulating expression of ndr2 and lft1. We first knocked down cdx1b with translation-blocking and splicing-blocking morpholinos (MOs). Most embryos injected with translation-blocking MOs showed absent ndr2, lft1 and pitx2c expression in the left dorsal diencephalon during segmentation and pharyngula stages accompanied by aberrant parapineal migration and habenular laterality at 72 ​h post fertilization (hpf). These defects were less frequent in embryos injected with splicing-blocking MO. To confirm the morphant phenotype, we next generated both zygotic (Z)cdx1b−/− and maternal zygotic (MZ)cdx1b−/− mutants by CRISPR-Cas9 mutagenesis. Expression of ndr2, lft1 and pitx2c was absent in the left dorsal diencephalon of a high proportion of MZcdx1b−/− mutants; however, aberrant dorsal diencephalic pitx2c expression patterns were observed at low frequency in Zcdx1b−/− mutant embryos. Correspondingly, dysregulated parapineal migration and habenular laterality were also observed in MZcdx1b−/− mutant embryos at 72 hpf. On the other hand, Kupffer’s vesicle cilia length and number, expression pattern of spaw in the lateral plate mesoderm and pitx2c in the gut as well as left-right patterning of various visceral organs were not altered in MZcdx1b−/− mutants compared to wild-type embryos. Chromatin immunoprecipitation revealed that Cdx1b directly regulates ndr2 and lft1 expression. Furthermore, injection of cdx1b-vivo MO1 but not cdx1b-vivo 4 ​mm MO1 in the forebrain ventricle at 18 hpf significantly downregulated lft1 expression in the left dorsal diencephalon at 23–24 ​s stages. Together, our results suggest that Cdx1b regulates transcription of ndr2 and lft1 to maintain proper Nodal activity in the dorsal diencephalon and epithalamic asymmetry in zebrafish embryos.

Alterations of monoamine transmission in mesocorticolimbic regions have been suggested in the pathophysiology of attention deficit/hyperactivity disorder (ADHD). The habenula is an important brain area in regulation of monoamine transmission. In this study, we investigated behavioral and electrophysiological alterations induced by neonatal habenula lesion (NHL) in rats. In NHL rats, age-dependent behavioral alterations relevant to the ADHD symptoms, such as hyperlocomotion, impulsivity, and attention deficit, were observed. Local field potentials (LFPs) in mesocorticolimbic regions of anesthetized rats were examined with in vivo electrophysiological recordings. Abnormally enhanced synchronization of slow (delta) and fast (gamma) LFP oscillations between the amygdala (AMY) and prefrontal cortex (PFC) was found in juvenile, but not in adult, NHL rats. We further examined the effects of an extract and the active compound from the perennial large brown algae Ecklonia stolonifera (ES), which have previously been demonstrated to modulate monoamine transmission, on these NHL-induced alterations. One week of ES extract treatments normalized the NHL-induced behavioral alterations, whereas the active compound fucosterol improved attention deficit and impulsivity, but not hyperlocomotion, in NHL rats. Consistent with the behavioral effects, ES extract treatments also normalized augmented AMY-PFC coupling. These results suggest that altered limbic-cortical information processing may be involved in ADHD-like behavioral alterations induced by NHL, which could be ameliorated by the natural substance, such as ES that affects monoamine transmission.

The review focuses on mast cells as resident cells in the central nervous system (CNS) of mammals, birds, reptiles, amphibians, and fish. Mast cell distribution in the brain of different vertebrate species is addressed in detail. In mammals, they are located most often in the choroid plexus, meninges, thalamus, medial nucleus of habenula, hypothalamus and circumventricular organs, usually in the immediate vicinity of blood vessels and cerebrospinal fluid (CSF)-containing spaces. Their number in brain is unstable, changing during postnatal ontogeny and under the influence of hormonal and immune factors. There are different ways of mediator release from cytoplasmic granules of mast cells, which provide targeted or diffuse effects on surrounding cells. Mast cells express a large number of receptors for immunoglobulins, neurotransmitters and hormones, which makes them highly sensitive to changes in the external and internal environments of the body. At the same time, numerous mediators contained in mast cells allow them to induce inflammatory, anti-inflammatory, immuno-, vasculo-, gliocyto- and/or neuromodulating effects, the intensity and direction of which relies on the composition and amount of secreted mediators and the ways of their release, which, in turn, depends on which receptors of mast cells are activated. Mast cells control the permeability of the blood–brain and blood–CSF barriers, contribute to angiogenesis and, along with glial cells, regulate neuroinflammation (which is a leading pathogenetic factor of some nervous system and mental diseases) and protect the CNS from other pathogenic factors. Mast cells in the brain play the role of neuroimmune centers with afferent and efferent functions that link peripheral organs to specialized CNS cells and are actively involved in maintaining CNS homeostasis under both physiological conditions and the influence of pathological factors.

There is emerging evidence implicating glucagon-like peptide-1 (GLP-1) in reward, including palatable food reinforcement and alcohol-based reward circuitry. While recent findings suggest that mesolimbic structures, such as the ventral tegmental area (VTA) and the nucleus accumbens (NAc), are critical anatomical sites mediating the role of GLP-1’s inhibitory actions, the present study focused on the potential novel impact of GLP-1 within the habenula, a region of the forebrain expressing GLP-1 receptors. Given that the habenula has also been implicated in the neural control of reward and reinforcement, we hypothesized that this brain region, like the VTA and NAc, might mediate the anhedonic effects of GLP-1. Rats were stereotaxically implanted with guide cannula targeting the habenula and trained on a progressive ratio 3 (PR3) schedule of reinforcement. Separate rats were trained on an alcohol two-bottle choice paradigm with intermittent access. The GLP-1 agonist exendin-4 (Ex-4) was administered directly into the habenula to determine the effects on operant responding for palatable food as well as alcohol intake. Our results indicated that Ex-4 reliably suppressed PR3 responding and that this effect was dose-dependent. A similar suppressive effect on alcohol consumption was observed. These findings provide initial and compelling evidence that the habenula may mediate the inhibitory action of GLP-1 on reward, including operant and drug reward. Our findings further suggest that GLP-1 receptor mechanisms outside of the midbrain and ventral striatum are critically involved in brain reward neurotransmission.

Allelic variation inCHRNA3, the gene encoding the α3 nicotinic acetylcholine receptor (nAChR) subunit, increases vulnerability to tobacco dependence and smoking-related diseases, but little is known about the role for α3-containing (α3*) nAChRs in regulating the addiction-related behavioral or physiological actions of nicotine. α3* nAChRs are densely expressed by medial habenula (mHb) neurons, which project almost exclusively to the interpeduncular nucleus (IPn) and are known to regulate nicotine avoidance behaviors. We found thatChrna3tm1.1Hwrthypomorphic mice, which express constitutively low levels of α3* nAChRs, self-administer greater quantities of nicotine (0.4 mg kg−1per infusion) than their wild-type littermates. Microinfusion of a lentivirus vector to express a short-hairpin RNA into the mHb or IPn to knock-downChrna3transcripts markedly increased nicotine self-administration behavior in rats (0.01–0.18 mg kg−1per infusion). Using whole-cell recordings, we found that the α3β4* nAChR-selective antagonist α-conotoxin AuIB almost completely abolished nicotine-evoked currents in mHb neurons. By contrast, the α3β2* nAChR-selective antagonist α-conotoxin MII only partially attenuated these currents. Finally, micro-infusion of α-conotoxin AuIB (10 μm) but not α-conotoxin MII (10 μm) into the IPn in rats increased nicotine self-administration behavior. Together, these data suggest that α3β4* nAChRs regulate the stimulatory effects of nicotine on the mHb-IPn circuit and thereby regulate nicotine avoidance behaviors. These findings provide mechanistic insights into howCHRNA3risk alleles can increase the risk of tobacco dependence and smoking-related diseases in human smokers.SIGNIFICANCE STATEMENTAllelic variation inCHRNA3, which encodes the α3 nicotinic acetylcholine receptor (nAChR) subunit gene, increases risk of tobacco dependence but underlying mechanisms are unclear. We report thatChrna3hypomorphic mice consume greater quantities of nicotine than wild-type mice and that knock-down ofChrna3gene transcripts in the habenula or interpeduncular nucleus (IPn) increases nicotine intake in rats. α-Conotoxin AuIB, a potent antagonist of the α3β4 nAChR subtype, reduced the stimulatory effects of nicotine on habenular neurons, and its infusion into the IPn increased nicotine intake in rats. These data suggest that α3β4 nAChRs in the habenula-IPn circuit regulate the motivational properties of nicotine.

The habenula (Hb) is an epithalamic structure that links multiple forebrain areas with the mid/hindbrain monoaminergic systems. As an anti-reward center, it has been implicated in the etiology of various neuropsychiatric disorders, particularly those associated with dysregulated reward circuitry. In this regard, Hb has been proposed as a therapeutic target for treatment-resistant depression associated with a higher risk of suicide. Therefore, we aimed to gain insight into the molecular signatures of the Hb in association with suicide in individuals with major depression. Postmortem gene expression analysis identified 251 differentially expressed genes (DEGs) in the Hb tissue of suicides in comparison with Hb tissues from neurotypical individuals. Subsequent bioinformatic analyses using single-cell transcriptome data from the mouse Hb showed that the levels of a subset of endothelial cell-enriched genes encoding cell–cell junctional complex and plasma membrane-associated proteins, as well as the levels of their putative upstream transcriptional regulators, were significantly affected in suicides. Although our findings are based on a limited number of samples, the present study suggests a potential association of endothelial dysfunction in the Hb with depression and suicidal behavior.

Introduction:The multiplicity and complexity of the neuronal connections in the central nervous system make it difficult to disentangle circuits that play an essential role in the development or treatment of (neuro)psychiatric disorders. By choosing the evolutionary development of the forebrain as a starting point, a certain order in the connections can be created. The dorsal diencephalic connection (DDC) system can be applied for the development of biomarkers that can predict treatment response.Materials and methods:After providing a brief introduction to the theory, we examined neuroanatomical publications on the connectivity of the DDC system. We then searched for neurochemical components that are specific for the habenula.Results and discussion:The best strategy to find biomarkers that reflect the function of the habenular connection is to use genetic variants of receptors, transporters or enzymes specific to this complex. By activating these with probes and measuring the response in people with different functional genotypes, the usefulness of biomarkers can be assessed.Conclusions:The most promising biomarkers in this respect are those linked to activation or inhibition of the nicotine receptor, dopamine D4 receptor, μ-opioid receptor and also those of the functioning of habenular glia cells (astrocytes and microglia).

Upon stress exposure a broad network of structures comes into play in order to provide adequate responses and restore homeostasis. It has been known for decades that the main structures engaged during the stress response are the medial prefrontal cortex, the amygdala, the hippocampus, the hypothalamus, the monoaminergic systems (noradrenaline, dopamine, serotonin), and the periaqueductal gray. The lateral habenula (LHb) is an epithalamic structure directly connected to prefrontal cortical areas and to the amygdala, whereas it functionally interacts with the hippocampus. Also, it is a main modulator of monoaminergic systems. The LHb is activated upon exposure to basically all types of stressors, suggesting it is also involved in the stress response. However, it remains unknown if and how the LHb functionally interacts with the broad stress response network. In the current study we performed in rats a restraint stress procedure followed by immunohistochemical staining of the c-Fos protein throughout the brain. Using Graph Theory-based functional connectivity analyses, we confirm the principal hubs of the stress network (e.g. prefrontal cortex, amygdala, periventricular hypothalamus), and show that the LHb is engaged during stress exposure in close interaction with the medial prefrontal cortex, the lateral septum, and the medial habenula. In addition, we performed DREADD-induced LHb inactivation during the same restraint paradigm in order to explore its consequences on the stress response network. This last experiment gave contrasting results as the DREADD ligand alone, clozapine-N-oxide, was able to modify the network.GRAPHICAL ABSTRACTGRAPHICAL ABSTRACT TEXTIn this study, using immunohistochemical staining of the immediate early gene c-fos and graph theory-based functional correlational analyses, we aimed at unravelling the possible engagement of the lateral habenula (LHb) within the stress response network during acute stress exposure (10-min restraint) in rats. We found that the medial part of the LHb (LHbM) was preferentially engaged, and that this engagement was concomitant to this of structures such as the medial prefrontal cortex (mPFC), the insular cortex (Ins), hypothalamic (PVH) and thalamic (PVT) paraventricular nuclei, the extended amygdala, comprising the Bed nucleus of the stria terminalis (BNST) and the entire amygdala (AMG), as well as the dopaminergic ventral tegmental area (VTA) and the serotonergic dorsal raphe nucleus (RD). This suggests upon stressful situations the LHbM serves as a relay of cortical, thalamic, hypothalamic and temporal information, further transmitted to midbrain monoaminergic systems to probably initiate coping strategies.

Postoperative cognitive dysfunction (POCD) is common and is associated with poor outcome. Neural circuit involvement in POCD is unknown. Lateral habenula (LHb) that regulates coping and depression-like behaviors after aversive stimuli is activated by surgery in the previous study. Here, surgery activated LHb and ventral tegmental area (VTA) are presented. VTA is known to receive projections from LHb and project to the prefrontal cortex and hippocampus. Direct chemogenetic inhibition of LHb or damaging LHb attenuates surgery-induced learning and memory impairment, N-methyl-d-aspartate (NMDA) receptor activation, endoplasmic reticulum stress, inflammatory responses and cell injury in the VTA, and activation of rostromedial tegmental nucleus, an intermediate station to connect LHb with VTA. LHb inhibition preserves dendritic spine density in the prefrontal cortex and hippocampus. Retrograde inhibition of LHb via its projections to VTA attenuated surgery-induced learning and memory dysfunction is observed. Retrograde activation of LHb induced learning and memory dysfunction is observed. Inhibition of NMDA receptors, dopamine synthesis, and endoplasmic reticulum stress in the VTA reduced surgery-induced learning and memory impairment, inflammatory responses, and cell injury are observed. These results suggest that surgery activates the LHb-VTA neural circuit, which contributes to POCD and neuropathological changes in the brain. These novel findings represent initial evidence for neural circuit involvement in surgery effects.

The habenula is a complex neuronal population integrated in a pivotal functional position into the vertebrate limbic system. Its main afference is the stria medullaris and its main efference the fasciculus retroflexus. This neuronal complex is composed by two main components, the medial and lateral habenula. Transcriptomic and single cell RNAseq studies have unveiled the morphological complexity of both components. The aim of our work was to analyze the relation between the origin of the axonal fibers and their final distribution in the habenula. We analyzed 754 tracing experiments from Mouse Brain Connectivity Atlas, Allen Brain Map databases, and selected 12 neuronal populations projecting into the habenular territory. Our analysis demonstrated that the projections into the medial habenula discriminate between the different subnuclei and are generally originated in the septal territory. The innervation of the lateral habenula displayed instead a less restricted distribution from preoptic, terminal hypothalamic and peduncular nuclei. Only the lateral oval subnucleus of the lateral habenula presented a specific innervation from the dorsal entopeduncular nucleus. Our results unveiled the necessity of novel sorts of behavioral experiments to dissect the different functions associated with the habenular complex and their correlation with the distinct neuronal populations that generate them., Work supported by MINECO/AEI/FEDER (BFU2013-48230) to E. Puelles and D. Echevarría; MINECO/AEI/FEDER (SAF2017-83702-R; PID2020-118171RB-I00), GVA (PROMETEO/2018/041), ISCIII (“RD16/001/0010”), co-funded by ERDF/ESF, “Investing in your future”, and FTPGB (FTPGB18/SM) to S. Martinez. The Institute of Neurosciences is a “Centre of Excellence Severo Ochoa (SEV-2017-0723)”.

The habenula (Hb), one of the hottest structures in depression, has been widely demonstrated to be involved in the neurobiology of depression. Although the structural and functional abnormalities of Hb have been reported in major depressive disorders (MDD) patients, the role of Hb in treatment response in MDD remains unclear. In this study, resting-state functional connectivity (RSFC) and Granger causality analysis (GCA) were performed to investigate the intrinsic and causal changes of Hb in MDD after ECT. Moreover, support vector classification was applied to find out whether the changed functional and causal connections of Hb can effectively distinguish the MDD patients from healthy controls. The RSFC and GCA identified increased RSFC strength between bilateral Hb and left angular gyrus (AG), decreased causal connectivity strength from left AG to left Hb, from right Hb to left AG, and bidirectional interactions between left and right Hb in MDD patients after ECT. The changed causal connectivities from left AG to left Hb, and from right Hb to left AG were correlated with the changed depression symptoms and impaired delay memory recall performances. Furthermore, the functional and causal connectivities between left AG and bilateral Hb could serve as a biomarker to differentiate MDD from HCs. These results provided new evidence for the importance of Hb in depression and revealed that the interactions between Hb and left AG contribute to ECT response in MDD. Our findings will facilitate the future treatment of depression with the target of Hb in MDD and other brain disorders.

Guanine nucleotide binding protein (G protein) gamma 8 (Gng8) is a subunit of G proteins and expressed in the medial habenula (MHb) and interpeduncular nucleus (IPN). Recent studies have demonstrated that Gng8 is involved in brain development; however, the roles of Gng8 on cognitive function have not yet been addressed. In the present study, we investigated the expression of Gng8 in the brain and found that Gng8 was predominantly expressed in the MHb-IPN circuit of the mouse brain. We generated Gng8 knockout (KO) mice by CRISPR/Cas9 system in order to assess the role of Gng8 on cognitive function. Gng8 KO mice exhibited deficiency in learning and memory in passive avoidance and Morris water maze tests. In addition, Gng8 KO mice significantly reduced long-term potentiation (LTP) in the hippocampus compared to that of wild-type (WT) mice. Furthermore, we observed that levels of acetylcholine (ACh) and choline acetyltransferase (ChAT) in the MHb and IPN of Gng8 KO mice were significantly decreased, compared to WT mice. The administration of nAChR α4β2 agonist A85380 rescued memory impairment in the Gng8 KO mice, suggesting that Gng8 regulates cognitive function via modulation of cholinergic activity. Taken together, Gng8 is a potential therapeutic target for memory-related diseases and/or neurodevelopmental diseases.

Studies of habenula (Hb) function and structure provided evidence of its involvement in psychiatric disorders, including schizophrenia and bipolar disorder. Previous studies using magnetic resonance imaging (manual/semiautomated segmentation) have reported conflicting results. Aiming to improve Hb segmentation reliability and the study of large datasets, we describe a fully automated protocol that was validated against manual segmentations and applied to 3 datasets (childhood/adolescence and adult bipolar disorder and schizophrenia). It achieved reliable Hb segmentation, providing robust volume estimations across a large age range and varying image acquisition parameters. Applying it to clinically relevant datasets, we found smaller Hb volumes in the adult bipolar disorder dataset and larger volumes in the adult schizophrenia dataset compared with healthy control subjects. There are indications that Hb volume in both groups shows deviating developmental trajectories early in life. This technique sets a precedent for future studies, as it allows for fast and reliable Hb segmentation and will be publicly available.

Treatment of fibromyalgia is an unmet medical need; however, its pathogenesis is still poorly understood. In a series of studies, we have demonstrated that some pharmacological treatments reverse generalized chronic pain but do not affect the lack of morphine analgesia in the intermittent cold stress (ICS)-induced fibromyalgia-like pain model in mice. Here we report that repeated intraperitoneal treatments with mirtazapine, which is presumed to disinhibit 5-hydroxytriptamine (5-HT) release and activate 5-HT1 receptor through mechanisms of blocking presynaptic adrenergic α2 and postsynaptic 5-HT2 and 5-HT3 receptors, completely reversed the chronic pain for more than 4 to 5 days after the cessation of treatments. The repeated mirtazapine treatments also recovered the morphine analgesia after the return of nociceptive threshold to the normal level. The microinjection of small interfering RNA (siRNA) adrenergic α2a receptor (ADRA2A) into the habenula, which showed a selective upregulation of α2 receptor gene expression after ICS, reversed the hyperalgesia but did not recover the morphine analgesia. However, both reversal of hyperalgesia and recovery of morphine analgesia were observed when siRNA ADRA2A was administered intracerebroventricularly. As the habenular is reported to be involved in the emotion/reward-related pain and hypoalgesia, these results suggest that mirtazapine could attenuate pain and/or augment hypoalgesia by blocking the habenular α2 receptor after ICS. The recovery of morphine analgesia in the ICS model, on the other hand, seems to be mediated through a blockade of α2 receptor in unidentified brain regions. SIGNIFICANCE STATEMENT: This study reports possible mechanisms underlying the complete reversal of hyperalgesia and recovery of morphine analgesia by mirtazapine, a unique antidepressant with adrenergic α2 and serotonergic receptor antagonist properties, in a type of intermittently repeated stress (ICS)-induced fibromyalgia-like pain model. Habenula, a brain region which is related to the control of emotional pain, was found to play key roles in the antihyperalgesia, whereas other brain regions appeared to be involved in the recovery of morphine analgesia in the ICS model.

The often-overlooked dorsal diencephalic conduction system (DDCS) is a highly conserved pathway linking the basal forebrain and the monoaminergic brainstem. It consists of three key structures; the stria medullaris, the habenula and the fasciculus retroflexus. The first component of the DDCS, the stria medullaris, is a discrete bilateral tract composed of fibers from the basal forebrain that terminate in the triangular eminence of the stalk of the pineal gland, known as the habenula. The habenula acts as a relay hub where incoming signals from the stria medullaris are processed and subsequently relayed to the midbrain and hindbrain monoaminergic nuclei through the fasciculus retroflexus. As a result of its wide-ranging connections, the DDCS has recently been implicated in a wide range of behaviors related to reward processing, aversion and motivation. As such, an understanding of the structure and connections of the DDCS may help illuminate the pathophysiology of neuropsychiatric disorders such as depression, addiction and pain. This is the first review of all three components of the DDCS, the stria medullaris, the habenula and the fasciculus retroflexus, with particular focus on their anatomy, function and development.

Background: Individual differences in behavioural traits influence susceptibility to addictive disorders. Drug addiction involves changes in gene expression, proposed to occur via DNA methylation (DNAm). Aims: To investigate DNAm changes in reward-related brain structures (nucleus accumbens (NAc), lateral habenula (LHb)) in response to cocaine exposure in rats differing in spontaneous exploratory activity. Methods: Rats were observed in the exploration box and categorised as high- (HE) or low explorers (LE). Rats were administered vehicle or cocaine (12 mg/kg, i.p.) for 7 days, followed by a 14-day withdrawal period and cocaine challenge (7 mg/kg); horizontal locomotor activity was recorded. Brain tissue was dissected after 24 h; we analysed messenger RNA (mRNA) and activity levels of epigenetic DNA modifiers (DNMTs and TETs) as well as mRNA and promoter methylation levels at selected genes previously linked to addictive behaviours. Results: The cocaine challenge dose stimulated locomotor activity in both LE- and HE rats only when administered after a repeated cocaine schedule, suggesting development of behavioural sensitisation. Quantitative polymerase chain reaction analyses demonstrated higher basal expression of Dnmt3a, Tet2 and Tet3 in the LHb of HE- vs. LE rats, and we observed differential effects of cocaine exposure on the expression and activity of epigenetic DNA modifiers in the NAc and LHb of HE- and LE rats. Furthermore, cocaine exposure differentially altered promoter methylation levels of A2AR, Ppp1cc, and Taar7b in the NAc and LHb of HE- and LE rats. Conclusions: DNAm might play a role in the HE- and LE phenotypes as well as mediate behavioural effects of LE- and HE rats in response to drugs of abuse.

Significance At high latitudes, about 10% of the population suffers from depression in winter. Although it has become a serious public health issue, its underlying mechanism remains unknown. Interestingly, animals also show depression-like behavior in winter, and small teleosts have emerged as powerful models for the study of complex brain disorders. Here, we show that medaka exhibit decreased sociability and increased anxiety-like behavior under winter-like conditions. Using metabolomic and transcriptomic analyses, we found changes in multiple signaling pathways involved in depression, including the NRF2 antioxidant pathway. Chemical genomics and targeted mutation of the NRF2 gene revealed that seasonal changes in the NRF2 pathway regulate winter depression-like behavior. This study provides insights into the understanding and treatment of seasonally regulated affective disorders., Seasonal changes in the environment lead to depression-like behaviors in humans and animals. The underlying mechanisms, however, are unknown. We observed decreased sociability and increased anxiety-like behavior in medaka fish exposed to winter-like conditions. Whole brain metabolomic analysis revealed seasonal changes in 68 metabolites, including neurotransmitters and antioxidants associated with depression. Transcriptome analysis identified 3,306 differentially expressed transcripts, including inflammatory markers, melanopsins, and circadian clock genes. Further analyses revealed seasonal changes in multiple signaling pathways implicated in depression, including the nuclear factor erythroid-derived 2-like 2 (NRF2) antioxidant pathway. A broad-spectrum chemical screen revealed that celastrol (a traditional Chinese medicine) uniquely reversed winter behavior. NRF2 is a celastrol target expressed in the habenula (HB), known to play a critical role in the pathophysiology of depression. Another NRF2 chemical activator phenocopied these effects, and an NRF2 mutant showed decreased sociability. Our study provides important insights into winter depression and offers potential therapeutic targets involving NRF2.

The past decade has witnessed exponentially growing interest in the lateral habenula (LHb) owing to new discoveries relating to its critical role in regulating negatively motivated behaviour and its implication in major depression. The LHb, sometimes referred to as the brain's 'antireward centre', receives inputs from diverse limbic forebrain and basal ganglia structures, and targets essentially all midbrain neuromodulatory systems, including the noradrenergic, serotonergic and dopaminergic systems. Its unique anatomical position enables the LHb to act as a hub that integrates value-based, sensory and experience-dependent information to regulate various motivational, cognitive and motor processes. Dysfunction of the LHb may contribute to the pathophysiology of several psychiatric disorders, especially major depression. Recently, exciting progress has been made in identifying the molecular and cellular mechanisms in the LHb that underlie negative emotional state in animal models of drug withdrawal and major depression. A future challenge is to translate these advances into effective clinical treatments.

Nicotine addiction, through smoking, is the principal cause of preventable mortality worldwide. Human genome-wide association studies have linked polymorphisms in theCHRNA5-CHRNA3-CHRNB4gene cluster, coding for the α5, α3, and β4 nicotinic acetylcholine receptor (nAChR) subunits, to nicotine addiction. β4*nAChRs have been implicated in nicotine withdrawal, aversion, and reinforcement. Here we show that β4*nAChRs also are involved in non-nicotine-mediated responses that may predispose to addiction-related behaviors. β4 knock-out (KO) male mice show increased novelty-induced locomotor activity, lower baseline anxiety, and motivational deficits in operant conditioning for palatable food rewards and in reward-based Go/No-go tasks. To further explore reward deficits we used intracranial self-administration (ICSA) by directly injecting nicotine into the ventral tegmental area (VTA) in mice. We found that, at low nicotine doses, β4KO self-administer less than wild-type (WT) mice. Conversely, at high nicotine doses, this was reversed and β4KO self-administered more than WT mice, whereas β4-overexpressing mice avoided nicotine injections. Viral expression of β4 subunits in medial habenula (MHb), interpeduncular nucleus (IPN), and VTA of β4KO mice revealed dose- and region-dependent differences: β4*nAChRs in the VTA potentiated nicotine-mediated rewarding effects at all doses, whereas β4*nAChRs in the MHb-IPN pathway, limited VTA-ICSA at high nicotine doses. Together, our findings indicate that the lack of functional β4*nAChRs result in deficits in reward sensitivity including increased ICSA at high doses of nicotine that is restored by re-expression of β4*nAChRs in the MHb-IPN. These data indicate that β4 is a critical modulator of reward-related behaviors.SIGNIFICANCE STATEMENTHuman genetic studies have provided strong evidence for a relationship between variants in theCHRNA5-CHRNA3-CHRNB4gene cluster and nicotine addiction. Yet, little is known about the role of β4 nicotinic acetylcholine receptor (nAChR) subunit encoded by this cluster. We investigated the implication of β4*nAChRs in anxiety-, food reward- and nicotine reward-related behaviors. Deletion of the β4 subunit gene resulted in an addiction-related phenotype characterized by low anxiety, high novelty-induced response, lack of sensitivity to palatable food rewards and increased intracranial nicotine self-administration at high doses. Lentiviral vector-induced re-expression of the β4 subunit into either the MHb or IPN restored a “stop” signal on nicotine self-administration. These results suggest that β4*nAChRs provide a promising novel drug target for smoking cessation.

Objectives: Lateral habenula (LHb) is a key brain structure for mediating behavioural responses to aversive stimuli. It receives presynaptic inputs from ventral pallidum (VP) which relates to rewar...

Opioid use disorder (OUD) is characterized by the development of a negative emotional state that develops after a history of long-term exposure to opioids. OUD represents a true challenge for treatment and relapse prevention. Human research has amply documented emotional disruption in individuals with an opioid substance use disorder, at both behavioral and brain activity levels; however, brain mechanisms underlying this particular facet of OUD are only partially understood. Animal research has been instrumental in elucidating genes and circuits that adapt to long-term opioid use or are modified by acute withdrawal, but research on long-term consequences of opioid exposure and their relevance to the negative affect of OUD remains scarce. In this article, we review the literature with a focus on two questions: 1) Do we have behavioral models in rodents, and what do they tell us? and 2) What do we know about the neuronal populations involved? Behavioral rodent models have successfully recapitulated behavioral signs of the OUD-related negative affect, and several neurotransmitter systems were identified (i.e., serotonin, dynorphin, corticotropin-releasing factor, oxytocin). Circuit mechanisms driving the negative mood of prolonged abstinence likely involve the 5 main reward-aversion brain centers (i.e., nucleus accumbens, bed nucleus of the stria terminalis, amygdala, habenula, and raphe nucleus), all of which express mu opioid receptors and directly respond to opioids. Future work will identify the nature of these mu opioid receptor-expressing neurons throughout reward-aversion networks, characterize their adapted phenotype in opioid abstinent animals, and hopefully position these primary events in the broader picture of mu opioid receptor-associated brain aversion networks.

Near threshold stochastic vestibular stimulation (SVS) enhances postural control and improves other symptoms in neurodegenerative disorders like Parkinson's disease (PD). Improvement of postural control can tentatively be explained by increased responsivity of the vestibular system, but the mechanism behind other effects of near threshold SVS, like improved motor symptoms and cognitive responsiveness in PD, are not known. To better understand the effect of vestibular stimulation on brain activity in PD, c-Fos expression was used as a marker of change in functional activity following SVS in 6-hydroxydopamine (6-OHDA) hemi-lesioned and in sham-lesioned rats. The results were compared with the effect of a single levodopa injection in 6-OHDA hemi-lesioned or saline in sham-lesioned rats. SVS was found to increase c-Fos expression more than levodopa as well as saline in the parvocellular medial vestibular nucleus (MVePC) and more in 6-OHDA hemi-lesioned than in sham-lesioned animals. Furthermore, c-Fos expression increased more in the habenula nucleus (LHb) after SVS than it did after levodopa in 6-OHDA hemilesioned animals and after saline in the sham-lesioned animals. SVS and levodopa induced similar c-Fos expression in several regions, e.g. the caudate putamen (CPu), where saline had no effect. In conclusion there was overlap between SVS-activated areas and levodopa-activated areas, but activation was more pronounced following SVS in the MVePC of 6-OHDA lesioned and in the LHb in both lesioned and sham-lesioned rats.

The habenula is an epithalamic structure implicated in negative reward mechanisms and plays a downstream modulatory role in regulation of dopaminergic and serotonergic functions. Human and animal studies show its hyperactivity in depression which is curtailed by the antidepressant response of ketamine. Deep brain stimulation of habenula (DBS) for major depression have also shown promising results. However, direct neuronal activity of habenula in human studies have rarely been reported. Here, in a cross-sectional design, we acquired both spontaneous resting state and emotional task-induced neuronal recordings from habenula from treatment resistant depressed patients undergoing DBS surgery. We first characterise the aperiodic component (1/f slope) of the power spectrum, interpreted to signify excitation-inhibition balance, in resting and task state. This aperiodicity for left habenula correlated between rest and task and which was significantly positively correlated with depression severity. Time-frequency responses to the emotional picture viewing task show condition differences in beta and gamma frequencies for left habenula and alpha for right habenula. Notably, alpha activity for right habenula was negatively correlated with depression severity. Overall, from direct habenular recordings, we thus show findings convergent with depression models of aberrant excitatory glutamatergic output of the habenula driving inhibition of monoaminergic systems., Medical Research Council Senior Clinical Fellowship (MR/P008747/1)

Up to 20% of women experience stress-related disorders during the postpartum period; however, little is known about the specific neural circuitry by which maternal stress exerts its negative impacts on mental health and maternal caregiving behavior. Theoretically, such a circuitry should serve as an interface between the stress response system and maternal neural network, transmitting stress signals to the neural circuitry that mediates maternal behavior. In this paper, I propose that the lateral habenula (LHb) serves this interface function. Evidence shows that the LHb plays a key role in encoding stress-induced effects and in the pathophysiology of major depression and stress-related anxiety, and thus may play a role in maternal behavior as part of the maternal brain network. I hypothesize that maternal stress acts upon the LHb and two of its major downstream targets, i.e., ventral tegmental area (VTA) and dorsal raphe nucleus (DRN), compromising the maternal care and contributing to postpartum mental disorders. This hypothesis makes three predictions: (1) maternal stress enhances LHb neuronal activity; (2) activation of DRN- and VTA-projecting neurons in the LHb mimics the detrimental effects of maternal stress on maternal behavior; and (3) suppression of DRN- and VTA-projecting neurons in the LHb attenuates the detrimental effects of maternal stress on maternal care in stressed mothers. Confirmation of this hypothesis is expected to enhance our understanding of the neurocircuit mechanisms mediating stress effects on maternal behavior.高达20%的女性在产后会经历应激相关的疾病，但是我们对母性应激引起自身心理健康及母性照顾行为不利影响的具体神经环路却知之甚少。理论上讲，这种环路应该作为应激反应系统和母性神经网络之间的连接点，将应激信号传递到母性行为的神经环路中。在该文中，我提出外侧缰核(Lateral habenula, LHb)起这个连接作用。有证据表明，外侧缰核在编码应激诱导效应、重度抑郁症和应激性焦虑的病理生理学中发挥着关键作用。作为母性神经网络的一部分，外侧缰核也在母性行为的表达中发挥作用。我提出如下假设：母性应激作用于外侧缰核及其两个主要下游靶点，即腹侧被盖区(ventral tegmental area, VTA)和中缝背核(dorsal raphe nucleus, DRN)，从而破坏了母本照顾行为，并导致产后精神障碍。 基于这一假说有三个预测：(1)母性应激增强了外侧缰核神经元的活性；(2)外侧缰核中DRN-和VTA-投射神经元的激活会起到母性应激对母性行为的不利影响相似的效果；(3)在应激的母亲中，外侧缰核中DRN-和VTA-投射神经元的抑制会缓解母性应激对母本照顾行为的不利影响。这一假说的证实有望增强我们对介导应激对母性行为影响的神经环路机制的理解。

The habenula (Hb) is central to adaptive reward- and aversion-driven behaviors, comprising a hub for higher-order processing networks involving the prefrontal cortex (PFC). Despite an established role in preclinical models of cocaine addiction, the translational significance of the Hb and its connectivity with the PFC in humans is unclear. Using diffusion tractography, we detailed PFC structural connectivity with the Hb and two control regions, quantifying tract-specific microstructural features in healthy and cocaine-addicted individuals. White matter was uniquely impaired in PFC-Hb projections in both short-term abstainers and current cocaine users. Abnormalities in this tract further generalized to an independent sample of heroin-addicted individuals and were associated, in an exploratory analysis, with earlier onset of drug use across the addiction subgroups, potentially serving as a predisposing marker amenable for early intervention. Importantly, these findings contextualize a plausible PFC-Hb circuit in the human brain, supporting preclinical evidence for its impairment in cocaine addiction.

The use of deep brain stimulation (DBS) in treatment resistant patients with schizophrenia is of considerable current interest, but where to site the electrodes is challenging. This article reviews rationales for electrode placement in schizophrenia based on evidence for localized brain abnormality in the disorder and the targets that have been proposed and employed to date. The nucleus accumbens and the subgenual anterior cingulate cortex are of interest on the grounds that they are sites of potential pathologically increased brain activity in schizophrenia and so susceptible to the local inhibitory effects of DBS; both sites have been employed in trials of DBS in schizophrenia. Based on other lines of reasoning, the ventral tegmental area, the substantia nigra pars reticulata and the habenula have also been proposed and in some cases employed. The dorsolateral prefrontal cortex has not been suggested, probably reflecting evidence that it is underactive rather than overactive in schizophrenia. The hippocampus is also of theoretical interest but there is no clear functional imaging evidence that it shows overactivity in schizophrenia. On current evidence, the nucleus accumbens may represent the strongest candidate for DBS electrode placement in schizophrenia, with the substantia nigra pars reticulata also showing promise in a single case report; the ventral tegmental area is also of potential interest, though it remains untried.

Fear is an extreme form of aversion that underlies pathological conditions such as panic or phobias. Fear conditioning (FC) is the best-understood model of fear learning. In FC the context and a cue are independently associated with a threatening unconditioned stimulus (US). The lateral habenula (LHb) is a general encoder of aversion. However, its role in fear learning remains poorly understood. Here we studied in rats the role of the LHb in FC using optogenetics and pharmacological tools. We found that inhibition or activation of the LHb during entire FC training impaired both cued and contextual FC. In contrast, optogenetic inhibition of the LHb restricted to cue and US presentation impaired cued but not contextual FC. In either case, simultaneous activation of contextual and cued components of FC, by the presentation of the cue in the training context, recovered the conditioned fear response. Our results support the notion that the LHb is required for the formation of independent contextual and cued fear memories, a previously uncharacterized function for this structure, that could be critical in fear generalization., This work was supported by the following grants: PICT 2016-0034 from the National Agency for Scientific and Technological Promotion of Argentina (ANPCyT) to JM; I-COOP + (CSIC) ref COOPA20198 to PM; NARSAD, Young Investigator Award 2015 (#23861) from the Brain and Behavior Foundation, PICT 2015-2609 and PICT 2017-4594 from the ANPCyT to JP. TS was supported by a predoctoral fellowship from the CONICET. CDP is supported by the Canadian Institutes of Health Research grant PJT169117 and the Natural Science and Engineering Research Council of Canada grant RGPIN-2017-06131, and receives Fonds de Recherche en Santé du Québec (FRQS) Junior-1 salary support.