GABAB receptors induce phasic release from medial habenula terminals through activity-dependent recruitment of release-ready vesicles.

GABAB receptor (GBR) activation inhibits neurotransmitter release in axon terminals in the brain, except in medial habenula (MHb) terminals, which show robust potentiation. However, mechanisms underlying this enigmatic potentiation remain elusive. Here, we report that GBR activation on MHb terminals induces an activity-dependent transition from a facilitating, tonic to a depressing, phasic neurotransmitter release mode. This transition is accompanied by a 4.1-fold increase in readily releasable vesicle pool (RRP) size and a 3.5-fold increase of docked synaptic vesicles (SVs) at the presynaptic active zone (AZ). Strikingly, the depressing phasic release exhibits looser coupling distance than the tonic release. Furthermore, the tonic and phasic release are selectively affected by deletion of synaptoporin (SPO) and Ca2+-dependent activator protein for secretion 2 (CAPS2), respectively. SPO modulates augmentation, the short-term plasticity associated with tonic release, and CAPS2 retains the increased RRP for initial responses in phasic response trains. The cytosolic protein CAPS2 showed a SV-associated distribution similar to the vesicular transmembrane protein SPO, and they were colocalized in the same terminals. We developed the "Flash and Freeze-fracture" method, and revealed the release of SPO-associated vesicles in both tonic and phasic modes and activity-dependent recruitment of CAPS2 to the AZ during phasic release, which lasted several minutes. Overall, these results indicate that GBR activation translocates CAPS2 to the AZ along with the fusion of CAPS2-associated SVs, contributing to persistency of the RRP increase. Thus, we identified structural and molecular mechanisms underlying tonic and phasic neurotransmitter release and their transition by GBR activation in MHb terminals.

A glutamatergic pathway between the medial habenula and the rostral ventrolateral medulla may regulate cardiovascular function in a rat model of post-traumatic stress disorder.

Post-traumatic stress disorder (PTSD) is a serious psychiatric disorder, and there is an association between it and the development of cardiovascular disease. The aim of this study was to explore whether there is a glutamatergic pathway connecting the medial habenula (MHb) with the rostral ventrolateral medulla (RVLM) that is involved in the regulation of cardiovascular function in a rat model of PTSD. Vesicular glutamate transporter 2 (VGLUT2)-positive neurons in the MHb region were retrogradely labeled with FluoroGold (FG) by the double-labeling technique of VGLUT2 immunofluorescence and FG retrograde tracing. Rats belonging to the PTSD model group were microinjected with artificial cerebrospinal fluid (ACSF) or kynurenic acid (KYN; a nonselective glutamate receptor blocker) into their RVLM. Subsequently, with electrical stimulation of MHb, the discharge frequency of the RVLM neurons, heart rate, and blood pressure were found to be significantly increased after microinjection of ACSF using an in vivo multichannel synchronous recording technology; however, this effect was inhibited by injection of KYN. The expression of N-methyl-D-aspartic acid (NMDA) and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor subunits was significantly increased in RVLM of PTSD model rats analyzed by the Western blotting technique. These findings suggest that there may be a glutamatergic pathway connection between MHb and RVLM and that this pathway may be involved in the regulation of cardiovascular function in the PTSD model rats, by acting on NMDA and AMPA receptors in the RVLM.

Unravelling the Role of Habenula Subnuclei on Avoidance Response: Focus on Activation and Neuroinflammation.

Understanding the mechanisms responsible for anxiety disorders is a major challenge. Avoidance behavior is an essential feature of anxiety disorders. The two-way avoidance test is a preclinical model with two distinct subpopulations-the good and poor performers-based on the number of avoidance responses presented during testing. It is believed that the habenula subnuclei could be important for the elaboration of avoidance response with a distinct pattern of activation and neuroinflammation. The present study aimed to shed light on the habenula subnuclei signature in avoidance behavior, evaluating the pattern of neuronal activation using FOS expression and astrocyte density using GFAP immunoreactivity, and comparing control, good and poor performers. Our results showed that good performers had a decrease in FOS immunoreactivity (IR) in the superior part of the medial division of habenula (MHbS) and an increase in the marginal part of the lateral subdivision of lateral habenula (LHbLMg). Poor performers showed an increase in FOS in the basal part of the lateral subdivision of lateral habenula (LHbLB). Considering the astroglial immunoreactivity, the poor performers showed an increase in GFAP-IR in the inferior portion of the medial complex (MHbl), while the good performers showed a decrease in the oval part of the lateral part of the lateral complex (LHbLO) in comparison with the other groups. Taken together, our data suggest that specific subdivisions of the MHb and LHb have different activation patterns and astroglial immunoreactivity in good and poor performers. This study could contribute to understanding the neurobiological mechanisms responsible for anxiety disorders.

Adhesion molecule Amigo2 is involved in the fasciculation process of the fasciculus retroflexus.

BACKGROUND: The fasciculus retroflexus is the prominent efferent pathway from the habenular complex. Medial habenular axons form a core packet whereas lateral habenular axons course in a surrounding shell. Both groups of fibers share the same initial pathway but differ in the final segment of the tract, supposedly regulated by surface molecules. The gene Amigo2 codes for a membrane adhesion molecule with an immunoglobulin-like domain 2 and is selectively expressed in the medial habenula. We present it as a candidate for controlling the fasciculation behavior of medial habenula axons. RESULTS: First, we studied the development of the habenular efferents in an Amigo2 lack of function mouse model. The fasciculus retroflexus showed a variable defasciculation phenotype. Gain of function experiments allowed us to generate a more condensed tract and rescued the Amigo2 knock-out phenotype. Changes in Amigo2 function did not alter the course of habenular fibers. CONCLUSION: We have demonstrated that Amigo2 plays a subtle role in the fasciculation of the fasciculus retroflexus.

Neuropeptide Y in the medial habenula alleviates migraine-like behaviors through the Y1 receptor.

BACKGROUND: Migraine is a highly disabling health burden with multiple symptoms; however, it remains undertreated because of an inadequate understanding of its neural mechanisms. Neuropeptide Y (NPY) has been demonstrated to be involved in the modulation of pain and emotion, and may play a role in migraine pathophysiology. Changes in NPY levels have been found in patients with migraine, but whether and how these changes contribute to migraine is unknown. Therefore, the purpose of this study was to investigate the role of NPY in migraine-like phenotypes. METHODS: Here, we used intraperitoneal injection of glyceryl trinitrate (GTN, 10 mg/kg) as a migraine mouse model, which was verified by light-aversive test, von Frey test, and elevated plus maze test. We then performed whole-brain imaging with NPY-GFP mice to explore the critical regions where NPY was changed by GTN treatment. Next, we microinjected NPY into the medial habenula (MHb), and further infused Y1 or Y2 receptor agonists into the MHb, respectively, to detect the effects of NPY in GTN-induced migraine-like behaviors. RESULTS: GTN effectively triggered allodynia, photophobia, and anxiety-like behaviors in mice. After that, we found a decreased level of GFP+ cells in the MHb of GTN-treated mice. Microinjection of NPY attenuated GTN-induced allodynia and anxiety without affecting photophobia. Furthermore, we found that activation of Y1-but not Y2-receptors attenuated GTN-induced allodynia and anxiety. CONCLUSIONS: Taken together, our data support that the NPY signaling in the MHb produces analgesic and anxiolytic effects through the Y1 receptor. These findings may provide new insights into novel therapeutic targets for the treatment of migraine.

Nectin-2 in general and in the brain.

Nectins are immunoglobulin-like cell adhesion molecules constituting a family with four members, nectin-1, nectin-2, nectin-3, and nectin-4. In the brain, nectin-2 as well as nectin-1 and nectin-3 are expressed whereas nectin-4 is hardly expressed. In the nervous system, physiological functions of nectin-1 and nectin-3, such as synapse formation, mossy fiber trajectory regulation, interneurite affinity, contextual fear memory formation, and stress-related mental disorders, have been revealed. Nectin-2 is ubiquitously expressed in non-neuronal tissues and various nectin-2 functions in non-nervous systems have been extensively investigated, but nectin-2 functions in the brain have not been revealed until recently. Recent findings have revealed that nectin-2 is expressed in the specific areas of the brain and plays important roles, such as homeostasis of astrocytes and neurons and the formation of synapses. Moreover, a single nucleotide polymorphism in the human NECTIN2 gene is associated with Alzheimer's disease. We here summarize recent progress in our understanding of nectin-2 functions in the brain.

TMEM16A expression in cholinergic neurons of the medial habenula mediates anxiety-related behaviors.

TMEM16A, a Ca2+ -activated Cl- channel, is known to modulate the excitability of various types of cells; however, its function in central neurons is largely unknown. Here, we show the specific expression of TMEM16A in the medial habenula (mHb) via RNAscope in situ hybridization, immunohistochemistry, and electrophysiology. When TMEM16A is ablated in the mHb cholinergic neurons (TMEM16A cKO mice), the slope of after-hyperpolarization of spontaneous action potentials decreases and the firing frequency is reduced. Reduced mHb activity also decreases the activity of the interpeduncular nucleus (IPN). Moreover, TMEM16A cKO mice display anxiogenic behaviors and deficits in social interaction without despair-like phenotypes or cognitive dysfunctions. Finally, chemogenetic inhibition of mHb cholinergic neurons using the DREADD (Designer Receptors Exclusively Activated by Designer Drugs) approach reveals similar behavioral phenotypes to those of TMEM16A cKO mice. We conclude that TMEM16A plays a key role in anxiety-related behaviors regulated by mHb cholinergic neurons and could be a potential therapeutic target against anxiety-related disorders.

The orphan receptor GPR139 signals via Gq/11 to oppose opioid effects.

The interplay between G protein-coupled receptors (GPCRs) is critical for controlling neuronal activity that shapes neuromodulatory outcomes. Recent evidence indicates that the orphan receptor GPR139 influences opioid modulation of key brain circuits by opposing the actions of the µ-opioid receptor (MOR). However, the function of GPR139 and its signaling mechanisms are poorly understood. In this study, we report that GPR139 activates multiple heterotrimeric G proteins, including members of the Gq/11 and Gi/o families. Using a panel of reporter assays in reconstituted HEK293T/17 cells, we found that GPR139 functions via the Gq/11 pathway and thereby distinctly regulates cellular effector systems, including stimulation of cAMP production and inhibition of G protein inward rectifying potassium (GIRK) channels. Electrophysiological recordings from medial habenular neurons revealed that GPR139 signaling via Gq/11 is necessary and sufficient for counteracting MOR-mediated inhibition of neuronal firing. These results uncover a mechanistic interplay between GPCRs involved in controlling opioidergic neuromodulation in the brain.

Genetic inactivation of the sigma-1 chaperone protein results in decreased expression of the R2 subunit of the GABA-B receptor and increased susceptibility to seizures.

There is a growing body of evidence demonstrating the significant involvement of the sigma-1 chaperone protein in the modulation of seizures. Several sigma-1 receptor (Sig1R) ligands have been demonstrated to regulate the seizure threshold in acute and chronic seizure models. However, the mechanism by which Sig1R modulates the excitatory and inhibitory pathways in the brain has not been elucidated. The aim of this study was to compare the susceptibility to seizures of wild type (WT) and Sig1R knockout (Sig1R-/-) mice in intravenous pentylenetetrazol (PTZ) and (+)-bicuculline (BIC) infusion-induced acute seizure and Sig1R antagonist NE-100-induced seizure models. To determine possible molecular mechanisms, we used quantitative PCR, Western blotting and immunohistochemistry to assess the possible involvement of several seizure-related genes and proteins. Peripheral tissue contractile response of WT and Sig1R-/- mice was studied in an isolated vasa deferentia model. The most important finding was the significantly decreased expression of the R2 subunit of the GABA-B receptor in the hippocampus and habenula of Sig1R-/- mice. Our results demonstrated that Sig1R-/- mice have decreased thresholds for PTZ- and BIC-induced tonic seizures. In the NE-100-induced seizure model, Sig1R-/- animals demonstrated lower seizure scores, shorter durations and increased latency times of seizures compared to WT mice. Sig1R-independent activities of NE-100 included downregulation of the gene expression of iNOS and GABA-A γ2 and inhibition of KCl-induced depolarization in both WT and Sig1R-/- animals. In conclusion, the results of this study indicate that the lack of Sig1R resulted in decreased expression of the R2 subunit of the GABA-B receptor and increased susceptibility to seizures. Our results confirm that Sig1R is a significant molecular target for seizure modulation and warrants further investigation for the development of novel anti-seizure drugs.

The blueprint of the vertebrate forebrain - With special reference to the habenulae.

The medial and lateral habenulae are conserved throughout vertebrate evolution, and form an integrated part in the forebrain control of behavior together with the basal ganglia, the dopamine and serotonin systems and cortex. The lateral habenula plays a role in the control of dopamine activity in the context of aversive behavior and the converse, a reward situation. These circuits are important for a value-based evaluation of the success of prior actions. The medial habenula is involved in mediating escape and freezing behavior. These structures are reviewed with a focus on the lamprey, belonging to the oldest group of now living vertebrate, showing that most aspects of the habenular structure and function have been conserved throughout vertebrate phylogeny.

Unique synaptic topography of crest-type synapses in the interpeduncular nucleus.

Neurons in the central nervous system display a great diversity of synaptic architecture. While much of our knowledge on the excitatory synapse morphology derives from the prototypical asymmetric synapses, little has been studied about the atypical crest-type synapse that exists in the restricted brain regions. Here, we used focused ion beam scanning electron microscopy (FIB/SEM) to image a neuropil volume of interpeduncular nucleus (IPN) and manually reconstructed several dendrites to obtain an insight about the topography and quantitative features of crest synapses. Three-dimensional reconstruction showed numerous U-shaped structures protruding from the IPN dendrites. On either faces of the U-shaped structure, a pair of crest synapses are aligned in parallel such that there exists a positive correlation between the postsynaptic density (PSD) area of synapses that participate in pair formation. Interestingly, mitochondria are excluded from the site of crest synapses. Several presynaptic axons run through the hollow, cylindrical space of the U-shape grooves such that the plasma membrane of the axon and the dendrite are organized in a tight opposition without any intervening glial membrane. Unlike the peculiar dendritic morphology, IPN neurons possess typical somatic morphology with an oval, centrally located nucleus. In conclusion, our data reveals a hitherto unknown unique topographical feature of crest synapses in the IPN.

Interaction of nectin-2α with the auxiliary protein of the voltage-gated A-type K+ channel Kv4.2 dipeptidyl aminopeptidase-like protein at the boundary between the adjacent somata of clustered cholinergic neurons in the medial habenula.

The medial habenula (MHb) receives septal inputs and sends efferents to the interpeduncular nucleus and is implicated in stress, depression, memory, and nicotine withdrawal syndrome. We previously showed by immunofluorescence microscopy that the cell adhesion molecule nectin-2α is expressed in the cholinergic neurons in the developing and adult mouse MHbs and localized at the boundary between the adjacent somata of clustered cholinergic neurons where the voltage-gated A-type K+ channel Kv4.2 is localized. We further showed by immunoelectron microscopy that Kv4.2 is localized at the membrane specializations (MSs) whereas nectin-2α is localized mostly outside of these MSs. In addition, we showed that genetic ablation of nectin-2 delays the localization of Kv4.2 at the MSs in the developing MHb. We investigated here how nectin-2α regulates this localization of Kv4.2 at the MSs. In vitro biochemical analysis revealed that nectin-2α interacted with the auxiliary protein of Kv4.2 dipeptidyl aminopeptidase-like protein 6 (DPP6), but not with Kv4.2 or another auxiliary protein Kv channel-interacting protein 1 (KChIP1). Immunofluorescence microscopy analysis showed that DPP6 was colocalized with nectin-2α at the boundary between the adjacent somata of the clustered cholinergic neurons in the developing and adult MHbs. Immunoelectron microscopy analysis on this boundary revealed that DPP6 was localized both at the inside and the outside of the MSs. Genetic ablation of nectin-2 did not affect the localization of DPP6 at the boundary between the adjacent somata of the clustered cholinergic neurons in the developing and adult MHbs. These results indicate that nectin-2α interacts with DPP6 but regulates the localization of Kv4.2 at the MSs in a DPP6-independent manner.

Medial habenula cholinergic signaling regulates cocaine-associated relapse-like behavior.

Propensity to relapse, even following long periods of abstinence, is a key feature in substance use disorders. Relapse and relapse-like behaviors are known to be induced, in part, by re-exposure to drug-associated cues. Yet, while many critical nodes in the neural circuitry contributing to relapse have been identified and studied, a full description of the networks driving reinstatement of drug-seeking behaviors is lacking. One area that may provide further insight to the mechanisms of relapse is the habenula complex, an epithalamic region composed of lateral and medial (MHb) substructures, each with unique cell and target populations. Although well conserved across vertebrate species, the functions of the MHb are not well understood. Recent research has demonstrated that the MHb regulates nicotine aversion and withdrawal. However, it remains undetermined whether MHb function is limited to nicotine and aversive stimuli or if MHb circuit regulates responses to other drugs of abuse. Advances in circuit-level manipulations now allow for cell-type and temporally specific manipulations during behavior, specifically in spatially restrictive brain regions, such as the MHb. In this study, we focus on the response of the MHb to reinstatement of cocaine-associated behavior, demonstrating that cocaine-primed reinstatement of conditioned place preference engages habenula circuitry. Using chemogenetics, we demonstrate that MHb activity is sufficient to induce reinstatement behavior. Together, these data identify the MHb as a key hub in the circuitry underlying reinstatement and may serve as a target for regulating relapse-like behaviors.

ErbB4 Receptors in the Medial Habenula Regulate Contextual Fear Memory.

The Medial Habenular (MHb) and the Lateral Habenular nuclei are 2 main parts of the habenular complex (Hb). Recent studies showed that MHb plays an important role in memory, and in the expression of ErbB4. However, the expression of MHb ErbB4 receptor and its role in fear memory is not well understood. In this study, western blotting and quantitative real-time polymerase chain reaction were used to assess the protein and mRNA levels of ErbB4 in the process of contextual fear conditioning. A pharmacological approach was used to block and stimulate the ErbB4 receptor. Contextual fear conditioning tests induced a significant increase on the expression of ErbB4 at various times in the Hb and the MHb. Moreover, the blockade and stimulation of MHb ErbB4 receptors did not affect the fear formation but impaired and improved the contextual-dependent fear expression. Furthermore, in vitro electrophysiological recordings showed that the blockade of the MHb ErbB4 receptor reduced the presynaptic gamma-amino butyric acid release. ErbB4 is a susceptible gene for schizophrenia and the above findings may provide new insights into the mechanisms of fear-related responses.

Neuropeptidomics of the Rat Habenular Nuclei.

Conserved across vertebrates, the habenular nuclei are a pair of small symmetrical structures in the epithalamus. The nuclei functionally link the forebrain and midbrain by receiving input from and projecting to several brain regions. Each habenular nucleus comprises two major asymmetrical subnuclei, the medial and lateral habenula. These subnuclei are associated with different physiological processes and disorders, such as depression, nicotine addiction, and encoding aversive stimuli or omitting expected rewarding stimuli. Elucidating the functions of the habenular nuclei at the molecular level requires knowledge of their neuropeptide complement. In this work, three mass spectrometry (MS) techniques-liquid chromatography (LC) coupled to Orbitrap tandem MS (MS/MS), LC coupled to Fourier transform (FT)-ion cyclotron resonance (ICR) MS/MS, and matrix-assisted laser desorption/ionization (MALDI) FT-ICR MS-were used to uncover the neuropeptide profiles of the rodent medial and lateral habenula. With the assistance of tissue stabilization and bioinformatics, a total of 262 and 177 neuropeptides produced from 27 and 20 prohormones were detected and identified from the medial and lateral habenula regions, respectively. Among these neuropeptides, 136 were exclusively found in the medial habenula, and 51 were exclusively expressed in the lateral habenula. Additionally, novel sites of sulfation, a rare post-translational modification, on the secretogranin I prohormone are identified. The results demonstrate that these two small brain nuclei have a rich and differentiated peptide repertoire, with this information enabling a range of follow-up studies.

Optical probing of acetylcholine receptors on neurons in the medial habenula with a novel caged nicotine drug analogue.

KEY POINTS: A new caged nicotinic acetylcholine receptor (nAChR) agonist was developed, ABT594, which is photolysed by one- and two-photon excitation. The caged compound is photolysed with a quantum yield of 0.20. One-photon uncaging of ABT594 elicited large currents and Ca2+ transients at the soma and dendrites of medial habenula (MHb) neurons of mouse brain slices. Unexpectedly, uncaging of ABT594 also revealed highly Ca2+ -permeable nAChRs on axons of MHb neurons. ABSTRACT: Photochemical release of neurotransmitters has been instrumental in the study of their underlying receptors, with acetylcholine being the exception due to its inaccessibility to photochemical protection. We caged a nicotinic acetylcholine receptor (nAChR) agonist, ABT594, via its secondary amine functionality. Effective photolysis could be carried out using either one- or two-photon excitation. Brief flashes (0.5-3.0 ms) of 410 nm light evoked large currents and Ca2+ transients on cell bodies and dendrites of medial habenula (MHb) neurons. Unexpectedly, photorelease of ABT594 also revealed nAChR-mediated Ca2+ signals along the axons of MHb neurons.

Development- and experience-dependent plasticity in the dorsomedial habenula.

Of the two major subdivisions of the habenula, the medial and lateral nuclei, the medial habenula is the least understood in terms of synaptic transmission, intrinsic properties and plasticity. The medial habenula (MHb) is composed of glutamatergic neurons which receive the majority of their inputs from the septal region and project predominantly to the interpeduncular nucleus (IPN). To understand the synaptic transmission, we studied both glutamatergic and GABAergic synaptic transmission in the dorsal region of the medial habenula (dMHb). While glutamatergic transmission dominates during early development, an attenuation of glutamatergic transmission and an enhancement of GABAergic transmission occur during development leading into adulthood. Furthermore, as reported previously, GABAA receptor-mediated transmission is excitatory in the adult dMHb, which is consistent with the reduced expression of the K-Cl co-transporter KCC2. Given the potential role of the dMHb in aversive behaviors, we examined whether fear conditioning or exposure to foot shock affects excitability in dMHb neurons. We observed a suppression of the excitability of dMHb neurons in mice that either underwent fear conditioning or were exposed to foot shock. Furthermore, we observed a suppression of GABAergic but not glutamatergic transmission in the dMHb neurons following fear conditioning. These results suggest that aversive experience produces a suppression of the dMHb neuronal activity. Given that the medial habenula is upstream of the median raphe nucleus which is believed to be involved in the negative regulation of aversive memory, the suppression of dMHb neurons following an aversive experience might play a role in strengthening of aversive memories.

Nicotine enhances excitability of medial habenular neurons via facilitation of neurokinin signaling.

The medial habenula (MHb) densely expresses nicotinic acetylcholine receptors (nAChRs) and participates in nicotine-related behaviors such as nicotine withdrawal and regulating nicotine intake. Although specific nAChR subunits are identified as being involved in withdrawal behavior, the cellular mechanisms through which nicotine acts to cause this aversive experience is unclear. Here, we demonstrate an interaction between the nicotinic and neurokinin signaling systems that may form the basis for some symptoms experienced during nicotine withdrawal. Using patch-clamp electrophysiology in mouse brain slices, we show that nicotine (1 µm) increases intrinsic excitability in MHb neurons. This nicotine-induced phenomenon requires α5-containing nAChRs and depends on intact neurokinin signaling. The effect is blocked by preincubation with neurokinin 1 (NK1; L-732138, 10 µm) and NK3 (SB222200, 2 µm) antagonists and mimicked by NK1 (substance P, 100 nm) and NK3 (neurokinin B [NKB], 100 nm) agonists. Microinjections (1 µl) of L-732138 (50 nm) and SB222200 (100 nm) into the MHb induces withdrawal behavior in chronic nicotine-treated (8.4 mg/kg/d, 2 weeks) mice. Conversely, withdrawal behavior is absent with analogous microinjections into the lateral habenula of nicotine-treated mice or in mice chronically treated with a vehicle solution. Further, chronic nicotine reduces nicotine's acute modulation of intrinsic excitability while sparing modulation by NKB. Our work elucidates the interplay between two neuromodulatory signaling systems in the brain through which nicotine acts to influence intrinsic excitability. More importantly, we document a neuroadaptation of this mechanism to chronic nicotine exposure and implicate these mechanisms collectively in the emergence of nicotine withdrawal behavior.

Role of the dorsal medial habenula in the regulation of voluntary activity, motor function, hedonic state, and primary reinforcement.

The habenular complex in the epithalamus consists of distinct regions with diverse neuronal populations. Past studies have suggested a role for the habenula in voluntary exercise motivation and reinforcement of intracranial self-stimulation but have not assigned these effects to specific habenula subnuclei. Here, we have developed a genetic model in which neurons of the dorsal medial habenula (dMHb) are developmentally eliminated, via tissue-specific deletion of the transcription factor Pou4f1 (Brn3a). Mice with dMHb lesions perform poorly in motivation-based locomotor behaviors, such as voluntary wheel running and the accelerating rotarod, but show only minor abnormalities in gait and balance and exhibit normal levels of basal locomotion. These mice also show deficits in sucrose preference, but not in the forced swim test, two measures of depression-related phenotypes in rodents. We have also used Cre recombinase-mediated expression of channelrhodopsin-2 and halorhodopsin to activate dMHb neurons or silence their output in freely moving mice, respectively. Optical activation of the dMHb in vivo supports intracranial self-stimulation, showing that dMHb activity is intrinsically reinforcing, whereas optical silencing of dMHb outputs is aversive. Together, our findings demonstrate that the dMHb is involved in exercise motivation and the regulation of hedonic state, and is part of an intrinsic reinforcement circuit.

Neuronal Excitability in the Medial Habenula and Ventral Tegmental Area Is Differentially Modulated by Nicotine Dosage and Menthol in a Sex-Specific Manner.

The medial habenula (MHb) has been identified as the limiting factor for nicotine intake and facilitating nicotine withdrawal. However, few studies have assessed MHb neuronal excitability in response to nicotine, and, currently, a gap in knowledge is present for finding behavioral correlates to neuronal excitability in the region. Moreover, no study to date has evaluated sex or nicotine dosage as factors of excitability in the MHb. Here, we utilized an e-vape self-administration (EVSA) model to determine differences between sexes with different nicotine dosages ± menthol. Following this paradigm, we employed patch-clamp electrophysiology to assess key metrics of MHb neuronal excitability in relation to behavioral endpoints. We observed female mice self-administered significantly more than males, regardless of dosage. We also observed a direct correlation between self-administration behavior and MHb excitability with low-dose nicotine + menthol in males. Conversely, a high dose of nicotine ± menthol yields an inverse correlation between excitability and self-administration behavior in males only. In addition, intrinsic excitability in the ventral tegmental area (VTA) does not track with the amount of nicotine self-administered. Rather, they correlate to the active/inactive discrimination of mice. Using fast-scan cyclic voltammetry, we also observed that dopamine release dynamics are linked to reinforcement-related behavior in males and motivation-related behaviors in females. These results point to a sex-specific difference in the activity of the MHb and VTA leading to distinct differences in self-administration behavior. His could lend evidence to clinical observations of smoking and nicotine-use behavior differing between males and females.