Whole-brain connections of glutamatergic neurons in the mouse lateral habenula in both sexes.

BACKGROUND: The lateral habenula (LHb) is an epithalamus nucleus that is evolutionarily conserved and involved in various physiological functions, such as encoding value signals, integrating emotional information, and regulating related behaviors. The cells in the LHb are predominantly glutamatergic and have heterogeneous functions in response to different stimuli. The circuitry connections of the LHb glutamatergic neurons play a crucial role in integrating a wide range of events. However, the circuitry connections of LHb glutamatergic neurons in both sexes have not been thoroughly investigated. METHODS: In this study, we injected Cre-dependent retrograde trace virus and anterograde synaptophysin-labeling virus into the LHb of adult male and female Vglut2-ires-Cre mice, respectively. We then quantitatively analyzed the input and output of the LHb glutamatergic connections in both the ipsilateral and contralateral whole brain. RESULTS: Our findings showed that the inputs to LHbvGlut2 neurons come from more than 30 brain subregions, including the cortex, striatum, pallidum, thalamus, hypothalamus, midbrain, pons, medulla, and cerebellum with no significant differences between males and females. The outputs of LHbvGlut2 neurons targeted eight large brain regions, primarily focusing on the midbrain and pons nuclei, with distinct features in presynaptic bouton across different brain subregions. While correlation and cluster analysis revealed differences in input and collateral projection features, the input-output connection pattern of LHbvGlut2 neurons in both sexes was highly similar. CONCLUSIONS: This study provides a systematic and comprehensive analysis of the input and output connections of LHbvGlut2 neurons in male and female mice, shedding light on the anatomical architecture of these specific cell types in the mouse LHb. This structural understanding can help guide further investigations into the complex functions of the LHb.

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

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

Relapse to cocaine seeking is regulated by medial habenula NR4A2/NURR1 in mice.

Drugs of abuse can persistently change the reward circuit in ways that contribute to relapse behavior, partly via mechanisms that regulate chromatin structure and function. Nuclear orphan receptor subfamily4 groupA member2 (NR4A2, also known as NURR1) is an important effector of histone deacetylase 3 (HDAC3)-dependent mechanisms in persistent memory processes and is highly expressed in the medial habenula (MHb), a region that regulates nicotine-associated behaviors. Here, expressing the Nr4a2 dominant negative (Nurr2c) in the MHb blocks reinstatement of cocaine seeking in mice. We use single-nucleus transcriptomics to characterize the molecular cascade following Nr4a2 manipulation, revealing changes in transcriptional networks related to addiction, neuroplasticity, and GABAergic and glutamatergic signaling. The network controlled by NR4A2 is characterized using a transcription factor regulatory network inference algorithm. These results identify the MHb as a pivotal regulator of relapse behavior and demonstrate the importance of NR4A2 as a key mechanism driving the MHb component of relapse.

The lateral habenula: A hub for value-guided behavior.

The habenula is an evolutionarily highly conserved diencephalic brain region divided into two major parts, medial and lateral. Over the past two decades, studies of the lateral habenula (LHb), in particular, have identified key functions in value-guided behavior in health and disease. In this review, we focus on recent insights into LHb connectivity and its functional relevance for different types of aversive and appetitive value-guided behavior. First, we give an overview of the anatomical organization of the LHb and its main cellular composition. Next, we elaborate on how distinct LHb neuronal subpopulations encode aversive and appetitive stimuli and on their involvement in more complex decision-making processes. Finally, we scrutinize the afferent and efferent connections of the LHb and discuss their functional implications for LHb-dependent behavior. A deepened understanding of distinct LHb circuit components will substantially contribute to our knowledge of value-guided behavior.

HCN1 in the lateral habenula contributes to morphine abstinence-induced anxiety-like behaviors in male mice.

Anxiety disorders, common symptoms during morphine withdrawal, are important negative reinforcement factors leading to relapse. Lateral habenula serves as a negative reinforcement center, however its role in morphine withdrawal-induced anxiety remains uncovered. The hyperpolarization activated cyclic nucleotide-gated (HCN) channels have been reported to be important in emotion processing and addiction, but the role of HCN in anxiety from drug protracted abstinence remains elusive. In this study, by using behavioral test, Western blot, immunofluorescence, electrophysiology and virus-mediated regulation of HCN, we found that: (1) Intra-LHb injection of selective HCN blocker ZD7288 alleviated anxiety-like behaviors in morphine protracted abstinent male mice. (2) The LHb neuronal activity was increased by morphine protracted abstinence. (3) LHb neurons were inhibited by ZD7288 and activated by 8-Br-cAMP respectively, which were enhanced by morphine withdrawal. (4) HCN1 in the LHb was upregulated by morphine withdrawal. (5) Virus-mediated overexpression of HCN1 in the LHb was sufficient to produce anxiety-like behaviors in male mice and virus-mediated knockdown of HCN1 in the LHb prevented the anxiety-like behaviors in male mice. The findings reveal that selective blockade of HCN1 channels in the LHb may represent a therapeutic approach to morphine withdrawal-induced anxiety.

A neural mechanism for conserved value computations integrating information and rewards.

Behavioral and economic theory dictate that we decide between options based on their values. However, humans and animals eagerly seek information about uncertain future rewards, even when this does not provide any objective value. This implies that decisions are made by endowing information with subjective value and integrating it with the value of extrinsic rewards, but the mechanism is unknown. Here, we show that human and monkey value judgements obey strikingly conserved computational principles during multi-attribute decisions trading off information and extrinsic reward. We then identify a neural substrate in a highly conserved ancient structure, the lateral habenula (LHb). LHb neurons signal subjective value, integrating information's value with extrinsic rewards, and the LHb predicts and causally influences ongoing decisions. Neurons in key input areas to the LHb largely signal components of these computations, not integrated value signals. Thus, our data uncover neural mechanisms of conserved computations underlying decisions to seek information about the future.

The preoptic area and dorsal habenula jointly support homeostatic navigation in larval zebrafish.

Animals must maintain physiological processes within an optimal temperature range despite changes in their environment. Through behavioral assays, whole-brain functional imaging, and neural ablations, we show that larval zebrafish, an ectothermic vertebrate, achieves thermoregulation through homeostatic navigation-non-directional and directional movements toward the temperature closest to its physiological setpoint. A brain-wide circuit encompassing several brain regions enables this behavior. We identified the preoptic area of the hypothalamus (PoA) as a key brain structure in triggering non-directional reorientation when thermal conditions are worsening. This result shows an evolutionary conserved role of the PoA as principal thermoregulator of the brain also in ectotherms. We further show that the habenula (Hb)-interpeduncular nucleus (IPN) circuit retains a short-term memory of the sensory history to support the generation of coherent directed movements even in the absence of continuous sensory cues. We finally provide evidence that this circuit may not be exclusive for temperature but may convey a more abstract representation of relative valence of physiologically meaningful stimuli regardless of their specific identity to enable homeostatic navigation.

Lateral Habenula Neurons Signal Cold Aversion and Participate in Cold Aversion.

The aversion to cold is a fundamental motivated behavior that contributes to the body temperature homeostasis. However, the involvement of the lateral habenula (LHb) as a regulatory hub for negative emotions in this physiological process remains uninvestigated. In this study, we demonstrate an elevation in the population activity of LHb neurons following exposure to cold stimuli. Additionally, we establish the necessity of Vglut2-expressing neurons within the LHb for the encoding of cold aversion behaviors. Furthermore, we have elucidated a neural circuit from excitatory neurons of the dorsomedial hypothalamus (DMH) to LHb that plays a crucial role in this progress. Manipulation of the DMH-LHb circuit has a significant impact on cold aversion behavior in mice. It is worth noting that this circuit does not exhibit any noticeable effects on autonomic thermoregulation or depression-like behavior. The identification of these neural mechanisms involved in behavioral thermoregulation provides a promising avenue for future research.

The thalamic reticular nucleus-lateral habenula circuit regulates depressive-like behaviors in chronic stress and chronic pain.

Chronic stress and chronic pain are two major predisposing factors to trigger depression. Enhanced excitatory input to the lateral habenula (LHb) has been implicated in the pathophysiology of depression. However, the contribution of inhibitory transmission remains unclear. Here, we dissect an inhibitory projection from the sensory thalamic reticular nucleus (sTRN) to the LHb, which is activated by acute aversive stimuli. However, chronic restraint stress (CRS) weakens sTRN-LHb synaptic strength, and this synaptic attenuation is indispensable for CRS-induced LHb neural hyperactivity and depression onset. Moreover, artificially inhibiting the sTRN-LHb circuit induces depressive-like behaviors in healthy mice, while enhancing this circuit relieves depression induced by both chronic stress and chronic pain. Intriguingly, neither neuropathic pain nor comorbid mechanical hypersensitivity in chronic stress is affected by this pathway. Altogether, our study demonstrates an sTRN-LHb circuit in establishing and modulating depression, thus shedding light on potential therapeutic targets for preventing or managing depression.

Lateral habenula deep brain stimulation alleviates depression-like behaviors and reverses the oscillatory pattern in the nucleus accumbens in an animal model of depression.

Depression is a series of symptoms that influence mood, thinking, and behavior and create unpleasant emotions like hopelessness and apathy. Treatment-resistant depression (TRD) affects 30 % of depression patients despite the availability of several non-invasive therapies. Deep brain stimulation (DBS) is a novel therapy for TRD. The aim of the current study was to evaluate the effect of LHb-DBS by recording local field potentials (LFP) and conducting behavioral experiments. Thirty-two mature male Wistar rats were randomly divided into four groups: control, chronic mild stress (CMS), CMS+DBS, and DBS. After surgery and electrode placement in the lateral habenula (LHb), nucleus accumbens (NAc), and prelimbic cortex (PrL), the CMS protocol was applied for 3 weeks to create depression-like models. The open field test (OFT), sucrose preference test (SPT), and forced swim test (FST) were also performed. In the DBS groups, the LHb area was stimulated for four consecutive days. Finally, on the 22nd day, LFP was recorded from the NAc and PrL and analyzed using MATLAB software. Analyzing the findings using ANOVA and P-values ≤ 0.05 was considered. LHb-DBS alleviated depression-like behaviors in chronic moderate stress model rats (P ≤ 0.05). Three weeks of CMS enhanced almost all band powers in the NAc, while LHb-DBS decreased the power of the theta, alpha, beta, and gamma bands in the NAc (P ≤ 0.05), and the low-gamma band in the PrL. CMS also boosted the NAc-PrL coherence in low-frequency bands, while LHb-DBS increased beta and low gamma band coherence (P ≤ 0.05). In sum, the results of the present study showed that depression enhances low-frequency coherence between NAc and PrL cortex. Depression also potentiates many brain oscillations in the NAc, which can be mainly reversed by LHb-DBS.

Investigating Habenula Functional Connectivity and Reward-Related Activity in Obesity Using Human Connectome Project Data.

Introduction: The habenula, a brain region involved in aversion, might negatively modulate caloric intake. Functional magnetic resonance imaging (fMRI) studies reported associations between weight loss and habenula functional connectivity. However, whether habenula resting-state functional connectivity (rsFC) and reward-related activity are altered in obesity is yet unknown. Methods: Using data from the Human Connectome Project, we included 300 subjects with various body mass indexes (BMIs) and a healthy long-term blood glucose (hemoglobin A1c [HbA1c]). In addition, we investigated a potential BMI × HbA1c interaction in a separate cohort including subjects with prediabetes (n = 72). Habenula rsFC was assessed using a region of interest (ROI)-to-ROI analysis. Furthermore, a separate analysis using gambling task fMRI data focused on reward-related habenula activity. Results: We did not find an association between BMI and habenula rsFC for any of the ROIs. For the exploratory analysis of the BMI × HbA1c effect, a significant interaction effect was found for the habenula-ventral tegmental area (VTA) connection, but this did not survive multiple comparisons correction. Monetary punishment compared with reward activated the bilateral habenula in the BMI sample, but this activity was not associated with BMI. Discussion: In conclusion, we did not find evidence for an association between BMI and habenula rsFC or reward-related activity. However, there might be an interaction between BMI and HbA1c for the habenula-VTA rsFC, suggestive of a role of the habenula in glucose regulation. Future studies should focus on metabolic parameters in their experimental design to confirm our findings and explore the precise role of the habenula in metabolism.

Feed-forward Activation of Habenula Cholinergic Neurons by Local Acetylcholine.

While the functional and behavioral role of the medial habenula (MHb) is still emerging, recent data indicate an involvement of this nuclei in regulating mood, aversion, and addiction. Unique to the MHb is a large cluster of cholinergic neurons that project to the interpeduncular nucleus and densely express acetylcholine receptors (AChRs) suggesting that the activity of these cholinergic neurons may be regulated by ACh itself. Whether endogenous ACh from within the habenula regulates cholinergic neuron activity has not been demonstrated. Supporting a role for ACh in modulating MHb activity, acetylcholinesterase inhibitors increased the firing rate of MHb cholinergic neurons in mouse habenula slices, an effect blocked by AChR antagonists and mediated by ACh which was detected via expressing fluorescent ACh sensors in MHb in vivo. To test if cholinergic afferents innervate MHb cholinergic neurons, we used anterograde and retrograde viral tracing to identify cholinergic inputs. Surprisingly, tracing experiments failed to detect cholinergic inputs into the MHb, including from the septum, suggesting that MHb cholinergic neurons may release ACh within the MHb to drive cholinergic activity. To test this hypothesis, we expressed channelrhodopsin in a portion of MHb cholinergic neurons while recording from non-opsin-expressing neurons. Light pulses progressively increased activity of MHb cholinergic neurons indicating feed-forward activation driven by MHb ACh release. These data indicate MHb cholinergic neurons may utilize a unique feed-forward mechanism to synchronize and increase activity by releasing local ACh.

Tet2 acts in the lateral habenula to regulate social preference in mice.

The lateral habenula (LHb) has been considered a moderator of social behaviors. However, it remains unknown how LHb regulates social interaction. Here, we show that the hydroxymethylase Tet2 is highly expressed in the LHb. Tet2 conditional knockout (cKO) mice exhibit impaired social preference; however, replenishing Tet2 in the LHb rescues social preference impairment in Tet2 cKO mice. Tet2 cKO alters DNA hydroxymethylation (5hmC) modifications in genes that are related to neuronal functions, as is confirmed by miniature two-photon microscopy data. Further, Tet2 knockdown in the glutamatergic neurons of LHb causes impaired social behaviors, but the inhibition of glutamatergic excitability restores social preference. Mechanistically, we identify that Tet2 deficiency reduces 5hmC modifications on the Sh3rf2 promoter and Sh3rf2 mRNA expression. Interestingly, Sh3rf2 overexpression in the LHb rescues social preference in Tet2 cKO mice. Therefore, Tet2 in the LHb may be a potential therapeutic target for social behavior deficit-related disorders such as autism.

Nuclei and tracts in the epithalamus of crocodiles.

The epithalamus, an area of the dorsal diencephalon found in all vertebrates, consists of the habenula, the subhabenular nuclei, and associated tracts. The habenula is itself divisible into two parts-a medial and a lateral nucleus differing in their inputs, outputs, and cellular morphology. The medial component is related to the limbic system and serotonergic raphe, while the lateral nucleus is more interconnected with the basal ganglia and midbrain dopamine systems. These findings, which come from experiments mainly done on mammals, serve as a basis for comparison with other vertebrates. However, similar studies in other amniotes, such as reptiles, are few. To fill this gap in knowledge, two species of crocodiles were examined utilizing a variety of histological methods in various planes of section. The following results were obtained. First, the habenula was divided into medial and lateral parts based on its cytoarchitecture. Neurons in the medial habenula were small, were closely packed, and had a limited dendritic arbor characterized by unusual distal dendritic appendages, whereas neurons in the lateral habenula were larger, were more loosely packed, and had longer dendritic processes that were commonly beaded. Second, the stria medullaris, the major input to the habenula, was identified by its immunoreactivity to parvalbumin. Third, the fasciculus retroflexus (habenulointerpeduncular tract), the primary output of the habenula, was visualized by staining with acetylcholinesterase. Fourth, nuclei associated with the habenula, the subhabenular nuclei, have been identified and characterized. These features provide a means to recognize the major nuclei and tracts in the epithalamus in crocodiles and are likely applicable to other reptiles.

Critical role of lateral habenula circuits in the control of stress-induced palatable food consumption.

Chronic stress fuels the consumption of palatable food and can enhance obesity development. While stress- and feeding-controlling pathways have been identified, how stress-induced feeding is orchestrated remains unknown. Here, we identify lateral habenula (LHb) Npy1r-expressing neurons as the critical node for promoting hedonic feeding under stress, since lack of Npy1r in these neurons alleviates the obesifying effects caused by combined stress and high fat feeding (HFDS) in mice. Mechanistically, this is due to a circuit originating from central amygdala NPY neurons, with the upregulation of NPY induced by HFDS initiating a dual inhibitory effect via Npy1r signaling onto LHb and lateral hypothalamus neurons, thereby reducing the homeostatic satiety effect through action on the downstream ventral tegmental area. Together, these results identify LHb-Npy1r neurons as a critical node to adapt the response to chronic stress by driving palatable food intake in an attempt to overcome the negative valence of stress.

Instantaneous antidepressant effect of lateral habenula deep brain stimulation in rats studied with functional MRI.

The available treatments for depression have substantial limitations, including low response rates and substantial lag time before a response is achieved. We applied deep brain stimulation (DBS) to the lateral habenula (LHb) of two rat models of depression (Wistar Kyoto rats and lipopolysaccharide-treated rats) and observed an immediate (within seconds to minutes) alleviation of depressive-like symptoms with a high-response rate. Simultaneous functional MRI (fMRI) conducted on the same sets of depressive rats used in behavioral tests revealed DBS-induced activation of multiple regions in afferent and efferent circuitry of the LHb. The activation levels of brain regions connected to the medial LHb (M-LHb) were correlated with the extent of behavioral improvements. Rats with more medial stimulation sites in the LHb exhibited greater antidepressant effects than those with more lateral stimulation sites. These results indicated that the antidromic activation of the limbic system and orthodromic activation of the monoaminergic systems connected to the M-LHb played a critical role in the rapid antidepressant effects of LHb-DBS. This study indicates that M-LHb-DBS might act as a valuable, rapid-acting antidepressant therapeutic strategy for treatment-resistant depression and demonstrates the potential of using fMRI activation of specific brain regions as biomarkers to predict and evaluate antidepressant efficacy.

Esr1+ hypothalamic-habenula neurons shape aversive states.

Excitatory projections from the lateral hypothalamic area (LHA) to the lateral habenula (LHb) drive aversive responses. We used patch-sequencing (Patch-seq) guided multimodal classification to define the structural and functional heterogeneity of the LHA-LHb pathway. Our classification identified six glutamatergic neuron types with unique electrophysiological properties, molecular profiles and projection patterns. We found that genetically defined LHA-LHb neurons signal distinct aspects of emotional or naturalistic behaviors, such as estrogen receptor 1-expressing (Esr1+) LHA-LHb neurons induce aversion, whereas neuropeptide Y-expressing (Npy+) LHA-LHb neurons control rearing behavior. Repeated optogenetic drive of Esr1+ LHA-LHb neurons induces a behaviorally persistent aversive state, and large-scale recordings showed a region-specific neural representation of the aversive signals in the prelimbic region of the prefrontal cortex. We further found that exposure to unpredictable mild shocks induced a sex-specific sensitivity to develop a stress state in female mice, which was associated with a specific shift in the intrinsic properties of bursting-type Esr1+ LHA-LHb neurons. In summary, we describe the diversity of LHA-LHb neuron types and provide evidence for the role of Esr1+ neurons in aversion and sexually dimorphic stress sensitivity.

Neural mechanism of acute stress regulation by trace aminergic signalling in the lateral habenula in male mice.

Stress management is necessary for vertebrate survival. Chronic stress drives depression by excitation of the lateral habenula (LHb), which silences dopaminergic neurons in the ventral tegmental area (VTA) via GABAergic neuronal projection from the rostromedial tegmental nucleus (RMTg). However, the effect of acute stress on this LHb-RMTg-VTA pathway is not clearly understood. Here, we used fluorescent in situ hybridisation and in vivo electrophysiology in mice to show that LHb aromatic L-amino acid decarboxylase-expressing neurons (D-neurons) are activated by acute stressors and suppress RMTg GABAergic neurons via trace aminergic signalling, thus activating VTA dopaminergic neurons. We show that the LHb regulates RMTg GABAergic neurons biphasically under acute stress. This study, carried out on male mice, has elucidated a molecular mechanism in the efferent LHb-RMTg-VTA pathway whereby trace aminergic signalling enables the brain to manage acute stress by preventing the hypoactivity of VTA dopaminergic neurons.

Lateral septum adenosine A2A receptors control stress-induced depressive-like behaviors via signaling to the hypothalamus and habenula.

Major depressive disorder ranks as a major burden of disease worldwide, yet the current antidepressant medications are limited by frequent non-responsiveness and significant side effects. The lateral septum (LS) is thought to control of depression, however, the cellular and circuit substrates are largely unknown. Here, we identified a subpopulation of LS GABAergic adenosine A2A receptors (A2AR)-positive neurons mediating depressive symptoms via direct projects to the lateral habenula (LHb) and the dorsomedial hypothalamus (DMH). Activation of A2AR in the LS augmented the spiking frequency of A2AR-positive neurons leading to a decreased activation of surrounding neurons and the bi-directional manipulation of LS-A2AR activity demonstrated that LS-A2ARs are necessary and sufficient to trigger depressive phenotypes. Thus, the optogenetic modulation (stimulation or inhibition) of LS-A2AR-positive neuronal activity or LS-A2AR-positive neurons projection terminals to the LHb or DMH, phenocopied depressive behaviors. Moreover, A2AR are upregulated in the LS in two male mouse models of repeated stress-induced depression. This identification that aberrantly increased A2AR signaling in the LS is a critical upstream regulator of repeated stress-induced depressive-like behaviors provides a neurophysiological and circuit-based justification of the antidepressant potential of A2AR antagonists, prompting their clinical translation.