Biology subjects

Kash, T. L.

Publications and source records attributed to Kash, T. L..

At least 19 recordsLinked to original sources

bioRxiv2026

Chronic Intermittent Ethanol Exposure Produces Physiological Tolerance and Distinct Behavioral Alterations Across Abstinence in Female Mice

Chronic ethanol consumption produces long-lasting effects on the brain that contribute to persistent behavioral changes. This has been modeled in animals using the chronic intermittent ethanol (CIE) paradigm, which induces escalation of consumption and alterations in affective and cognitive behavior. While this model has been well characterized in male subjects, the effects of CIE in females and long-term effects that persist beyond the acute withdrawal remain relatively underexplored. Here, female C57BL/6J mice were exposed to four weeks of chronic intermittent ethanol (CIE) vapor to induce dependence. Following CIE, mice were evaluated for physiological tolerance to ethanol and alterations in anxiety-like behaviors and memory processing. Using the ethanol responsivity battery (ERB) assay to assess physiological tolerance, we found blunted responsiveness to the intoxicating effects of ethanol in the CIE-exposed females, indicating heightened ethanol tolerance that persists beyond the acute withdrawal phase. A cohort underwent behavioral testing following CIE to characterize changes in anxiety-like behavior, exploration, and memory across abstinence. In early abstinence (<5 days post-CIE), CIE-exposed females showed increased exploratory behavior compared with Air controls. After one week of abstinence, CIE-exposed females displayed impaired object recognition memory. CIE-exposed females also showed increased anxiety-like behavior at protracted abstinence time points, exhibiting hyponeophagia in the novelty-suppressed feeding test at 2 weeks post-CIE. In addition, following cued fear conditioning, we found reduced extinction recall at 4 weeks post-CIE. Together, this work indicates that chronic alcohol exposure leads to increased alcohol tolerance and distinct, persistent alterations in memory and anxiety-like behavior in female mice.

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bioRxiv2026

ABEL: an active-learning behavior estimation and labeling platform

Detailed behavior analysis is essential for thorough characterization of ethologically relevant behaviors in model organisms, yet manual annotation of the full behavioral repertoire remains subjective, time intensive, and susceptible to observer error. Advances in machine learning have enabled high-throughput pose estimation on recorded video, but tools for behavior classification from pose and video data are still developing. Instead of hand-scoring every frame of video, experimenters can instead label a small subset of video frames and software trained through machine learning makes predictions on the rest. Here, we present an Active-learning Behavior Estimation and Labeling (ABEL) platform that uses clip-level active learning (i.e., human labeling of short video snippets) with multimodal features (pose, video, context/ROI) to train robust behavior classifiers. We rigorously validated ABEL-derived behavior predictions against expert human observers and field-standard automated software, across diverse rodent behavioral assays. Across eight assays and 45 behaviors, model training required 19.5 hours of human annotation in total, with the reviewer scoring ~8% of available video. Models trained in ABEL achieved a mean precision-recall area under the curve (PR-AUC - a 0-1 score of how well a model balances missed detections against false alarms, with 1 being perfect) of 0.90 (SD 0.09, range 0.60-0.99), with no association between performance and behavior prevalence (r = 0.20). This was aided by custom tools, Essence Extractor and UMAP Interactive Selection, for targeted discovery of high probability clips which reduce the clip review needed to find a rare behavior 6-fold relative to random sampling and 10-fold relative to labeling whole videos. As a biological validation, we assessed how ABEL-derived behaviors relate to underlying neuronal calcium dynamics. Behavior labels were tightly synced with neuronal signatures distinct from ambiguous behavior and randomly chosen, behavior-unrelated time windows (shuffle control). Together, these data indicate that ABEL provides an efficient platform for frame-precise classification of distinct ethologically relevant behaviors.

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bioRxiv2026

The role of serotonin in the orbitofrontal cortex in alcohol consumption

Binge alcohol drinking is a public health concern that can dramatically increase the risk for development of alcohol use disorder (AUD). Continued alcohol drinking in the face of negative consequences is another key feature of AUD. A better understanding of the neural circuitry that regulates these behaviors could provide insight as to novel treatments for AUD. Serotonin is a neurotransmitter that has been implicated in alcohol consumption in both human studies and animal models. The orbitofrontal cortex (OFC) is a brain region that both receives serotonergic input from the dorsal raphe and has been implicated in AUD. However, how volitional alcohol consumption impacts serotonin signaling within the OFC and how this contributes to alcohol related behaviors is unknown. Here, we show that a history of alcohol consumption alters the ability of 5-HT to hyperpolarize OFC pyramidal neurons in mice and monkeys. Consistent with this, a history of binge alcohol consumption decreases the expression of the 5-HT1A but not 5-HT2A receptor in the OFC from mice. Next, we show that deletion of the 5-HT1A receptor from the OFC increased alcohol intake and preference in male, but not female mice. Finally, we found that 5-HT1A receptor deletion led to increased quinine-adulterated alcohol intake, a measure of aversion-resistant drinking, in both male and female mice. Altogether, we identified serotonin signaling in the OFC as key target for modulation of binge and compulsive alcohol consumption.

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bioRxiv2026

Chronic oral fentanyl consumption produces dynamic behavioral adaptations during abstinence in female mice

RationaleThe opioid epidemic continues to be driven by synthetic opioids, particularly fentanyl, yet the long-term behavioral manifestations of withdrawal remain poorly characterized. Women exhibit unique vulnerabilities to opioid use disorder, including greater susceptibility to withdrawal-related symptoms and relapse, suggesting that defining the behavioral adaptations that emerge during prolonged fentanyl abstinence may identify mechanisms underlying female-specific relapse vulnerability. Thus, we used an oral fentanyl drinking model to longitudinally examine nociceptive, affective, and exploratory behaviors across chronic exposure and abstinence in female mice. MethodsAdult female C57BL/6J mice underwent a modified 5-week Drinking-in-the-Dark paradigm with 4-hour daily access to fentanyl (30 {micro}g/mL) or water. Somatic withdrawal was assessed following naloxone-precipitated withdrawal. Thermal and mechanical nociception, sucrose preference, exploratory behavior, acoustic startle, nest building, and locomotor activity were evaluated during fentanyl exposure and throughout one month of abstinence. ResultsFemale mice exhibited robust naloxone-precipitated somatic withdrawal, confirming physiological effects of opioid drinking. Chronic fentanyl exposure produced transient thermal hyperalgesia during weeks 2-4 of drinking that resolved by the final week, but re-emerged after 30 days of abstinence. After 30 days of abstinence, we also found increased mechanical sensitivity. During early abstinence, fentanyl-exposed mice exhibited increased sucrose consumption and greater exploration of anxiogenic environments without alterations in general locomotion. Increased exploratory behavior persisted into prolonged abstinence and was partially rescued in mice who previously received naloxone. Additionally, fDID mice exhibited impaired nesting behavior, while prior naloxone-precipitated withdrawal improved nest-building performance. ConclusionsWe demonstrate that chronic opioid exposure, precipitated withdrawal trials, and duration of abstinence interact to shape protracted withdrawal phenotypes in female mice that extend well beyond drug exposure. These results provide insight into persistent withdrawal symptoms that may shape relapse vulnerability using a novel translationally relevant framework.

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bioRxiv2026

Divergent ethanol drinking phenotypes are linked to region-specific dysregulation of serotonin systems in the mouse brain

Excessive alcohol drinking is a leading cause of preventable death in the United States. High alcohol consumption and persistent drinking despite adverse events, also known as compulsive drinking, are key criteria that contribute to the development and progression of alcohol use disorder (AUD). There is a clear need to better understand the mechanisms that support these related but distinct behaviors. The serotonin (5-HT) system has been associated with alcohol consumption and risk of alcohol dependence, however given the complexity of this system, there remains much to discover regarding specific alcohol related phenotypes. The current study uses a combination of volitional home-cage drinking and operant conditioning to phenotype mice based on ethanol intake and persistence of alcohol drinking following quinine adulteration, a model to study compulsive drinking. Brain tissue of 10 regions known to be implicated in regulating executive function, reward, and stress was collected, and gene expression of serotonergic receptors, transporters, and enzymes was quantified. Three opioid receptors were included given their well-established roles in alcohol-related behaviors and interactions with the 5HT system. Region-specific gene expression patterns emerged, with serotonergic and opioid receptor expression differentially associated with alcohol drinking phenotype. 5-HT and opioid receptors displayed opposing directionality across regions, consistent with functional heterogeneity within the system. These findings identify region-specific molecular alterations following chronic alcohol that may contribute to individual differences in alcohol drinking phenotypes, highlighting candidate targets for biomarkers of increased alcohol use disorder susceptibility or as interventions aimed at preventing the progression of AUD.

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bioRxiv2025

Selective dysregulation of serotonin dynamics in the anterior cingulate cortex and central amygdala following binge alcohol consumption

Serotonin (5HT) is a critical modulator of brain function and behavior that is dysregulated in alcohol use disorder (AUD). The anterior cingulate cortex (ACC) and central nucleus of the amygdala (CeA) play distinct roles in AUD and undergo functional changes in 5HT signaling following binge drinking, but our understanding of real-time 5HT dynamics in these structures is lacking. We hypothesized that binge drinking would elicit brain-region specific dysfunction in 5HT dynamics during appetitive and aversive stimuli processing. Using fiber photometry with the GRAB5HT sensor, we identified distinct reward and aversion 5HT signaling motifs in the ACC and CeA. Consumption of alcohol and other tastants elicited a suppression of GRAB5HT signal in the ACC and an increase in 5HT signal in the CeA. In contrast, aversive stimuli similarly increased 5HT in both structures. The effect of binge drinking on 5HT function was surprisingly non-uniform, producing brain-region, sex-, and stimulus-specific dysfunction in 5HT signaling that dramatically shifted across weeks of alcohol experience. This suggests that adaptations in 5HT signaling are specific to neural circuits underlying discrete functions. Optogenetically stimulating 5HT terminals in the ACC and CeA increased avoidance behavior without being overtly rewarding or aversive, and stimulating 5HT release in the ACC blunted alcohol drinking. Together, these data identify distinct 5HT reward and aversion signaling motifs in the ACC and CeA and highlight early binge drinking as a critical stage of 5HT adaptation and a potential window for therapeutic intervention.

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bioRxiv2025

Adolescent alcohol exposure disrupts extinction learning and retrosplenial cortex physiology in adult males

Adolescent binge drinking can lead to a myriad of issues in adulthood, including psychiatric comorbidities that worsen clinical prognosis. Efforts to understand the effects of adolescent alcohol exposure on later health outcomes have unveiled lasting effects on physiology of brain regions critical for associative learning processes, including the hippocampus and prefrontal cortex (PFC). Here, we tested the hypothesis that adolescent alcohol exposure would produce lasting alterations in physiology within the retrosplenial cortex (RSC), another region critical for associative learning that is reciprocally connected to the hippocampus and PFC. In support of this hypothesis, we found that adolescent intermittent ethanol (AIE) vapor exposure resulted in lasting reductions in intrinsic excitability in RSC pyramidal cells of adult mice. Importantly, these changes were sex-specific, occurring in males but not females. This work suggests that the RSC may be a key, vulnerable locus to the detrimental effects of adolescent alcohol.

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bioRxiv2025

Lateral hypothalamus CRFR1 regulation of chronic binge drinking: divergence along anterior-posterior axis

Binge alcohol drinking increases the risk of developing an alcohol use disorder (AUD) and comorbid psychopathology. The lateral hypothalamus (LH) is a brain structure that integrates cognitive and sensory information to tightly regulate motivated behavior, including binge drinking. Importantly, LH function is vulnerable to modulation by the pro-stress neuropeptide corticotropin-releasing factor (CRF), and acute antagonism of CRF receptor 1 (CRFR1) in the LH blunts binge drinking. However, the role of LH CRFR1 in chronic binge drinking is unknown. We used genetically targeted knockdown (KD) of CRFR1 in the LH of male and female mice followed by three weeks of binge drinking using the "Drinking in the Dark" (DID) model. CRFR1 KD in the posterior LH increased alcohol consumption, independent of sex, with no effect of KD in the anterior LH. Consistent with this, total alcohol consumption was negatively correlated with the location of CRFR1 KD in the LH along the anterior-posterior axis. CRFR1 KD did not alter water consumption or body weight, suggesting the effects of CRFR1 KD on alcohol consumption were not due to broad disruption of fluid intake or homeostatic function. In contrast to the observed effects on binge drinking, CRFR1 KD increased anxiety-like behavior and blunted sucrose preference, independent of KD location in the LH. Our findings provide foundational insight into LH function in the context of AUD and prompt further investigation into the divergent roles that distinct circuitry or cell populations along the anterior-posterior axis of the LH may play in binge drinking. HighlightsO_LICRFR1 knockdown in posterior LH increased alcohol drinking independent of sex C_LIO_LIAlcohol intake negatively correlated with AP location of CRFR1 knockdown in LH C_LIO_LICRFR1 knockdown was anxiogenic with more pronounced effects in females C_LIO_LISucrose preference was blunted by CRFR1 knockdown C_LI

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bioRxiv2024

Chronic alcohol consumption alters sex-dependent BNST neuron function in rhesus macaques

Repeated alcohol drinking contributes to a number of neuropsychiatric diseases, including alcohol use disorder and co-expressed anxiety and mood disorders. Women are more susceptible to the development and expression of these diseases with the same history of alcohol exposure as men, suggesting they may be more sensitive to alcohol-induced plasticity in limbic brain regions controlling alcohol drinking, stress responsivity, and reward processing, among other behaviors. Using a translational model of alcohol drinking in rhesus monkeys, we examined sex differences in the basal function and plasticity of neurons in the bed nucleus of the stria terminalis (BNST), a brain region in the extended amygdala shown to be a hub circuit node dysregulated in individuals with anxiety and alcohol use disorder. We performed slice electrophysiology recordings from BNST neurons in male and female monkeys following daily "open access" (22 hr/day) to 4% ethanol and water for more than one year or control conditions. We found that BNST neurons from control females had reduced overall current density, hyperpolarization-activated depolarizing current (Ih), and inward rectification, as well as higher membrane resistance and greater synaptic glutamatergic release and excitatory drive, than those from control males, suggesting that female BNST neurons are more basally excited than those from males. Chronic alcohol drinking produced a shift in these measures in both sexes, decreasing current density, Ih, and inward rectification and increasing synaptic excitation. In addition, network activity-dependent synaptic inhibition was basally higher in BNST neurons of males than females, and alcohol exposure increased this in both sexes, a putative homeostatic mechanism to counter hyperexcitability. Altogether, these results suggest that the rhesus BNST is more basally excited in females than males and chronic alcohol drinking produces an overall increase in excitability and synaptic excitation. These results shed light on the mechanisms contributing to the female-biased susceptibility to neuropsychiatric diseases including co-expressed anxiety and alcohol use disorder.

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bioRxiv2024

Dynamic regulation of CeA gene expression during acute and protracted abstinence from chronic binge drinking of male and female C57BL/6J mice.

Binge alcohol consumption is a major risk factor for developing Alcohol Use Disorder (AUD) and is associated with alcohol-related problems like accidental injury, acute alcohol poisoning, and black-outs. While there are numerous brain regions that have been shown to play a role in this AUD in humans and animal models, the central nucleus of the amygdala (CeA) has emerged as a critically important locus mediating binge alcohol consumption. In this study, we sought to understand how relative gene expression of key signaling molecules in the CeA changes during different periods of abstinence following bouts of binge drinking. To test this, we performed drinking in the dark (DID) on two separate cohorts of C57BL/6J mice and collected CeA brain tissue at one day (acute) and 7 days (protracted) abstinence after DID. We used qRTPCR to evaluate relative gene expression changes of 25 distinct genes of interest related to G protein-coupled receptors (GPCRs), neuropeptides, ion channel subunits, and enzymes that have been previously implicated in AUD. Our findings show that during acute abstinence CeA punches collected from female mice had upregulated relative mRNA expression of the gamma-aminobutyric acid receptor subunit alpha 2 (Gabra2), and the peptidase, angiotensinase c (Prcp). CeA punches from male mice at the same time point in abstinence had upregulated relative mRNA encoding for neuropeptide-related molecules, neuropeptide Y (Npy) and somatostatin (Sst), as well as the neuropeptide Y receptor Y2 (Npyr2) but downregulated, Glutamate ionotropic receptor NMDA type subunit 1 (Grin1). After protracted abstinence CeA punches collected from female mice had increased mRNA expression of corticotropin releasing hormone (Crh) and Npy. While CeA punches collected from male mice at the same timepoint had upregulated relative mRNA expression of Npy2r and downregulated mRNA expression of Gabra2, Grin1 and opioid receptor kappa 1 (Oprk1). Our findings support that there are differences in how the CeA of male and female respond to binge-alcohol exposure, highlighting the need to understand the implications of such differences in the context of AUD and binge drinking behavior.

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bioRxiv2023

Bed Nucleus of the Stria Terminalis (BNST) neurons containing the serotonin 5HT2c receptor modulate operant alcohol self-administration behavior in mice

The serotonin 5HT2c receptor has been widely implicated in the pathophysiology of alcohol use disorder (AUD), particularly alcohol seeking and the affective consequences of chronic alcohol consumption. However, little is known about the brain sites in which 5HT2c exerts its effects on specific alcohol-related behaviors, especially in females. Here, we investigated the effects of site-specific manipulation of the 5HT2c receptor system in the BNST on operant alcohol self-administration behaviors in adult mice of both sexes, including the acquisition and maintenance of fixed-ratio responding, motivation for alcohol (progressive ratio), and quinine-adulterated responding for alcohol on a fixed-ratio schedule (punished alcohol seeking). Knockdown of 5HT2c in the BNST did not affect the acquisition or maintenance of operant alcohol self-administration, nor did it affect progressive ratio responding for alcohol. This manipulation had only a subtle effect on responding for quinine alcohol selectively in females. On the other hand, chemogenetic inhibition of BNST 5HT2c-containing neurons (BNST5HT2c) increased operant alcohol self-administration behavior in both sexes on day 2, but not day 9, of testing. It also increased operant responding for 1000 M quinine-adulterated alcohol selectively in males. Importantly, chemogenetic inhibition of BNST5HT2c did not alter operant sucrose responding or motivation for sucrose in either sex. We then performed cell-type specific anterograde tracing, which revealed that BNST5HT2c project to similar regions in males and females, many of which have been previously implicated in AUD. We next used chemogenetics and quantification of the immediate early gene cFos to characterize the functional influence of BNST5HT2c inhibition on vlPAG activity. We show that chemogenetic inhibition of BNST5HT2c reduces vlPAG cFos in both sexes, but that this reduction is more robust in males. Together these findings suggest that BNST5HT2c neurons, and to a small extent the BNST 5HT2c receptor, serve to promote aversive responses to alcohol consumption, potentially through sex-dependent disinhibition of vlPAG neurons.

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bioRxiv2023

Dopamine D2 receptors in the bed nucleus of the stria terminalis modulate alcohol-related behaviors.

Dysregulation of the dopamine (DA) system is a hallmark of substance abuse disorders, including alcohol use disorder (AUD). Of the DA receptor subtypes, the DA D2 receptors (D2Rs) play a key role in the reinforcing effects of alcohol. D2Rs are expressed in numerous brain regions associated with the regulation of appetitive behaviors. One such region is the bed nucleus of the stria terminalis (BNST), which has been linked to the development and maintenance of AUD. Recently, we identified alcohol withdrawal-related neuroadaptations in the periaqueductal gray/dorsal raphe to BNST DA circuit in male mice. However, the role of D2R-expressing BNST neurons in voluntary alcohol consumption is not well characterized. In this study, we used a CRISPR-Cas9-based viral approach, to selectively reduce the expression of D2Rs in BNST VGAT neurons and interrogated the impact of BNST D2Rs in alcohol-related behaviors. In male mice, reduced D2R expression potentiated the stimulatory effects of alcohol and increased voluntary consumption of 20% w/v alcohol in a two-bottle choice intermittent access paradigm. This effect was not specific to alcohol, as D2R deletion also increased sucrose intake in male mice. Interestingly, cell-specific deletion of BNST D2Rs in female mice did not alter alcohol-related behaviors but lowered the threshold for mechanical pain sensitivity. Collectively, our findings suggest a role for postsynaptic BNST D2Rs in the modulation of sex-specific behavioral responses to alcohol and sucrose.

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bioRxiv2023

Impact and role of hypothalamic corticotropin releasing hormone neurons in withdrawal from chronic alcohol consumption in female and male mice.

Worldwide, alcohol use and abuse are a leading risk of mortality, causing 5.3% of all deaths (W.H.O., 2022). The endocrine stress system, initiated by the peripheral release of corticotropin releasing hormone (CRH) from primarily glutamatergic neurons in the paraventricular nucleus of the hypothalamus (PVN), is profoundly linked with alcohol use, abuse, and relapse (Blaine & Sinha, 2017). These PVN CRH-releasing (PVNCRH) neurons are essential for peripheral and central stress responses (Rasiah et al., 2023), but little is known about how alcohol affects these neurons. Here, we show that two- bottle choice alcohol consumption blunts the endocrine mediated corticosterone response to stress during acute withdrawal in female mice. Conversely, using slice electrophysiology, we demonstrate that acute withdrawal engenders a hyperexcitable phenotype of PVNCRH neurons in females that is accompanied by increased glutamatergic transmission in both male and female mice. Only male mice show a concurrent increase in GABAergic synaptic transmission. We then tested whether chemogenetic inhibition of PVNCRH neurons would restore stress response in female mice with a history of alcohol drinking in the looming disc test, which mimics an approaching predator threat. Accordingly, inhibition of PVNCRH neurons reduced active escape in hM4Di alcohol history mice only. This study indicates that stress responsive PVNCRH neurons in females are particularly affected by a history of alcohol consumption. Interestingly, women have indicated an increase in heavy alcohol use to cope with stress (Rodriguez et al., 2020), perhaps pointing to a potential underlying mechanism in alcohol mediated changes to PVNCRH neurons that alter stress response.

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bioRxiv2022

Sex-specific regulation of binge drinking and affective behaviors by subcortical serotonin 5HT2c receptors

Serotonin 5HT2c receptors have been implicated in the pathophysiology of both mood disorders and alcohol use disorder, but the circuits mediating the effects of systemic pharmacological manipulations of this receptor on behavior have not been identified. Binge alcohol consumption induces discrete social and arousal disturbances in human populations, which are thought to promote increased drinking. However, whether models of binge drinking in rodents can induce these same long-term negative behavioral symptoms is unknown. In this study, we employed multiple anatomical, physiological, and behavioral approaches to identify two populations of neurons expressing serotonin 5HT2c receptors, one in the lateral habenula (LHb5HT2c) and one in the bed nucleus of the stria terminalis (BNST5HT2c), that display coordinated in-vivo responses to social, arousal, and alcohol-related stimuli and are physiologically modulated by binge alcohol consumption in a sex-specific manner. Critically, these physiological changes were associated with sex-specific behavioral disturbances that closely mirror social and arousal symptoms observed in humans during abstinence from binge drinking. Specifically, we observed that one week of abstinence from three weeks of binge alcohol drinking induced social recognition deficits in females and potentiated acoustic startle responses in males. While both populations of neurons (BNST and LHb) and the 5HT2c receptor itself contribute to the sex-specific effects of alcohol on social and arousal behaviors to some degree, the primary causal mechanism underlying these phenomena appears to be excessive activation of LHb5HT2c neurons. These findings may have implications for the development of sex-specific treatments for mood and alcohol use disorders targeting the brains serotonin system.

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bioRxiv2022

Activation of the dorsal septum increases alcohol consumption in male C57BL/6J mice.

Binge drinking is a common pattern of excessive alcohol consumption associated with Alcohol Use Disorder (AUD) and unraveling the neurocircuitry that promotes this type of drinking is critical to the development of novel therapeutic interventions. The septal region was once a focal point of alcohol research yet has seen limited study over the last decade in relation to binge drinking. Numerous studies point to involvement of the dorsal septum (dSep) in excessive drinking and withdrawal, but few studies have manipulated this region in the context of binge drinking behavior. The present experiments were primarily designed to determine the effect of chemogenetic manipulation of the dSep on binge-like alcohol drinking in male and female C57BL/6J mice. Mice received bilateral infusion of AAVs harboring hM4Di, hM3Dq, or mCherry into the dSep and subjects were challenged with systemic administration of clozapine-N-oxide (CNO; 3 mg/kg) and vehicle (saline; 0.9%) in the context of binge-like alcohol consumption, locomotor activity, and sucrose drinking. CNO-mediated activation (hM3Dq) of the dSep resulted in a significant increase in binge-like alcohol consumption, locomotor activity, and sucrose intake in male mice. DSep activation promoted sucrose drinking in female mice, but alcohol intake and locomotor activity were unaffected. Conversely, silencing (hM4Di) of the dSep modestly decreased locomotor activity in males and did not influence alcohol or sucrose intake in either sex. Lastly, CNO was without effect in mCherry-expressing control groups. These data support a role for the dSep in promoting excessive, binge-like drinking behavior in a sex-dependent fashion and suggests a broad role for the region in the modulation of locomotor activity as well as general appetitive behavior such as sucrose drinking.

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bioRxiv2021

Tumor necrosis factor-α modulates GABAergic and Dopaminergic neurons in the ventral periaqueductal gray of female mice.

Neuroimmune signaling is increasingly identified as a critical component of various illnesses, including chronic pain, substance use disorder, and depression. However, the underlying neural mechanisms remain unclear. Proinflammatory cytokines, such as tumor necrosis factor- (TNF-), may play a key role by modulating synaptic function and long-term plasticity. The midbrain structure periaqueductal gray (PAG) plays a well-established role in pain processing, and while TNF- inhibitors have emerged as a potential therapeutic strategy for pain-related disorders, the impact of TNF- on PAG neuronal activity has not been thoroughly characterized. Recent studies have identified subpopulations of ventral PAG (vPAG) with opposing effects on nociception, with DA neurons driving pain relief in contrast to GABA neurons. Therefore, we used ex vivo slice physiology to examine the effects of TNF- on neuronal activity of both subpopulations. We selectively targeted GABA and dopamine neurons using a vGAT-reporter and a TH-eGFP reporter mouse line, respectively. Following exposure to TNF-, the intrinsic properties of GABA neurons were altered, resulting in increased excitability along with a reduction in glutamatergic synaptic drive. In DA neurons, TNF- exposure resulted in a robust decrease in excitability along with a modest reduction in glutamatergic synaptic transmission. Furthermore, the effect of TNF- was specific to excitatory transmission onto DA neurons as inhibitory transmission was unaltered. Collectively, these data suggest that TNF- differentially affects the basal synaptic properties of GABA and DA neurons and enhances our understanding of how TNF- mediated signaling modulates vPAG function. New & NoteworthyThe present study describes the effects of tumor necrosis factor- (TNF-) on two distinct subpopulations of neurons in the ventral periaqueductal gray (vPAG). We show that TNF- alters both neuronal excitability and glutamatergic synaptic transmission on GABA neurons and dopamine neurons within the vPAG. This provides critical new information on the role of TNF- in the potential modulation of pain since activation of vPAG GABA neurons drives nociception, whereas activation of DA neurons drives analgesia.

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bioRxiv2020

Corticotropin-Releasing Factor Neurons in the Bed Nucleus of the Stria Terminalis Differentially Influence Pain Processing and Modulation in Male and Female Mice

The bed nucleus of the stria terminalis (BNST) plays an emerging yet understudied role in pain. Corticotropin-releasing factor (CRF) is an important source of pain modulation in the BNST, with local pharmacological inhibition of CRF receptors conditionally impacting the sensory and affective-motivational components of pain. Knowledge on how pain dynamically engages CRF neurons in the BNST and is influenced by intra-BNST production of CRF, however, remains unknown. In the present study, we utilized in vivo calcium imaging to show robust and synchronized recruitment of BNSTCRF+ neurons during acute exposure to noxious heat. Distinct patterns of recruitment were observed by sex, as the magnitude and timing of heat responsive activity in BNSTCRF+ neurons differed for male and female mice. We then established the necessity of CRF for intact pain behaviors by genetically deleting Crf in the BNST, which reduced thermal and mechanical nociceptive sensitivity for both sexes, and increased paw attending in female mice, suggesting a divergent role for CRF with respect to active coping responses to pain. Together, these findings demonstrate that CRF in the BNST contributes to multiple facets of the pain experience and may play a key role in the sex-specific expression of pain-related behaviors.

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bioRxiv2020

The paraventricular thalamus provides a polysynaptic brake on limbic CRF neurons to sex-dependently blunt binge alcohol drinking and avoidance behavior

Bed nucleus of the stria terminalis (BNST) neurons that synthesize and release the stress neuropeptide corticotropin-releasing factor (CRF) drive binge alcohol drinking and anxiety, behaviors that are primary risk factors for alcohol use disorder (AUD) and comorbid neuropsychiatric diseases more common in women than men. Here, we show that female C57BL/6J mice binge drink more than males and have greater basal BNSTCRF neuron excitability and synaptic excitation. We identified a dense VGLUT2+ glutamatergic synaptic input from the paraventricular thalamus (PVT) that is anatomically similar in males and females. These PVTBNST neurons release glutamate directly onto BNSTCRF neurons but also engage a large BNST interneuron population to ultimately provide a net inhibition of BNSTCRF neurons, and both components of this polysynaptic PVTVGLUT2-BNSTCRF circuit are more robust in females than males. Chemogenetic inhibition of the PVTBNST projection promoted binge alcohol drinking in females without affecting males, and chemogenetic activation of the pathway was sufficient to reduce avoidance behavior in both sexes in anxiogenic contexts. Lastly, we show that withdrawal from repeated binge drinking produces a female-like phenotype in the male PVT-BNSTCRF excitatory synapse without altering the function of PVTBNST neurons per se. Our data describe a complex feedforward inhibitory PVTVGLUT2-BNSTCRF glutamatergic circuit that is more robust in females, plays sex-dependent roles in alcohol drinking and avoidance behavior, and undergoes sex-dependent alcohol-induced plasticity.

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