Bed nucleus of the stria terminalis (BNST) neurons that synthesize corticotropin-releasing factor (CRF) drive binge alcohol drinking and anxiety. Here, we found 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 + synaptic input from the paraventricular thalamus (PVT) that releases glutamate directly onto BNSTCRF neurons but also engages a large BNST interneuron population to ultimately inhibit BNSTCRF neurons, and this polysynaptic PVTVGLUT2-BNSTCRF circuit is more robust in females than males. Chemogenetic inhibition of the PVTBNST projection promoted binge alcohol drinking only in female mice, while activation reduced avoidance behavior in both sexes. Lastly, repeated binge drinking produced 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 circuit that is sex-dependent in its function, behavioral roles, and alcohol-induced plasticity.

Chronic stress during the developmental period of adolescence increases susceptibility to many neuropsychiatric diseases in adulthood, including anxiety, affective, and alcohol/substance use disorders. Preclinical rodent models of adolescent stress have produced varying results that are species, strain, sex, and laboratory-dependent. However, adolescent social isolation is a potent stressor in humans that has been reliably modeled in male rats, increasing adult anxiety-like and alcohol drinking behaviors, among others. In this study, we examined the generalizability and sex-dependence of this model in C57BL/6J mice, the most commonly used rodent strain in neuroscience research. We also performed a parallel study using social isolation in adulthood to understand the impact of adult social isolation on basal behavioral phenotypes. We found that 6 weeks of social isolation with minimal handling in adolescence through early adulthood [postnatal day (PD) 28-70] produced a hypersocial phenotype in both male and female mice and an anxiolytic phenotype in the elevated plus-maze in female mice. However, it had no effects in other assays for avoidance behavior or on fear conditioning, alcohol drinking, reward or aversion sensitivity, or novel object exploration in either sex. In contrast, 6 weeks of social isolation in adulthood beginning at PD77 produced an anxiogenic phenotype in the light/dark box but had no effects on any other assays. Altogether, our results suggest that: (1) adolescence is a critical period for social stress in C57BL/6J mice, producing aberrant social behavior in a sex-independent manner; and (2) chronic individual housing in adulthood does not alter basal behavioral phenotypes that may confound interpretation of behavior following other laboratory manipulations.

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 BNST CRF 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 PVT BNST neurons release glutamate directly onto BNST CRF neurons but also engage a large BNST interneuron population to ultimately provide a net inhibition of BNST CRF neurons, and both components of this polysynaptic PVT VGLUT2 -BNST CRF circuit are more robust in females than males. While chemogenetic inhibition of the general PVT VGLUT2 neuron population suppressed binge alcohol drinking in both sexes, chemogenetic inhibition specifically of the PVT BNST projection promoted this behavior in females without affecting males; 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-BNST CRF excitatory synapse without altering the function of PVT BNST neurons per se. Our data describe a complex and unique behavioral role of the feedforward inhibitory PVT VGLUT2 -BNST CRF glutamatergic circuit that is more robust in females and undergoes sex-dependent alcohol-induced plasticity. Fig. 1: Females display higher binge alcohol drinking and have greater BNST CRF neuron excitation. a,Average 2-hr alcohol consumption across cycles of the Drinking in the dark (DID) binge drinking paradigm showing higher binge drinking in females than males (n = 10 male mice, 10 female mice). Two-way RM-ANOVA: main effect of sex (F1,18 = 21.62, *** P = 0.0002) and cycle (F4,73 = 6.34, P = 0.0002, not indicated), but no interaction (P > 0.05); direct comparisons within-cycle show sex differences on cycle 2 (t15.8 = 1.58, adjusted *P = 0.037), cycle 5 (t16.3 = 4.71, adjusted **P = 0.001), and cycle 6 (t14.5 = 4.96, adjusted **P = 0.001), with trends on cycle 3 (t17.9 = 2.32, adjusted $ P = 0.063) and cycle 4 (t13.9 = 2.67, adjusted $ P = 0.054). b, Schematic of whole-cell patch clamp recordings of CRF+ neurons in the BNST (BNST CRF neurons, left) and representative 10x confocal image of a coronal BNST section from a CRF-Cre x Ai9 reporter (CRF-reporter) mouse. c, Proportion of BNST CRF neurons sampled that fire in their basal state is greater in females than males (n = 10 male mice, 18 cells; 8 female mice, 19 cells). d-f, spontaneous excitatory and inhibitory synaptic transmission in male and female BNST CRF neurons (males: n = 8 mice, 17 cells; females: n = 8 mice, 21 cells; unpaired ttests). d, Top: representative traces of spontaneous excitatory postsynap...

The anterior and posterior subregions of the paraventricular thalamus (aPVT and pPVT, respectively) play unique roles in learned behaviors, from fear conditioning to alcohol/drug intake, potentially through differentially organized projections to limbic brain regions including the nucleus accumbens medial shell (mNAcSh). Here, we found that the aPVT projects broadly to the mNAcSh and that the aPVT-mNAcSh circuit encodes positive valence, such that in vivo manipulations of the circuit modulated both innately programmed and learned behavioral responses to positively and negatively valenced stimuli, particularly in females. Further, the endogenous activity of aPVT presynaptic terminals in the mNAcSh was greater in response to positively than negatively valenced stimuli, and the probability of synaptic glutamate release from aPVT neurons in the mNAcSh was higher in females than males. In contrast, we found that the pPVT-mNAcSh circuit encodes stimulus salience regardless of valence. While pPVT-mNAcSh circuit inhibition suppressed behavioral responses in both sexes, circuit activation increased behavioral responses to stimuli only in males. Our results point to circuit-specific stimulus feature encoding by parallel PVT-mNAcSh circuits that have sex-dependent biases in organization and function.