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.

SUMMARY Sensory inputs activate sparse neuronal ensembles in the dentate gyrus of the hippocampus, but how eligibility of individual neurons to recruitment is determined remains elusive. We identify thousands of largely bistable (CpG methylated or unmethylated) regions within neuronal gene bodies, established during mouse dentate gyrus development. Reducing DNA methylation and the proportion of the methylated epialleles at bistable regions compromises novel context-induced neuronal activation. Conversely, increasing methylation and the frequency of the methylated epialleles at bistable regions enhances intrinsic excitability. Single-nucleus profiling reveals enrichment of specific epialleles related to a subset of primarily exonic, bistable regions in activated neurons. Genes displaying both differential methylation and expression in activated neurons define a network of proteins regulating neuronal excitability and structural plasticity. We propose a model in which bistable regions create neuron heterogeneity and constellations of exonic methylation, which may contribute to cell-specific gene expression, excitability, and eligibility to a coding ensemble.

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...

Episodic memories are stored in distributed neurons but how eligibility of individual neurons to coding ensembles is determined remains elusive. We identified thousands of predominantly bistable (CpG methylated or unmethylated) regions within neuronal gene bodies, established during the development of the mouse hippocampal dentate gyrus. Reducing DNA methylation and the proportion of the methylated epialleles at bistable regions compromised novel context-induced neuronal activation and spatial memory. Conversely, increasing methylation and the frequency of the methylated epialleles at bistable regions enhanced intrinsic excitability and spatial memory but impaired spatial working memory, indicating that the developmentally established methylated-unmethylated epiallelic balance at bistable regions is essential for proper neuronal excitability and hippocampal cognitive functions. Single-nucleus profiling revealed the enrichment of specific epialleles from a subset of bistable regions, primarily exonic, in encoding neurons. We propose a model in which epigenetically bistable regions create neuron heterogeneity, and specific constellations of exonic epialleles dictate, via modulating neuronal excitability, eligibility to a coding ensemble.Bidirectional manipulation of the relative frequency of the methylated epialleles in vivo reduced and increased neuronal intrinsic excitability, novel context-induced neuronal activation, and hippocampal functions, linking methylation states and the combinations of these states, to neuronal eligibility to coding. Single-nucleus profiling of recently recruited neurons revealed enrichment for a set of epialleles that, via gene expression, may underlie eligibility for coding environmental inputs. Through these findings, our work implicates an epigenetic mechanisms dictating neuronal eligibility to recruitment, the first step of memory formation. Results:We found evidence for the developmental emergence of epigenetic heterogeneity in otherwise morphologically homogenous and genetically identical dorsal (d) DGCs in C57BL/6 male mice 15 .DGCs or their progenitors were microdissected from the granule cell layer of d (d) hippocampal slices at either embryonic day (E) 10.5, postnatal day (P) 6 or at 10-12 weeks of age, followed by enhanced RRBS methylation profiling of 1.5-2.5 million cytosines 16 (Fig. 1a). Loss and gain of methylation during the transition of P6 young DGCs (yDGCs) to adult DGCs produced 170,198 differentially methylated (DM) CpG sites (q<0.01, RRBS at >10x coverage, average change 22.23%). In adult DGCs, these DM CpGs were by and large not fully methylated and unmethylated but rather were in the state of intermediate methylation (IM, i.e. were methylated across the entire 0% to 100% range, Fig. 1c, pink). Many of these sites were already IM at P6 ( Fig. 1c, blue) indicating that the partial methylation/demethylation process began earlier.Indeed, comparing adult DGCs with cells from an earlier developmental stage, E10.5 hippocampal progenitors (HPs), still yielded DM sites ...