Probes for INS

Stress is a precipitating agent in neuropsychiatric disease and initiates relapse to drug-seeking behavior in addicted patients. Targeting the stress system in protracted abstinence from drugs of abuse with anxiolytics may be an effective treatment modality for substance use disorders. α2A-adrenergic receptors (α2A-ARs) in extended amygdala structures play key roles in dampening stress responses. Contrary to early thinking, α2A-ARs are expressed at non-noradrenergic sites in the brain. These non-noradrenergic α2A-ARs play important roles in stress-responses, but their cellular mechanisms of action are unclear. In humans, the α2A-AR agonist guanfacine reduces overall craving and uncouples craving from stress yet minimally affects relapse, potentially due to competing actions in the brain. Here we show that heteroceptor α2A-ARs postsynaptically enhance dorsal BNST (dBNST) neuronal activity in mice of both sexes. This effect is mediated by hyperpolarization-activated cyclic nucleotide-gated cation (HCN) channels, as inhibition of these channels is necessary and sufficient for excitatory actions. Finally, this excitatory action is mimicked by clozapine-N-oxide activation of the Gi-coupled DREADD hM4Di in dBNST neurons, and its activation elicits anxiety-like behavior in the elevated plus maze. Together, this data provides a framework for elucidating cell-specific actions of GPCR signaling and provides a potential mechanism whereby competing anxiogenic and anxiolytic actions of guanfacine may affect its clinical utility in the treatment of addiction.SIGNIFICANCE STATEMENTStress impacts the development of neuropsychiatric disorders including anxiety and addiction. Guanfacine is an α2A-adrenergic receptor (α2A-AR) agonist with actions in the bed nucleus of the stria terminalis (BNST) that produces antidepressant actions and uncouples stress from reward-related behaviors. Here we show that guanfacine increases dBNST neuronal activity through actions at postsynaptic α2A-ARs via a mechanism that involves hyperpolarization-activated cyclic nucleotide gated cation (HCN) channels. This action is mimicked by activation of the designer receptor hM4Di expressed in the BNST, which also induces anxiety-like behaviors. Together, these data suggest 1) that postsynaptic α2A-ARs in BNST have excitatory actions on BNST neurons, and 2) these actions can be phenocopied by the so-called "inhibitory" DREADDs, suggesting care must be taken regarding interpretation of data obtained with these tools.

Most people exposed to stress do not develop depression. Animal models have shown that stress resilience is an active state that requires broad transcriptional adaptations, but how this homeostatic process is regulated remains poorly understood. In this study, we analyze upstream regulators of genes differentially expressed after chronic social defeat stress. We identify estrogen receptor α (ERα) as the top regulator of pro-resilient transcriptional changes in the nucleus accumbens (NAc), a key brain reward region implicated in depression. In accordance with these findings, nuclear ERα protein levels are altered by stress in male and female mice. Further, overexpression of ERα in the NAc promotes stress resilience in both sexes. Subsequent RNA-sequencing reveals that ERα overexpression in NAc reproduces the transcriptional signature of resilience in male, but not female, mice. These results indicate that NAc ERα is an important regulator of pro-resilient transcriptional changes, but with sex-specific downstream targets.

The medial preoptic area (mPOA) differs between males and females in nearly all species examined to date, including humans. Here, using fiber photometry recordings of Ca2+ transients in freely behaving mice, we show ramping activities in the mPOA that precede and correlate with sexually dimorphic display of male-typical mounting and female-typical pup retrieval. Strikingly, optogenetic stimulation of the mPOA elicits similar display of mounting and pup retrieval in both males and females. Furthermore, by means of recording, ablation, optogenetic activation, and inhibition, we show mPOA neurons expressing estrogen receptor alpha (Esr1) are essential for the sexually biased display of these behaviors. Together, these results underscore the shared layout of the brain that can mediate sex-specific behaviors in both male and female mice and provide an important functional frame to decode neural mechanisms governing sexually dimorphic behaviors in the future.