Parabrachial Calcitonin Gene-Related Peptide Neurons Sex-Specifically Modulate Anxiety in Alcohol Withdrawal

1. Parabrachial nucleus (PBN) neurons expressing Calcitonin Gene-Related Peptide (CGRP) modulate fear- and anxiety-like behavior. It is unknown if PBN(CGRP) neurons play a role in anxiety during withdrawal from alcohol or after repeated stress. First, to investigate the anxiogenic role of activating PBN(CGRP) neurons in naïve conditions, Calca ^CRE female and male mice expressing CRE-dependent hM3D(Gq) DREADDs in the PBN were tested on the elevated plus maze (EPM). PBN(CGRP) neurons drive phasic activity in the bed nucleus of the stria terminalis (BNST) that synchronizes to anxiety-like behavior. Therefore, a transsynaptic anterograde AAV-based strategy was used in C57BL/6J female and male mice to activate BNST neurons innervated by the PBN projections (BNST ^PBN ) during EPM. Additionally, PBN(CGRP) neurons and CGRP-innervated BNST cells were measured in experimentally-naïve CGRP-DTR ^GFP female and male mice, to investigate baseline sex-differences. Next, to investigate the impact of PBN(CGRP) inhibition on anxiety-like behavior following chronic intermittent ethanol vapor exposure (CIE), Calca ^CRE female and male mice expressing CRE-dependent hM4D(Gi) DREADDs in the PBN were tested in EPM during acute withdrawal. Additionally, mice were exposed to repeated forced swim stress (FSS) paired with PBN(CGRP) inhibition followed by testing in the novelty suppressed feeding task (NSFT), to investigate the role of PBN(CGRP) on anxiety-like behavior after stress in prolonged withdrawal. Activating PBN(CGRP) or BNST ^PBN neurons did not change behavior in EPM in either sex. Total PBN(CGRP) and CGRP-innervated BNST cells did not differ between females and males. In acute withdrawal, inhibiting PBN(CGRP) neurons decreased time in the open arm and increased time in the closed arm selectively in females. In prolonged withdrawal, inhibiting PBN(CGRP) neurons during FSS did not affect immobility in either sex, but did subsequently decrease approach frequency and average speed selectively in males. Altogether, the data report the anxiogenic effect of manipulating PBN(CGRP) neurons is stress and sex-specific during alcohol withdrawal.