Figure 1: Sex-dependent current responses to membrane hyperpolarization in monkey BNST neurons during voltage clamp recordings is modulated by a history of chronic voluntary alcohol intake.

A) Voluntary alcohol access paradigm in male and female rhesus monkeys, in which animals were singly housed and introduced to interactive panels to deliver water (~6 months) and then alcohol to achieve escalating intake up to 1.5 g/kg (three months), followed by open access to alcohol for 12 months prior to necropsy and electrophysiological recordings. B) Average daily 4% EtOH intake across the open access period in monkeys used in this study was similar between sexes (t₁₃ = 0.45, p > 0.65). C) Schematic of a monkey brain on the coronal plane containing BNST, with blue dashed outline representing the approximate dissected tissue used for slice electrophysiology recordings. D) Stimulus waveform for electrophysiological recordings measuring the current-voltage relationship in BNST neurons from control males (CON M), alcohol males (EtOH M), control females (CON F), and alcohol females (EtOH F), in which cells held under voltage clamp at a holding potential of −70 mV were underwent increasingly hyperpolarizing steps of 10 mV to −120 mV. E) Representative traces for current responses to the stimulus in D. F) Both sex and chronic alcohol drinking altered the raw current response to hyperpolarization of the membrane potential. 3xRM-ANOVA: main effects of sex (F_1,51 = 5.15, ^@p = 0.028) and voltage step (F_5,255 = 65.78, ****p < 0.0001) and a trend of EtOH (F_1,51 = 3.10, p = 0.085); interactions between sex × step (F_5,255 = 11.30, ^&&&&p < 0.0001) and EtOH × step (F_5,255 = 7.70, ^####p < 0.0001) but no others (ps > 0.10). Multiple t-tests with H-S corrections across all 6 voltage steps showed that CON M and CON F differed on the −50 mV step (t₁₂₆ = 2.95, ^&p = 0.023) and CON M and EtOH M differed on the −50 mV step (t₁₆₂ = 3.03, ^#p = 0.017); all other comparisons, including between CON F and EtOH F, did not differ (ps > 0.10). G) Membrane capacitance was unaffected by sex or EtOH. 2xANOVA:no effects of sex, EtOH, or their interaction (ps > 0.10). H) Current density responses to membrane hyperpolarization (change in current normalized to cell capacitance) were affected by sex and EtOH. 3xRM-ANOVA: main effects of EtOH (F_1,51 = 4.77, ^$p = 0.034) and voltage step (F_5,255 = 21.11, ****p < 0.0001) and a trend of sex (F_1,51 = 2.94, p = 0.093); interactions between sex × step (F_5,255 = 3.39, ^&&p = 0.006) and EtOH × step (F_5,255 = 4.86, ^###p =0.0003) but no others (ps > 0.25). Multiple t-tests with H-S corrections across all six voltage steps showed that CON M and EtOH M differed on the −50 mV step (t₁₆₂ = 3.10, ^#p = 0.013) with a trend on the −40 mV step (t₁₆₂ = 2.41, p = 0.083); CON F and EtOH F differed on the −50 mV step (t₁₄₄ = 3.41, ^##p = 0.005) and −40 mV step (t₁₄₄ = 2.76, ^#p = 0.033); all other comparisons, including between CON M and CON F, did not differ (ps > 0.10).