Sex-dependent differences in water and alcohol Drinking in the Dark (DID)

(a) Representation of viral strategy and expression to record presynaptic calcium activity of AIC inputs within the DLS. (b) Schematic of Drinking in the Dark (DID) protocol. (c) Group and individual animals’ water consumption by DID session (n = 14 animals). (d) Group and individual animals’ water consumption by DID session and by sex (mixed ANOVA, Sex F(1,12) = 0.1032, p = 0.7535; male n = 9, female n = 5). (e) Group and individual animals’ water consumption by DID week (rmANOVA, Week F(2,26) = 2.4712, p = 0.1041). (f) Group and individual animals’ water consumption by DID week and by sex (mixed ANOVA, Sex F(1,7) = 0.1032, p = 0.7535; male n = 9, female n = 5). (g) Group and individual animals’ alcohol consumption by DID session (n = 14 animals). (h) Group and individual animals’ alcohol consumption by DID session and by sex (mixed ANOVA, Sex F(1,7) = 0.4714, p = 0.5144; male n = 9, female n = 5). (i) Group and individual animals’ alcohol consumption by DID week (rmANOVA, Week F(2,26) = 7.8861, p = 0.002, np2 = 0.0992, Power (1 - b) = 0.9999; Week 4 to Week 5, p = 0.0012; male n = 9, female n = 5). (j) Group and individual animals’ alcohol consumption by DID week and by sex (mixed ANOVA, Sex F(1,7) = 0.4714, p = 0.5144; male n = 9, female n = 5). (k) Group and individual animals’ inter-session water consumption by DID week and by sex (mixed ANOVA, Sex x Drinking Week F(5,60) = 2.411, p = 0.0466, np2 = 0.1673, Power (1 - b) = 1.0; male n = 9, female n = 5). (l) Previous inter-session water intakes negatively correlate with next DID session water intake (Shepherd’s pi correlation, r = -0.2383, p = 0.0002). (m) Previous inter-session water intakes do not correlate with next DID session water intake by sex (ANCOVA, Sex F(1,257) = 0.3048, p = 0.5814; male n = 9, female n = 5). (n) Previous inter-session water intakes do not correlate with next DID session alcohol intake (Shepherd’s pi correlation, r = -0.0547, p = 0.4537). (o) Previous inter-session water intakes do not correlate with next DID session alcohol intake by sex (ANCOVA, Sex F(1,315) = 0.0101, p = 0.9201; male n = 9, female n = 5), but when a priori splitting by sex females, but not males, future alcohol intakes are negatively correlated with increasing amounts of inter-session water intakes (males only r = 0.1554, p = 0.0916; females only r = -0.4133, p = 0.0007, Power (1 - b) = 0.9328).

Locations of anterior insular cortex (AIC) injections and dorsolateral striatum (DLS) injections and fiber optic cannula placements. Bilateral injection centers of AAV-FLEX-GCaMP in the AIC and bilateral injection centers of AAVrg-cre-mCherry and fiber optic cannula placements as verified by histology (male: n = 9 animals; female n = 5 animals).

Microstructure features measured from lickometers correlate with alcohol and water intakes in a sex-dependent manner.

(a) Water events (Shepherd’s pi correlation, r = 0.3352, p = 1.1e-07; ANCOVA, Sex F(1, 252) = 0.3203, p = 0.5719; male n = 9, female n = 5), (b) total event duration (Shepherd’s pi correlation, r = 0.2726, p = 2.001e-05; ANCOVA, Sex F(1, 252) = 0.0757, p = 0.7834; male n = 9, female n = 5), and (c) drinks (Shepherd’s pi correlation, r = 0.4622, p = 9.884e-14; ANCOVA, Sex F(1, 252) = 0.9928, p = 0.32; male n = 9, female n = 5) correlate with water intakes in a non sex-dependent manner. (d) Alcohol events (Shepherd’s pi correlation, r = 0.274, p = 0.0001902; ANCOVA, Sex F(1, 191) = 2.13, p = 0.1461; male n = 9, female n = 5) correlate with alcohol intakes in a non-sex dependent manner. Yet, alcohol (e) event duration (Shepherd’s pi correlation, r = 0.434, p = 1.16e-09; ANCOVA, Sex F(1, 191) = 5.689, p = 0.01805, np2 = 0.02893, Power (1 - β) = 0.6934; male n = 9, female n = 5) and (f) drinks (Shepherd’s pi correlation, r = 0.5869, p = 4.799e-18; ANCOVA, Sex F(1, 191) = 9.7152, p = 0.00211, np2 = 0.0484, Power (1 - β) = 0.8965; male n = 9, female n = 5) correlate with alcohol intakes and females display a sex-dependent increase in intake for these metrics. Individual datapoints represent a microstructure feature and intake taken from a single DID session. Data were fit to a linear model represented by a line of best fit with shading indicating ± SEM. Axes histograms display distribution of microstructure features and intakes across all DID sessions.

Alterations in drinking microstructure represent different strategies used to consume similar levels of alcohol, but not water intake in a sex-dependent manner.

Water (a) events (mixed ANOVA, Sex F(1,11) = 0.6164, p = 0.4490; male n = 9, female n = 5), (b) total event duration (mixed ANOVA, Sex F(1,11) = 1.422, p = 0.2582; male n = 9, female n = 5), (c) drinks (mixed ANOVA, Sex F(1,11) = 0.3957, p = 0.5422; male n = 9, female n = 5), (d) latency to drink (mixed ANOVA, Sex F(1,11) = 0.3911, p = 0.5746; male n = 9, female n = 5) (e), mean inter-drink interval (mixed ANOVA, Sex F(1,11) = 0.0339, p = 0.8572; male n = 9, female n = 5), and (f) events in the first 30 minutes of a DID session (mixed ANOVA, Sex F(1,11) = 0.538, p = 0.4786; male n = 9, female n = 5) do not differ by sex across drinking week. Alcohol (g) events do not differ by sex across week (mixed ANOVA, Sex F(1,12) = 3.183, p = 0.0997; male n = 9, female n = 5), but alcohol (h) event duration (mixed ANOVA, Sex F(1,12) = 7.286, p = 0.0193, np2 = 0.3778, Power (1 - β) = 1.0; male n = 9, female n = 5), and (i) drinks are decreased in female compared to male binge alcohol drinkers (mixed ANOVA, Sex F(1,12) = 6.681, p = 0.0239, np2 = 0.3576, Power (1 - β) = 1.0; male n = 9, female n = 5). There were no sex differences in alcohol (j) latency to drink (mixed ANOVA, Sex F(1,12) = 1.914, p = 0.1917; male n = 9, female n = 5) (k), but alcohol mean inter-drink interval is increased in females compared to males (mixed ANOVA, Sex x Week F(2,24) = 3.623, p = 0.0422, np2 = 0.2319, Power (1 - β) = 1.0; Week 6 t(6.81) = 3.623, p = 0.01882; male n = 9, female n = 5). There are no sex-dependent changes in (l) events in the first 30 minutes of an alcohol DID session by week (mixed ANOVA, Sex F(1,12) = 1.972, p = 0.1856; male n = 9, female n = 5).

Sex-dependent calcium dynamics of AIC inputs into the left and right DLS during water and drinking bouts.

(a) AIC inputs to the DLS are most strongly engaged by alcohol at AIC inputs into the left DLS (ANOVA, Sex x Fluid x Time x Input Side F(149, 200.7) = 1.383, p = 0.001383, np2 = 0.000786, Power (1 - β) = 1.0; * = Time from Drink Start x AIC Input Side, -0.0369128 (s) – 5 (s), p < 0.05; = Time from Drink Start x Sex-Fluid, -1.308725 (s) – 4.95733 (s), p < 0.05) and (b) peak amplitudes ΔF/F changes are strongly associated with changes in AIC inputs to the left DLS than with the right DLS (ANOVA, Sex x Fluid x Input Side F(3,122.6) = 31.15, p = 1.298e-19, np2 = 0.05083, Power (1 - β) = 1.0). (c) For left AIC inputs into the DLS, there were both sex and fluid interactions by time for changes in ΔF/F (ANOVA, Sex x Fluid x Time F(149,221.9) = 1.404, p = 8.419e-4, np2 = 0.05083, Power (1 - β) = 1.0; * = Time from Drink Start x Sex, -1.107383 (s) – 5 (s), p < 0.05; = Time from Drink Start x Fluid, -2.181208 (s) – 5 (s), p < 0.05) and (d) increases in peak amplitude for ΔF/F for alcohol compared to water were observed, but no interaction was present between sex and fluid (ANOVA, Fluid F(1,32.45) = 19.46, p = 1.152e-5; Sex F(1,132.2) = 79.27, p = 2.919e-18, np2 = 0.05083, Power (1 - β) = 1.0). (e) Time to peak values for males increased due to alcohol exposure, yet the opposite occurred for females (ANOVA, Sex x Fluid F(1,14.25) = 19.44, p = 0.000012, np2 = 0.02688, Power (1 - β) = 0.9929; paired t-test, males t(375.5) = 4.0864, p = 0.000107; paired t-test, females, t(280.5) = -2.532 p = 0.02361). (f) Left AUC were increased in males in comparison to females, but no interaction was present between sex and fluid (ANOVA, Fluid F(1,332.3) = 56.58, p = 1.652e-13; Sex F(1,149.8) = 25.51, p = 5.613e-7, np2 = 0.074388, Power (1 - β) = 1.0). (g) For right AIC inputs into the DLS, there were both sex by time and fluid by time for changes in ΔF/F, but no three-way interaction (ANOVA, Sex x Time F(149, 772.6) = 5.881, p = 6.559e-103, np2 = 0.006891, Power (1 - β) = 1.0; = Time from Drink Start x Sex, - 1.778523 (s) – 5 (s), p < 0.05; Fluid x Time F(149,340.7) = 2.594, p = 5.977e-23, np2 = 0.003051, Power (1 - β) = 1.0). (h) Decreases in peak amplitude ΔF/F for alcohol compared to water were observed for males, but not females (ANOVA, Sex x Fluid F(1,3.652) = 3.925, p = 0.04787, np2 = 0.004641, Power (1 - β) = 0.5096; paired t-test, males t(407.1) = 4.490, p = 0.000009; paired t-test, females, t(271.9) = -2.248 p = 0.05015). (i) For females, time to peak decreased when transitioning from water to alcohol exposure, but there was no effect in males (ANOVA, Sex x Fluid F(1,15.02) = 20.99, p = 0.000005, np2 = 0.02852, Power (1 - β) = 0.9957; paired t-test, males t(410) = 1.288, p = 0.1985; paired t-test, females, t(289.6) = - 4.558 p = 0.000008). (j) For AUC, there was only a main effect of sex, with females displaying larger AUCs across both water and alcohol compared to males (ANOVA, Sex F(1,61.39) = 11.41, p = 0.00077, np2 = 0.01571, Power (1 - β) = 0.9226). GCaMP activity is aligned to the initiation of the water drinking bout with 5 seconds before and 5 seconds after the bout plotted. Solid lines represent means, and shading or error bars represent the standard error of the mean.

Calcium dynamics of AIC inputs into the left and right DLS during water drinking.

(a) Left AIC inputs to the DLS do not display a sex by time dependent change in ΔF/F (mixed ANOVA, Sex x Time F(149,1341) = 0.6601, p = 0.9993) (b) but for peak amplitude, males show larger maximum ΔF/F after water drinking events (MWU, U = 4465, p = 1.339e-8, Cohen’s d = 0.5607, Power (1 - β) = 0.9999). (c) There were no differences in time to peak (MWU, U = 16410, p = 0.7269), (d) but the AUC for males was larger than females for left AIC inputs (MWU, U = 20100, p = 0.001031, Cohen’s d = 0.3061, Power (1 - β) = 0.8289). (e) Right AIC inputs to the DLS also do not display a sex by time dependent change in ΔF/F (mixed ANOVA, Sex x Time F(149,1192) = 0.5882, p = 0.9996. (f) but like left AIC inputs, peak amplitudes in males were increased in males (MWU, U = 27902, p = 0.02137, Cohen’s d = 0.2912, Power (1 - β) = 0.8625). (g) Female time to peak values also increased in comparison to males (MWU, U = 989, p = 5.238e-8, Cohen’s d = 0.5953, Power (1 - β) = 0.9998), (h) but no changes in AUC were observed by sex (MWU, U = 13242, p = 0.06568) GCaMP activity is aligned to the initiation of the water drinking bout with 5 seconds before and 5 seconds after the bout plotted. Solid lines represent means and shading or error bars represent the standard error of the mean.

Calcium dynamics of AIC inputs into the left and right DLS across water drinking weeks.

(a) For males, left AIC inputs to the DLS do not display a by week dependent change in ΔF/F (rmANOVA, DID Week x Time from Drink F(298,298) = 0.6953, p = 0.5575), but (b) peak amplitudes increased in the final week of water drinking compared to the first two (ANOVA, DID Week F(2,163.8) = 7.235, p = 0.000975, np2 = 0.05062, Power (1 - β) = 0.9999; Week 3 – Week 1, p = 0.02478; Week 3 – Week 2, p = 0.000557). Yet, no difference in (c) time to peak (ANOVA, DID Week F(2,127.4) = 1.556, p = 0.215) or (d) AUC were observed by week (ANOVA, DID Week F(2,129.9) = 1.405, p = 0.2491) for male, left AIC inputs. I Right AIC inputs to the DLS did also not display a by week dependent change in ΔF/F (rmANOVA, DID Week x Time from Drink F(298,596) = 2.166, p = 0.2743), but (f) a decrease in peak amplitudes in the final water drinking week was observed in comparison to the first week (ANOVA, DID Week F(2,192) = 3.5774, p = 0.02983, np2 = 0.03593, Power (1 - β) = 0.9978; Week 3 – Week 1, p = 0.02206). (g) No change in time to peak was observed by week (ANOVA, DID Week F(2,104.6) = 2.139, p = 0.1229), but (h) a decrease in AUC was observed with the final week of water drinking displaying a deceased off-kinetic of the calcium signal in comparison to the initial drinking week (ANOVA, DID Week F(2,113.9) = 5.301, p = 0.006291, np2 = 0.06266, Power (1 - β) = 0.9999; Week 3 – Week 1, p = 0.004061). (i) For females, left AIC inputs to the DLS do not display a by week dependent change in ΔF/F (rmANOVA, DID Week x Time from Drink F(298,298) = 1.308, p = 0.5817), but (j) like males, peak amplitudes increased in the final week of water drinking (ANOVA, DID Week F(2,78.83) = 7.529, p = 0.001017, np2 = 0.060465, Power (1 - β) = 1.0; Week 3 – Week 1, p = 0.00108). No difference in (k) time to peak (ANOVA, DID Week F(2,30.37) = 1.591, p = 0.2203) was observed, but (l) AUC increased in the final week of water drinking compared to the initial week (ANOVA, DID Week F(2,34.22) = 7.0389, p = 0.002751, np2 = 0.07152, Power (1 - β) = 0.9999; Week 3 – Week 1, p = 0.000801). (m) Right AIC inputs to the DLS did also not display a by week dependent change in ΔF/F (rmANOVA, DID Week x Time from Drink F(298,596) = 1.022, p = 0.4966), but (n) an increase in peak amplitudes in the final water drinking week was observed in comparison to the initial week (ANOVA, DID Week F(2,93.65) = 4.533, p = 0.01321, np2 = 0.03957, Power (1 - β) = 0.9999; Week 3 – Week 1, p = 0.01778). (o) A decline in time to peak was observed in the final week of water drinking (ANOVA, DID Week F(2,58.75) = 10.26, p = 0.000151, np2 = 0.07598, Power (1 - β) = 0.9999; Week 3 – Week 1, p = 0.00066), but (p) no change in AUC was observed by week (ANOVA, DID Week F(2,64.13) = 0.7386, p = 0.4818). GCaMP activity is aligned to the initiation of the water drinking bout with 5 seconds before and 5 seconds after the bout plotted. Solid lines represent means, and shading or error bars represent the standard error of the mean.

Left AIC inputs into the DLS encode binge alcohol drinking in males, but not females.

(a) Left AIC inputs to the DLS display an increase in sex by time dependent change in ΔF/F, with males displaying an increased calcium signal after alcohol drinking initiation compared to (mixed ANOVA, Sex x Time F(149,1788 = 3.125, p = 4.006e-29, np2 = 0.2066, Power (1 - β) = 1.0; Time from Drink Start, 0.0369128 (s) – 1.989866 (s), p <= 0.05) and (b) male peak amplitudes are increased compared to females (MWU, U = 22385, p = 2.379e-9, Cohen’s d = 0.6703, Power (1 - β) = 0.9999). (c) There were no differences in time to peak (MWU, U = 18174.5, p = 0.2148), (d) but the AUC for males was larger than females for left AIC inputs during binge alcohol drinking (MWU, U = 19025, p = 1.281e-11, Cohen’s d = 0.4987, Power (1 - β) = 0.9924). (e) Right AIC inputs to the DLS also display a sex by time dependent change in ΔF/F, but no post hoc significance for any individual timepoint was discovered (mixed ANOVA, Sex x Time F(149,1788) = 1.2821, p = 0.00151, np2 = 0.09653, Power (1 - β) = 1.0) (f) nor were right AIC peak amplitudes different by sex (MWU, U = 17217, p = 0.5484). (g) No change in time to peak values were observed (MWU, U = 16045, p = 0.8015), (h) but female AUC measurements were greater than males (MWU, U = 12561.5, p = 0.001092, Cohen’s d = 0.3601, Power (1 - β) = 0.9108). GCaMP activity is aligned to the initiation of the water drinking bout with 5 seconds before and 5 seconds after the bout plotted. Solid lines represent means, and shading or error bars represent the standard error of the mean.

Calcium dynamics of AIC inputs into the left and right DLS across binge alcohol drinking.

(a) For males, left AIC inputs to the DLS do not display a week dependent change in ΔF/F (rmANOVA, DID Week x Time from Drink F(298,298) = 2.271, p = 0.3729), nor (b) did peak amplitudes change by binge week (ANOVA, DID Week F(2,246) = 0.05842, p = 0.9433). No difference in (c) time to peak were seen (ANOVA, DID Week F(2,113.7) = 1. 359, p = 0.2609) but (d) decreases in AUC were observed in the final binge week compared to the first two (ANOVA, DID Week F(2,91.76) = 3.481, p = 0.03491, np2 = 0.04435, Power (1 - β) = 0.9998: Week 4 – Week 6, p = 0.04111; Week 5 – Week 6, p = 0.03777) for male, left AIC inputs. (e) Right AIC inputs to the DLS did also not display a by week dependent change in ΔF/F (rmANOVA, DID Week x Time from Drink F(298,596) = 2.166, p = 0.2743), but (f) an increase in peak amplitudes in the second week of binge drinking week was observed in comparison to the final week (ANOVA, DID Week F(2,256) = 4.8388, p = 0.008655, np2 = 0.03643, Power (1 - β) = 0.9996; Week 5 – Week 6, p = 0.007429). (g) No change in time to peak was observed by week (ANOVA, DID Week F(2,109.8) = 2.933, p = 0.05744) (h) and no change in AUC was observed across binge drinking weeks for male, right AIC inputs into the DLS (ANOVA, DID Week F(2,87.91) = 0.5946, p = 0.5539). (i) For females, left AIC inputs to the DLS do not display a by week dependent change in ΔF/F (rmANOVA, DID Week x Time from Drink F(298,596) = 1.544, p = 0.3716). (j) No change in peak amplitudes, (ANOVA, DID Week F(2,128) = 2.961, p = 0.05534) (k) time to peak, (ANOVA, DID Week F(2,70.21) = 1.238, p = 0.2962) or (l) AUC were observed (ANOVA, DID Week F(2,69.62) = 2.089, p = 0.1315). (m) Right AIC inputs to the DLS did also not display a by week dependent change in ΔF/F (rmANOVA, DID Week x Time from Drink F(298,596) = 0.8997, p = 0.4538). Similar to female, left AIC inputs, there were no changes in (n) peak amplitudes, (ANOVA, DID Week F(2,135) = 1.606, p = 0.2045) (o) time to peak, (ANOVA, DID Week F(2,70.11) = 1.067, p = 0.3495), or (p) AUC by binge drinking week either (ANOVA, DID Week F(2,73.17) = 0. 1216, p = 0.8857). GCaMP activity is aligned to the initiation of the water drinking bout with 5 seconds before and 5 seconds after the bout plotted. Solid lines represent means, and shading or error bars represent the standard error of the mean.

Intra DID session calcium signals and global quantiles of drink length and total number of drinks per session.

Example male (a) left and (b) right AIC input to DLS calcium recordings from a single 2-hour binge alcohol DID recording (animal “P7”, session 17), with dashed vertical lines indicating the start of an alcohol drinking event overlayed on the time-resolved GCaMP signal. A histogram of all measured (c) drink lengths and (d) number of drinks observed across all water and alcohol drinking sessions. On the x-axis, the quartile ranges are printed and colored to visualize the quartile ranges of drink length and total number of drinks that occurred across all water and alcohol DID sessions.

Intra DID session calcium signals do not differ by the duration of each drinking event.

For water drinking, neither (a) left (rmANOVA, Drink Duration x Time from Drink F(596,1192) = 0.4524, p = 0.9999) or (b) right (rmANOVA, Drink Duration x Time from Drink F(596,596) = 0.06445, p = 0.9999) male AIC inputs calcium signals are altered by the length of the drinking event. Nor, do females display any difference in (c) left (rmANOVA, Drink Duration x Time from Drink F(596,1788) = 0.1412, p = 0.9999) or (d) right (rmANOVA, Drink Duration x Time from Drink F(596,1788) = 0.05649, p = 0.9493) AIC calcium signals by the length of the drinking event. For alcohol drinking events, neither (e) left (rmANOVA, Drink Duration x Time from Drink F(447,2682) = 1.594, p = 0.2461) or (f) right (rmANOVA, Drink Duration x Time from Drink F(596,596) = 0.8884, p = 0.9557) male AIC inputs calcium signals are altered by the length of the drinking event. Nor, do females display any difference in (g) left (rmANOVA, Drink Duration x Time from Drink F(447,1788) = 0.4118, p = 0.9999) or (h) right (rmANOVA, Drink Duration x Time from Drink F(447,1788) = 0.5659, p = 0.9999) AIC calcium signals by the length of the alcohol drinking event. GCaMP activity is aligned to the initiation of the water drinking bout with 5 seconds before and 5 seconds after the bout plotted. Solid lines represent means, and shading or error bars represent the standard error of the mean.

Intra DID session calcium signals do not differ by the number of drinking events within a single session.

For water drinking, neither (a) left (rmANOVA, Drink number x Time from Drink F(596,2980) = 0.8228, p = 0.4185) or (b) right (rmANOVA, Drink number x Time from Drink F(447,1788) = 1.016, p = 0.3921) male AIC inputs calcium signals are altered by the number of water drinking events within a DID session. Females do not display any difference in (c) left (rmANOVA, Drink number x Time from Drink F(447,1341) = 3.346, p = 0.1347) or (d) right (rmANOVA, Drink number x Time from Drink F(447,1341) = 2.598, p = 0.1741) AIC calcium signals by the increasing number of the drinking events. For alcohol drinking events, neither (e) left (rmANOVA, Drink number x Time from Drink F(447,1788) = 1.608, p = 0.2557) or (f) right (rmANOVA, Drink number x Time from Drink F(447,2235) = 1.664, p = 0.2435) male AIC inputs calcium signals are altered by increasing number of drinking events. Nor, do females display any difference in (g) left (rmANOVA, Drink number x Time from Drink F(447,447) = 0.8444, p = 0.5269) or (h) right (rmANOVA, Drink number x Time from Drink F(447,894) = 0.7267, p = 0.5078) AIC calcium signals by the number of alcohol drinking events. GCaMP activity is aligned to the initiation of the water drinking bout with 5 seconds before and 5 seconds after the bout plotted. Solid lines represent means, and shading or error bars represent the standard error of the mean.