Acute and chronic alcohol modulation of extended amygdala calcium dynamics

Abstract

The central amygdala (CeA) and bed nucleus of the stria terminalis (BNST) are reciprocally connected nodes of the extended amygdala thought to play an important role in alcohol consumption. Studies of immediate-early genes indicate that BNST and CeA are acutely activated following alcohol drinking and may signal alcohol reward in nondependent drinkers, while stress signaling in the extended amygdala following chronic alcohol exposure drives increased drinking via negative reinforcement. However, the temporal dynamics of neuronal activation in these regions during drinking behavior are poorly understood. In this study, we used fiber photometry and the genetically encoded calcium sensor GCaMP6s to assess acute changes in neuronal activity during alcohol consumption in BNST and CeA before and after a chronic drinking paradigm. Activity was examined in the pan-neuronal population and separately in dynorphinergic neurons. BNST and CeA showed increased pan-neuronal activity during acute consumption of alcohol and other fluid tastants of positive and negative valence, as well as highly palatable chow. Responses were greatest during initial consummatory bouts and decreased in amplitude with repeated consumption of the same tastant, suggesting modulation by stimulus novelty. Dynorphin neurons showed similar consumption-associated calcium increases in both regions. Following three weeks of continuous alcohol access (CA), calcium increases in dynorphin neurons during drinking were maintained, but pan-neuronal activity and BNST-CeA coherence were altered in a sex-specific manner. These results indicate that BNST and CeA, and dynorphin neurons specifically, are engaged during drinking behavior, and activity dynamics are influenced by stimulus novelty and chronic alcohol.

Keywords: alcohol; coherence; drinking; dynorphin; extended amygdala; fiber photometry.

Figure 1.. Overview of experimental design.

(A) Wildtype (C57BL/6J) mice were stereotaxically injected with AAV5-hsyn-GCaMP6s to drive pan-neuronal expression of the calcium indicator GCaMP6s in the central amygdala (CeA) and contralateral bed nucleus of the stria terminalis (BNST) and implanted with optical fibers above each injection site. A separate cohort of mice expressing cre under control of the preprodynorphin promoter (pdyn-cre) were injected with AAV5-hsyn-FLEX-GCaMP6s to target GCaMP expression to dynorphin-expressing neurons in the CeA and contralateral BNST with corresponding optical fiber placement. (B) Fiber photometry recordings of GCaMP in CeA and BNST were made during consumption of alcohol and other tastants in the home cage over 3–4 separate recording sessions. Mice were then provided with three weeks of continuous access to 20% w/v alcohol in the home cage. 24 hours following the end of continuous access, fiber photometry recordings were repeated to determine if chronic alcohol access affected calcium dynamics. (C,D) Representative images of virus and fiber placements in CeA and BNST in C57BL/6J (C) and preprodynorphin-cre (D) mice.

Figure 2.. BNST and CeA neurons exhibit acute calcium increases time-locked to drinking and eating behavior.

Data are shown for 15% ethanol (A-D), 5% sucrose (E-H), 100 μM quinine (I-L), 0.1% saccharin (M-P), water (Q-T), and high-fat chow (U-X). Representative traces from BNST and CeA demonstrate acute increases in activity during bouts of consumption of alcohol (A), sucrose (E), quinine (I), saccharin (M), water (Q) and high-fat chow (U) (bouts indicated by shading). Heatmaps show all 5s consumption bouts recorded in BNST and CeA for alcohol (B), sucrose (F), quinine (J), saccharin (N), water (R), and high-fat chow (V), with the onset of each 5s bout aligned to the dotted line. Data from male (n=6) and female mice (n=8) are indicated on the right side of the heatmaps. Peri-event plots show the mean±SEM GCaMP6s activity during the first, last, and average consumption bouts in BNST and CeA for alcohol (C), sucrose (G), quinine (K), saccharin (O), water (S), and high-fat chow (W). Time zero represents the start of consumption bouts. For both regions, the mean Z score significantly increased during 5-second consumption bouts compared to the 5 seconds prior to bout onset for alcohol (D), sucrose (H), quinine (L), saccharin (P), water (T), and high-fat chow (X). (paired t-test, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001) (For quinine, drinking bouts were only 2 seconds in duration.) The mean Z score during consumption decreased from the first to last bout in BNST and CeA for alcohol (D), sucrose (H), quinine (L), saccharin (P), and high-fat chow (X) (paired t-test, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001). There was no change from the first to last bout for water for either region (T).

Figure 3.. BNST and CeA dynorphin neurons exhibit acute calcium increases time-locked to drinking and eating behavior.

Data are shown for 15% ethanol (A-C), 5% sucrose (D-F), 100 μM quinine (G-I), 0.1% saccharin (J-L), water (M-O), and high-fat chow (P-R). Heatmaps show all 5s consumption bouts recorded in BNST and CeA for alcohol (A), sucrose (D), quinine (G), saccharin (J), water (M), and high-fat chow (P) from n=16 mice, with the onset of each 5s bout aligned to the dotted line. Data from male (n=11) and female mice (n=5) are indicated on the right side of the heatmaps. Peri-event plots show the mean±SEM GCaMP6s activity during the first, last, and average consumption bouts in BNST and CeA for alcohol (B), sucrose (E), quinine (H), saccharin (K), water (N), and high-fat chow (Q). Time zero represents the start of consumption bouts. The mean Z score significantly increased in BNST and CeA during 5-second consumption bouts compared to the 5 seconds prior to bout onset for alcohol (C), sucrose (F), quinine (I), saccharin (L), water (O), and high-fat chow (R) (paired t-test, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001). (For quinine, drinking bouts were 2 seconds in duration.) There was no change in mean Z score in the first compared to last bout of consumption for any tastant.

Figure 4.. BNST and CeA pan-neuronal activity during initial alcohol drinking bouts is reduced in females following 3 weeks of continuous access (CA).

(A) Alcohol intake and preference ratio were significantly greater in female than male mice. (B) There was a main effect of CA to reduce the frequency of peaks in calcium activity in BNST, while peak frequency was unchanged following CA in CeA. (C) Representative traces from BNST (top) and CeA (bottom) showing acute increases in neuronal activity during bouts of alcohol consumption (indicated by green shading) pre-CA and 24 hours following the end of CA in the same animal. D) 24 hours post CA, there was no change in the mean Z score from the first to last bout of alcohol drinking in BNST or CeA. (E) 3 weeks post CA, there was an increase in the mean Z score from the first to last bout of alcohol drinking in BNST (paired t-test, **p<0.01), and a decrease in the mean Z score from the first to last bout of alcohol drinking in CeA (paired t-test, *p<0.05). (F-I) Heatmaps showing alcohol drinking bouts 24 hours (F,H) and 3 weeks (G,I) following the end of CA. (J,K) Peri-event plots showing the mean±SEM GCaMP6s activity for average consumption bouts in male and female mice pre-CA (green), 24 hours post CA (red), and 3 weeks post CA (grey). Time zero represents the start of consumption bouts. (L) Following CA, there was no change in the peak Z score in BNST in either sex. 24 hours post CA in females, the mean Z score increase from baseline during alcohol drinking bouts was smaller compared to pre-CA. (M) 24 hours post CA, there was a main effect of CA (p<0.05) on the peak Z score, and in females, there was a significantly smaller peak Z score in CeA during alcohol drinking (mixed model ANOVA with Tukey post-hoc test, *p<0.05, **p<0.01). 24 hours post CA, there was a main effect of CA and a trend for a reduction in the Z score change acute alcohol drinking bouts in females in CeA.

Figure 5.. BNST-CeA coherence is increased during alcohol drinking in males.

(A-D) Coherence spectra for 0–2 Hz for males pre- (A) and post- (B) continuous access (CA), and females pre- (C) and post-CA (D) for the 10-second period prior to alcohol drinking bouts (gray) and 10 seconds following bout onset (green). Alcohol drinking-induced coherence changes were greatest between 1 and 1.25 Hz. (E) BNST-CeA coherence between 1 and 1.25 Hz was increased in the 10 s post bout onset compared to the 10 s prior to bout onset for alcohol drinking in males both before and after CA. There was a main effect of CA to reduce coherence (3-way ANOVA with Sidak’s post hoc test, p<0.05). (F-I) Coherence spectra for 0–2 Hz for males pre- (F) and post- (G) continuous access (CA), and females pre- (H) and post-CA (I) for the 10-second period prior to sucrose drinking bouts (gray) and 10 seconds following bout onset (purple). (J) BNST-CeA coherence between 1 and 1.25 Hz was unchanged during sucrose drinking.