Kappa opioid receptor and dynorphin signaling in the central amygdala regulates alcohol intake

Abstract

Excessive alcohol drinking has been shown to modify brain circuitry to predispose individuals for future alcohol abuse. Previous studies have implicated the central nucleus of the amygdala (CeA) as an important site for mediating the somatic symptoms of withdrawal and for regulating alcohol intake. In addition, recent work has established a role for both the Kappa Opioid Receptor (KOR) and its endogenous ligand dynorphin in mediating these processes. However, it is unclear whether these effects are due to dynorphin or KOR arising from within the CeA itself or other input brain regions. To directly examine the role of preprodynorphin (PDYN) and KOR expression in CeA neurons, we performed region-specific conditional knockout of these genes and assessed the effects on the Drinking in the Dark (DID) and Intermittent Access (IA) paradigms. Conditional gene knockout resulted in sex-specific responses wherein PDYN knockout decreased alcohol drinking in both male and female mice, whereas KOR knockout decreased drinking in males only. We also found that neither PDYN nor KOR knockout protected against anxiety caused by alcohol drinking. Lastly, a history of alcohol drinking did not alter synaptic transmission in PDYN neurons in the CeA of either sex, but excitability of PDYN neurons was increased in male mice only. Taken together, our findings indicate that PDYN and KOR signaling in the CeA plays an important role in regulating excessive alcohol consumption and highlight the need for future studies to examine how this is mediated through downstream effector regions.

Figure 1-. PDYN and KOR expression in CeA neurons is unaltered by a history of ethanol drinking.

(a) Timeline for experimental procedures. (b) Representative image of Pdyn and Oprk1 mRNA expression in the CeA (20x objective, NA=0.8) (c) Quantification of PDYN+ and KOR+ cell counts following experimental manipulations. Ethanol treatment did not affect the proportion of PDYN+, KOR+, and colocalized neurons X²(2)=1.494, p=0.474 (d) Ethanol treatment did not affect Oprk1 gene expression t(9.04)=0.64, p= 0.534 or (e) Pdyn gene expression t(11,899)=0.108, p=0.916. (c) n=12 images from N=3 ethanol or N= 2 water drinking mice. Individual dots reflect counts within a single image which were not pooled within subjects (d-e) punches from n=8 water and n=8 ethanol drinking animals

Figure 2-. Knockout of KOR in CeA decreases ethanol consumption in male, but not female mice.

(a) Timeline for experimental procedures. (b) Hypothesized actions of KOR knockout in CeA. (c) Representative images of control virus expression (left) and knockout virus (right). (d-j) Ethanol consumption in male animals. (d) Group averages for individual sessions over the course of four weeks of DID. Consumption at both the 2-hr and 4-hr time points are plotted separately on day 4. (e) Cumulative averages by group for four weeks of DID; knockout of KOR in CeA significantly decreased alcohol consumption in male animals, main effect of condition F(1,12)=7.100, p=0.021; condition X cycle interaction F(3,36)=0.324, p=0.808. (f) Cumulative totals from individual mice. (g) KOR knockout resulted in a significant reduction in the amount of alcohol consumed during IA; main effect of genotype: F(1,12)=13.628, p=0.003. (h) KOR knockout did not affect total fluid intake during IA; main effect of genotype: F(1,12)=1.652, p=0.223 (i) KOR knockout resulted in a significant reduction in ethanol preference; F(1,12)=6.616, p=0.024. (j) KOR knockout significantly reduced the cumulative amount of ethanol consumed during IA; main effect of genotype: F(1,12)=13.694, p<0.001. (k-q) Ethanol consumption in female mice. (k) Group averages for individual sessions over the course of four weeks of DID. Consumption at both the 2-hr and 4-hr time points are plotted separately on day 4. (l) Cumulative averages by group for four weeks of DID; KOR knockout in CeA did not significantly alter alcohol consumption in female animals, main effect of condition F(1,17)=0.909, p=0.353; cycle X condition interaction F(3,51)=0.614, p=0.609. (m) Cumulative totals from individual mice. (n) KOR knockout did not alter the amount of alcohol consumed during IA; main effect of genotype: F(1,17)=0.196, p=0.663. (o) KOR knockout did not affect total fluid intake during IA; main effect of genotype: F(1,17)=0.232, p=0.636. (p) KOR knockout did not alter ethanol preference; main effect of genotype: F(1,17)=0.512, p=0.484. (q) KOR knockout did not affect cumulative ethanol consumption during IA; main effect of genotype: F(1,17)= 0.263, p=0.614. (d,k) round dots indicate values at 2-hr time points and square dots indicate values at 4-hr time points (d-j) n=8 control males and n=7 knockout males (k-q) n=9 control females and n=10 knockout females

Figure 3-. PDYN knockout in CeA decreases ethanol consumption in male and female mice.

(a) Timeline for experimental procedures. (b) Hypothesized actions of PDYN knockout mice. (c) Representative images of control virus expression (left) and knockout virus (right). (d-j) Ethanol consumption in male animals. (d) Group averages for individual sessions over the course of 4 wk of DID; consumption at both 2-hr and 4-hr time points are plotted separately on day 4. (e) Cumulative averages by group for 4 wk of DID. PDYN knockout in CeA significantly decreased alcohol consumption in male animals; main effect of genotype: F(1,11)=6.899, p=0.024; genotype X cycle interaction F(3,33)=0.753, p=0.528. (f) Cumulative totals from individual mice. (g) PDYN knockout resulted in a significant reduction in the amount of alcohol consumed during IA; main effect of genotype: F(1,11)=4.860, p=0.0497. (h) PDYN knockout did not affect total fluid intake during IA; main effect of genotype: F(1,11)=1.485, p=0.248. (i) PDYN knockout did not affect ethanol preference; main effect of genotype: F(1,11)=3.788, p=0.078. (j) PDYN knockout did not significantly reduce the cumulative amount of ethanol consumed during IA F(1,11)=3.943, p=0.073. (k-q) Ethanol consumption in female animals. (k) Group averages for individual sessions over the course of 4 wk of DID. Consumption at both the 2-hr and 4-hr points are plotted separately on day 4. (l) Cumulative averages by group for 4 wk of DID; knockout of PDYN in CeA did not significantly alter alcohol consumption in female animals; main effect of genotype: F(1,17)=2.707, p=0.118; genotype X cycle interaction F(3,51)=1.643, p=0.191. (m) Cumulative totals from individual mice. (n) PDYN knockout significantly reduced the amount of alcohol consumed during IA; main effect of genotype: F(1,17)=4.490, p=0.049. (o) PDYN knockout did not affect total fluid intake during IA; main effect of genotype: F(1,17)=4.450, p=0.501. (p) PDYN knockout significantly reduced ethanol preference; main effect of genotype: F(1,17)=6.766, p=0.019. (q) PDYN knockout did not affect cumulative ethanol consumption during IA; main effect of genotype: F(1,17)=4.135, p=0.579 (d,k) round dots indicate values at 2-hr time points and square dots indicate values at 4-hr time points (d-j) n=7 control males and n=6 knockout males. (k-q) n=10 control females and n=9 knockout females

Figure 4-. Ethanol Drinking Does Not Alter Synaptic Transmission Onto CeA PDYN Neurons.

(a) Experimental timeline. (b) U69593 inhibition of eIPSCs is unaltered by a history of alcohol drinking in males t(9.391)=0.687, p=0.508. (c) U69593 inhibition of eIPSCs is also unaltered by a history of alcohol drinking in females t(14.880)=0.054, p=0.957. (d-g) Synaptic transmission in male animals. (d) Representative traces of excitatory and inhibitory events onto CeA dynorphin neurons. (e) Ethanol drinking did not alter the amplitude of excitatory t(11.152)=0.334, p=0.745 or inhibitory transmission t(14.704)=1.644, p=0.121. (f) Ethanol drinking did not alter the frequency of excitatory transmission t(13.390)=0.075, p=0.941 or inhibitory transmission t(11.767)=0.815, p=0.431. (g) synaptic drive was also unaltered after alcohol drinking t(10.982)=0.153, p=0.881. (h-k) Synaptic transmission in female animals. (h) Representative traces of excitatory and inhibitory events onto CeA dynorphin neurons. (i) Ethanol drinking did not alter the amplitude of excitatory transmission t(9.164)= 1.170, p=0.272 or inhibitory transmission t(12.847)=0.046, p=0.964. (j) Ethanol drinking did not alter the frequency of excitatory transmission t(10.198)=0.966, p=0.356 or inhibitory transmission t(12.731)=0.950, p=0.359. (k) synaptic drive was also unaltered after alcohol drinking t(11.806)=1.182, p=0.260. (b) Water male n=9 cells, N=6 mice, ethanol male n=7 cells, n=4 mice; (c) water female n=7 cells, N=5 mice, ethanol female n=6 cells, n=4 mice; (d-g) water male n=10 cells, n=6 mice, ethanol male n=9 cells, n=4 mice; (h-k) water female n=7 cells, n=4 mice, ethanol female n=8 cells, n=5 mice

Figure 5-. Ethanol Drinking Alters the Excitability of PDYN Neurons in A Sex-Specific Manner

(a-e) Data collected from male mice. (a)There was no effect of ethanol drinking on resting membrane potential t(16.833)=0.076, p=0.940. (b) Ethanol drinking did not change action potential threshold t(16.947)=0.111, p=0.912. (c) There was no significant difference in the amount of current injected needed to elicit an action potential t(13.279)=0.860, p=0.405. (d) Representative traces of PDYN-neuron firing in CeA. (e) There was a significant interaction between ethanol drinking and the number of action potentials fired in the VI plot F(10,160)=2.004, p=0.036. (f-j) Data collected from female mice. (f) There was no effect of ethanol drinking on resting membrane potential t(13.681)=0.101, p=0.920. (g) Action potential threshold t(13.248)=1.120, p=0.283 or (h) rheobase t(13.386)=0.568, p=0.579. (i) Representative traces of CeA PDYN-neuron firing. (j) Ethanol drinking resulted in a nonsignificant decrease in the number of action potentials fired in the VI plot F(1,16)=2.523, p=0.132. (a-e) Water males: n=10 cells from n=6 mice; ethanol male: n=9 cells from N=6 mice; (f-j) water females: n=8 cells from n=5 mice; ethanol female n=9 from n=5 mice