Differential potassium channel gene regulation in BXD mice reveals novel targets for pharmacogenetic therapies to reduce heavy alcohol drinking

Abstract

Alcohol (ethanol) dependence is a chronic relapsing brain disorder partially influenced by genetics and characterized by an inability to regulate harmful levels of drinking. Emerging evidence has linked genes that encode K_V7, K_IR, and K_Ca2 K⁺ channels with variation in alcohol-related behaviors in rodents and humans. This led us to experimentally test relations between K⁺ channel genes and escalation of drinking in a chronic-intermittent ethanol (CIE) exposure model of dependence in BXD recombinant inbred strains of mice. Transcript levels for K⁺ channel genes in the prefrontal cortex (PFC) and nucleus accumbens (NAc) covary with voluntary ethanol drinking in a non-dependent cohort. Transcripts that encode K_V7 channels covary negatively with drinking in non-dependent BXD strains. Using a pharmacological approach to validate the genetic findings, C57BL/6J mice were allowed intermittent access to ethanol to establish baseline consumption before they were treated with retigabine, an FDA-approved K_V7 channel positive modulator. Systemic administration significantly reduced drinking, and consistent with previous evidence, retigabine was more effective at reducing voluntary consumption in high-drinking than low-drinking subjects. We evaluated the specific K⁺ channel genes that were most sensitive to CIE exposure and identified a gene subset in the NAc and PFC that were dysregulated in the alcohol-dependent BXD cohort. CIE-induced modulation of nine genes in the NAc and six genes in the PFC covaried well with the changes in drinking induced by ethanol dependence. Here we identified novel candidate genes in the NAc and PFC that are regulated by ethanol dependence and correlate with voluntary drinking in non-dependent and dependent BXD mice. The findings that Kcnq expression correlates with drinking and that retigabine reduces consumption suggest that K_V7 channels could be pharmacogenetic targets to treat individuals with alcohol addiction.

Keywords: Alcohol use disorder; BXD mice; K(+) channels; Nucleus accumbens; Pharmacogenetics; Prefrontal cortex.

Figure 1. Schematic of the drinking models used in BXD RI strains of mice and the C57BL/6J parental strain

(A) The two-bottle choice limited-access voluntary drinking and chronic intermittent ethanol (CIE) exposure paradigm used in BXD RI strains of mice. Mice were given access to ethanol (15% v/v) for 2 hr using a limited-access 2-bottle choice (LA-2BC) procedure for 6 weeks to establish baseline ethanol intake. Mice then received either CIE vapor in inhalation chambers or air in control chambers. After chamber exposure, mice entered a 72 hr abstinence (Abs) period and were then tested for ethanol drinking for 5 days. This pattern of chamber exposure, abstinence, and voluntary drinking was repeated for 4 cycles. Brain tissue was collected 72 hr after the final chamber exposure. The percent change in drinking (Δ) was calculated as the percent change between Intake Test 4 and the 6^th week of baseline drinking. (B) Schematic of the intermittent access 2-bottle choice (IA-2BC) paradigm used in C57BL/6J mice. In this model, mice had access to 20% ethanol (v/v) for 24 hr three days a week with a 24 hr forced abstinence period in between drinking sessions. Mice were then treated with retigabine after 7 weeks of baseline drinking.

Figure 2. The Kv7 channel positive modulator, retigabine, reduced ethanol consumption in high-drinking, but not low-drinking male C57BL/6J mice

(A) Average weekly ethanol consumption and (B) intra-strain variability of 24 hr ethanol consumption in C57BL/6J mice during week 7 of the intermittent access model. (C) Despite the large between-subjects variation, the coefficient of variance during 7 weeks of access to ethanol in the intermittent model indicates increased within-subject stability (n = 19 mice, * p < .05 vs week 1, ** p < .05 vs week 1, ⁺ p < .0001 vs week 1, ⁺⁺ p < 0.001 vs week 3). (D) Overall, retigabine (10 mg/kg, IP) reduced drinking when measured at 2, 4 and 24 hr in C57BL/6J mice (*p < .005 vs vehicle, main effect of treatment). (E, F) Retigabine (5 and 10 mg/kg) significantly reduced drinking in high-drinking, but not low-drinking C57BL/6J mice (*p < .01 vs vehicle, main effect of treatment). (G) HLM analyses revealed a strong negative linear relationship (p = 0.025) between vehicle drinking and RTG-induced alteration of ethanol intake across time, in that the greater the vehicle drinking, the more K_V7 channel activation reduced ethanol consumption.

Figure 3. K⁺ channel gene expression changes in the nucleus accumbens induced by chronic intermittent ethanol exposure of BXD strains of mice

Strain heat maps of significantly different gene expression responses based on S-score analysis in the nucleus accumbens (n = 21 strains). Positive S-scores (shown in red) indicate up-regulation by CIE exposure and negative S-scores (shown in green) indicate down-regulation by CIE exposure.

Figure 4. Intrinsic excitability changes revealed by computational modeling of K⁺ channel gene expression adaptations in the prefrontal cortex of ethanol dependent BXD mice

(A) Strain heat maps of significantly different gene expression responses based on S-score analysis in the prefrontal cortex (n = 23 strains). Positive S-scores (shown in red) indicate up-regulation by CIE exposure and negative S-scores (shown in green) indicate down-regulation by CIE exposure. (B) Traces showing simulated evoked action potentials in response to depolarizing current steps (300, 400, 500 pA) under control conditions (black traces), 0.5-fold down-regulation of channel activity (green traces), and 1.5-fold increase in channel conductance (red traces). (C) Input-output relationship for each simulation. The number of action potentials produced by each current step is compared across the simulations. The 1.5-fold model revealed a reduction in the number of action potentials elicited by current injection at the soma. (D, E) The rheobase current and ISI are increased in the 1.5-fold model simulations, resulting in fewer action potentials evoked by current injection (*p < 0.05 vs. CTL stimulations).