Sex Specific Alterations in α4*Nicotinic Receptor Expression in the Nucleus Accumbens

Abstract

Background: The mechanisms leading from traumatic stress to social, emotional and cognitive impairment and the development of mental illnesses are still undetermined and consequently there remains a critical need to develop therapies for preventing the adverse consequences of traumatic stress. Research indicates nicotinic acetylcholine receptors containing α4 subunits (α4*nAChRs) are both impacted by stress and capable of modulating the stress response. In this study, we investigated whether varenicline, a partial α4β2*nAChR agonist which reduces nicotine, alcohol and sucrose consumption, can reduce stress, a driving factor in substance use disorders. We also examined the effect of stress on nucleus accumbens (NAc) α4*nAChR expression. Methods: Transgenic mice with fluorescent tags attached to α4*nAChRs were administered varenicline and/or yohimbine (a pharmacological stressor) and plasma corticosterone and NAc α4*nAChR expression were measured. A separated group of mice were exposed to maternal separation (MS) during post-natal day (P) 2⁻14, then restraint stressed (30 min) at six weeks of age. Body weight, anxiety-like behaviours (elevated plus maze), plasma corticosterone and NAc α4*nAChR levels were measured. Results: Varenicline attenuated yohimbine-induced plasma corticosterone increases with no effect on NAc α4*nAChR expression. MS reduced unrestrained plasma corticosterone levels in both sexes. In females, MS increased body weight and NAc α4*nAChR expression, whereas, in males, MS and restraint caused a greater change in anxiety-like behaviours and plasma corticosterone levels. Restraint altered NAc α4*nAChR expression in both male and female MS mice. Conclusions: The effects of stress on NAc α4*nAChR are sex-dependent. While varenicline attenuated acute stress-induced rises in corticosterone levels, future studies are required to determine whether varenicline is effective for relieving the effects of stress.

Keywords: maternal separation; nicotinic receptors; nucleus accumbens; stress; varenicline.

Figure 1

Varenicline attenuates yohimbine-induced increases in plasma corticosterone levels: (A) treatment with yohimbine caused a significant increase in plasma corticosterone levels compared to vehicle (p < 0.001), varenicline alone (p < 0.001) and varenicline with yohimbine (p < 0.05); (B) none of the varenicline/yohimbine treatment combinations had an effect on NAc α4*nAChR expression; and (C) representative Western blot image showing the effect of vehicle, varenicline alone, yohimbine alone and varenicline with yohimbine on tallow fluorescent protein (YFP) and Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) expression. n = 5–12. One-way ANOVA with Bonferroni’s post hoc test. * p < 0.05, *** p < 0.001 compared to vehicle plus yohimbine. Veh: vehicle, Var: varenicline, Yoh: yohimbine.

Figure 2

Effect of restraint stress on anxiety-like behaviours in female mice exposed to MS. In male and female mice, following exposure to maternal separation (MS, grey), 30 min of restraint stress had no effect on the (A) percentage of time spent on the open arm or (C) total distance travelled compared to control mice (white). In female mice, restraint reduced the (B) number of entries to the open arm (p = 0.485) in MS mice. Unrestrained female control and MS mice made more entries to the open arm than unrestrained male control and MS mice and restrained male MS mice. n = 7–11 per group. Three-way ANOVA with BKY’s post hoc test. * p < 0.05 compared to unrestrained female control mice. ^# p < 0.05, ^## p < 0.01 compared to unrestrained female MS mice.

Figure 2

Effect of restraint stress on anxiety-like behaviours in female mice exposed to MS. In male and female mice, following exposure to maternal separation (MS, grey), 30 min of restraint stress had no effect on the (A) percentage of time spent on the open arm or (C) total distance travelled compared to control mice (white). In female mice, restraint reduced the (B) number of entries to the open arm (p = 0.485) in MS mice. Unrestrained female control and MS mice made more entries to the open arm than unrestrained male control and MS mice and restrained male MS mice. n = 7–11 per group. Three-way ANOVA with BKY’s post hoc test. * p < 0.05 compared to unrestrained female control mice. ^# p < 0.05, ^## p < 0.01 compared to unrestrained female MS mice.

Figure 3

Sex-specific effects of MS and restraint stress on plasma corticosterone levels. Exposure to restraint increased plasma corticosterone levels compared to unrestrained mice in female control (white) and MS mice but had no effect in male mice (A). However, expressing plasma corticosterone levels as the percentage change from basal levels showed a significantly greater plasma corticosterone rise in restrained male MS mice compared to all other male groups (B). In females, unrestrained controls displayed a small change from basal plasma corticosterone levels than all other female groups. n = 6–19 per group. Three-way ANOVA with BKY’s post hoc test. * p < 0.05, ** p < 0.01 and *** p < 0.001 compared to unrestrained female control mice. ^# p < 0.05, ^## p < 0.01, and ^### p < 0.001 compared to unrestrained female MS mice. ^$$ p < 0.01, ^$$$ p < 0.001, and ^$$$$ p < 0.0001 compared to restrained female control mice. ^& p < 0.05, ^&& p < 0.01 and ^&&& p < 0.001 compared to restrained female MS mice. ^% p < 0.05 compared to unrestrained male control mice. ^@ p < 0.05 compared to unrestrained male MS mice. ^{^} p < 0.05 compared to restrained male control mice.

Figure 4

MS attenuates restraint-induced reductions in NAc α4*nAChR expression in male mice. (A) In male control (white) but not MS (grey) mice, exposure to restraint caused a decrease in NAc α4*nAChR expression. No effects were identified in female mice. (B) The percentage change in NAc α4*nAChR expression from unrestrained conditions was greatest in female control mice. (C) Representative Western blot image showing the effect of sex, MS and restraint on YFP and GAPDH expression. n = 4–11 per group. Three-way ANOVA with BKY’s post hoc test. * p < 0.05 compared to female unrestrained control mice. ^%% p < 0.01 compared to unrestrained male control mice. ** p < 0.01, *** p < 0.001 compared to female control mice.