SNARE Complex-Associated Proteins in the Lateral Amygdala of Macaca mulatta Following Long-Term Ethanol Drinking

Abstract

Background: Recent work with long-term ethanol (EtOH) self-administration in nonhuman primate models has revealed a complex array of behavioral and physiological effects that closely mimic human alcohol abuse. Detailed neurophysiological analysis in these models suggests a myriad of pre- and postsynaptic neurobiological effects that may contribute to the behavioral manifestations of long-term EtOH drinking. The molecular mechanisms regulating presynaptic effects of this chronic EtOH exposure are largely unknown. To this end, we analyzed the effects of long-term EtOH self-administration on the levels of presynaptic SNARE complex proteins in Macaca mulatta basolateral amygdala, a brain region known to regulate both aversive and reward-seeking behaviors.

Methods: Basolateral amygdala samples from control and EtOH-drinking male and female monkeys were processed. Total basolateral amygdala protein was analyzed by Western blotting using antibodies directed against both core SNARE and SNARE-associated proteins. We also performed correlational analyses between protein expression levels and a number of EtOH drinking parameters, including lifetime grams of EtOH consumed, preference, and blood alcohol concentration.

Results: Significant interactions or main effects of sex/drinking were seen for a number of SNARE core and SNARE-associated proteins. Across the range of EtOH-drinking phenotypes, SNAP25 and Munc13-1 proteins levels were significantly different between males and females, and Munc13-2 levels were significantly lower in animals with a history of EtOH drinking. A separate analysis of very heavy-drinking individuals revealed significant decreases in Rab3c (females) and complexin 2 (males).

Conclusions: Protein expression analysis of basolateral amygdala total protein from controls and animals following long-term EtOH self-administration suggests a number of alterations in core SNARE or SNARE-associated components that could dramatically alter presynaptic function. A number of proteins or multiprotein components were also correlated with EtOH drinking behavior, which suggest a potentially heritable role for presynaptic SNARE proteins.

Keywords: Complexin 1/2; Macaque; Munc13-1/2; Munc18-1; Rab3a/c.

Figure 1. Ethanol drinking and sex alter expression levels for some proteins involved in vesicle priming

Exemplar blots for each protein are shown beneath the bar graphs with approximate molecular weights of the target indicated to the left. “C” and “E” denote lateral amygdala samples prepared from non-drinking controls (also denoted as “CON”) and ethanol drinkers (also “EtOH”), respectively. Protein expression level is denoted as ‘arbitrary units’ (see Methods). (A) Rab3a protein expression is significantly reduced in samples from ethanol drinkers (two-way ANOVA; main effect of ethanol, p<0.05, see text) regardless of sex. (B) No significant effects of either ethanol drinking or sex on Rab3c protein expression. (C) Munc13-1 protein expression is significantly higher in samples prepared from females compared to males (main effect of sex, p<0.001, see text). Dashed line separates male and female samples in an exemplar blot. (D) Ethanol drinking significantly decreases protein expression of Munc13-2 (main effect of ethanol, p<0.05). (E) No significant effect of ethanol drinking or sex on vGluT1 protein expression. (F) No significant effect of ethanol drinking or sex on vGluT2 protein expression.

Figure 2. Correlational analysis between protein expression and drinking variables for proteins involved with vesicle priming

Correlations were performed between protein expression levels (arbitrary units) and lifetime ethanol intake during the voluntary consumption periods (in grams, left column; see Methods) or average blood alcohol concentration (mg%; right column) only for those proteins without a significant main effect of ethanol drinking (Fig. 1). Protein levels from male (red lines and symbols) and female samples (green lines and symbols) are shown separately. Statistically significant correlations are denoted by R² and P values; and 90% confidence limits are indicated by the dashed lines. (A) Protein levels of Rab3c were significantly correlated (slope = −0.33±0.13, F=6.115) with total voluntary ethanol intake but not average BAC in females. (B) Munc13-1 protein expression levels were significantly correlated with average BAC (slope = −3.12±1.14, F=7.48) but not total voluntary intake in males. (C) There is no significant correlation between vGluT1 protein levels and either voluntary consumption or BAC. (D) vGluT2 protein levels are significantly correlated with BAC (slope=−27.2±10.6, F=6.6), but not ethanol intake, in lateral amygdala from male drinkers.

Figure 3. Ethanol drinking and sex alter the expression of lateral amygdala proteins involved with presynaptic vesicle release

Notations for the exemplar blots are the same as for Figure 1. (A) Total protein levels for Munc18-1 (A₁) and for Munc18-1 phosphorylated on Ser515 (A₂) were not significantly altered by either sex or ethanol drinking. However, when ‘active’ Munc18-1 was calculated as the ratio between pSer515 Munc18-1 divided by total Munc18-1 (A₃), there was a trend for an interaction between the ethanol drinking and sex main factors. (B & C) No significant effect of either sex or ethanol drinking on VAMP1 or VAMP2 protein levels. (D) SNAP25 protein levels were significantly higher in female lateral amygdala samples compared to male samples (two-way ANOVA, P<0.001 across the sex main factor). (E & F) Ethanol drinking significantly reduced protein levels for Complexin 2 (P<0.05) with a trend for Complexin 1 levels. (G) No significant effect of either sex or ethanol drinking on Syntaxin 1 protein levels in lateral amygdala samples.

Figure 4. Correlational analyses for protein expression levels versus ethanol drinking variables reveals significant relationships for VAMP2 and Syntaxin 1

(A & B) No significant relationship between total Munc18-1 protein levels or phosphorylated Munc18-1 and either total voluntary consumption or BAC. (C) No correlation between VAMP1 levels and either ethanol intake of BAC. (D) VAMP2 protein expression was significantly correlated with voluntary consumption (slope = 1.96±0.57, F=11.67), but not BAC, in lateral amygdala samples from male drinkers. (E) No correlation between SNAP25 protein levels and ethanol intake or BAC. (F) Level of Syntaxin 1 were significantly correlated with BAC (slope = −2.83±1.27, F = 4.934), but not voluntary intake, in male lateral amygdala.

Figure 5. Functional relationships between presynaptic vesicle priming and release proteins from lateral amygdala samples examined in the current manuscript

Parenthetical statements indicate significant main effects, either sex or ethanol drinking, sound in the western analysis. Asterisks denote proteins significantly correlated with either total voluntary ethanol consumption or average blood alcohol concentration (see text).