Inhibition of fatty acid amide hydrolase in the central amygdala alleviates co-morbid expression of innate anxiety and excessive alcohol intake

Abstract

Fatty acid amide hydrolase (FAAH) is an enzyme that prominently degrades the major endocannabinoid N-arachidonoylethanolamine (anandamide). Inhibition of this enzyme leads to increased anandamide levels in brain regions that modulate stress and anxiety. Recently, we found that genetically selected Marchigian Sardinian alcohol-preferring (msP) rats display hyperactive FAAH in amygdalar regions that was associated with increased stress sensitivity and a hyper-anxious phenotype. Our previous work has also demonstrated that msPs display an innate preference for and excessive consumption of alcohol, potentially reflecting a form of self-medication to gain relief from hyper-anxious states. Here, we expand on our previous work by microinjecting the selective FAAH inhibitor URB597 (vehicle, 0.03, 0.1 and 1.0 μg per rat) into the central amygdala (CeA) and basolateral amygdala in msP versus non-selected Wistar rats to evaluate the effects of localized FAAH inhibition on operant alcohol self-administration and restraint-induced anxiety using the elevated plus maze. Intra-CeA URB597 significantly reduced alcohol self-administration in msP but not in Wistar rats. Intra-basolateral amygdala URB597 also attenuated alcohol drinking in msPs, although the effect was less pronounced relative to CeA treatment. In contrast, control experiments administering URB597 into the ventral tegmental area produced no genotypic differences in drinking. We also found that URB597 treatment in the CeA significantly reduced the anxiogenic effects of restraint stress in msPs, although no effects were detected in Wistars. Dysregulation of FAAH regulated systems in the major output region of the amygdala may drive the propensity for co-morbid expression of anxiety and excessive alcohol use.

Keywords: abuse; addiction; alcoholism; ethanol; stress.

Figure 1:

Effect of intra-CeA injections of URB597 on alcohol self-administration in a) msP rats and b) Wistar rats. URB597 at the doses of 0.1 and 1.0 μg/rat decreased alcohol-reinforced active lever responding in msP but not Wistar rats. Values represent the mean ± (SEM) of number of rewards obtained by active lever responding (top panel) and responses on the inactive lever (bottom panel). Significant differences from vehicle-treated rats (Veh): *p<0.05, **p<0.01.

Figure 2:

Effect of intra-BLA injections of URB597 on alcohol self-administration in a) msP rats and b) Wistar rats. URB597 at the dose of 1.0 μg/rat decreased alcohol-reinforced responding in msP but not Wistar rats. Values represent the mean ± (SEM) of number of rewards obtained by active lever responding (top panels) and responses on the inactive lever (bottom panels). Significant differences from vehicle-treated rats (Veh), **p<0.01

Figure 3:

Effect of intra-VTA injections of URB597 on alcohol self-administration in msP rats. URB597 did not alter alcohol-reinforced active lever responding. Values represent the mean ± (SEM) of number of rewards obtained by active lever responding and responses on the inactive lever.

Figure 4:

Effect of intra-CeA injections of URB597 (1.0 μg/rat) in msP rats on: a) % time spent in open arms; b) % number of open arm entries, c) % number of closed arm entries, d) total arm entries. URB597 exerted an anxiolytic-like effect in rats subjected to restraint stress as indicated by a higher percentage of time spent in the open arms. The effect of URB597 on open and closed arm entries are consistent with evidence of increased time spent exploring the open arms. Data are represented as the mean ± SEM. ###p<0.001 statistical differences versus V-STR (vehicle, restrained animals); **p<0.01***p<0.001 statistical differences versus V-V (vehicle, non-restrained animals).

Figure 5:

Effect of intra-CeA injections of URB597 (1.0 μg/rat) in Wistar rats on a) % time spent in open arms; b) % number of open arm entries, c) % number of closed arm entries and d) total arm entries. As opposed to the data collected from msP rats, URB597 did not differentially alter EPM behaviors in Wistars. Data are represented as the mean ± SEM.