Our findings provide evidence for a PKCε-dependent signaling pathway within the amygdala that is important for ethanol consumption in mice. These results provide a rationale for the development of PKCε inhibitors to treat alcohol use disorders.
For this study, we adapted a dark-phase, limited access single bottle paradigm (Lopez & Becker, 2005
; Rhodes et al., 2005
) to include a free-choice between water and ethanol using two bottles. With the conditions used here, hybrid mice consumed amounts of ethanol associated with mild intoxication in humans. Moreover, the mice showed a high preference (> 75%) for the ethanol solution over water. By comparison, ethanol preference is approximately 40% in C57BL/6J × 129S4/SvJae mice when given 24-h access to water and ethanol (Hodge et al., 1999
). This striking increase in preference for ethanol was not due to a preference for one side of the cage since the bottle position was changed every day. Therefore, restricting access to ethanol and providing ethanol during the early dark phase of the light-dark cycle increases the motivation of mice to consume ethanol.
PKCε null mice show reduced ethanol consumption and reward in unlimited access (Besheer et al., 2006
; Hodge et al., 1999
; Wallace et al., 2007
), operant self-administration (Olive et al., 2000
) and place preference (Newton & Messing, 2007
) paradigms. Here, using RNA interference, we identify the amygdala as an anatomical site of action for PKCε that regulates ethanol intake and preference. Heterozygous mice with a ~55% reduction in the abundance of PKCε in the amygdala, equivalent to that obtained using the lentiviral vector (Lesscher et al., 2008
), also showed a reduction in ethanol intake thus supporting our findings with the lentiviral vector. The size and pattern of effect is however different from the behavioral changes observed for PKCε knockdown mice. While local knockdown of PKCε in the amygdala reduces ethanol intake and preference and prevented the development of high ethanol intake and ethanol preference, heterozygous mice show an overall reduction in ethanol intake and preference but increase their drinking behavior over time. This apparent discrepancy can likely be explained by the fact that the reduction in PKCε abundance in PKCε+/−
mice is not confined to the amygdala. Thus far we have observed similar reductions in prefrontal cortex and whole brain lysates (P.M.N and R.O.M., unpublished observations), suggesting that PKCε+/−
mice have a reduced abundance of PKCε throughout the central nervous system. Our findings suggest that PKCε signaling, particularly in the amygdala, is important for ethanol intake and ethanol preference in mice.
Ethanol stimulates GABA release in the amygdala and we have recently shown that this requires PKCε (Bajo et al., 2008
). GABA antagonists infused in the amygdala reduce operant responding for ethanol (Hyytia & Koob, 1995
) suggesting that ethanol stimulation of GABA release in the amygdala acts as a feed-forward signal to promote ethanol self-administration. PKCε signaling in the amygdala may therefore facilitate ethanol intake through regulation of GABA release in the amygdala.
PKCε could also regulate alcohol intake through its actions at GABAA
receptors containing γ2 subunits. Chronic ethanol intake by nonhuman primates is associated with a decrease in the abundance of mRNA for γ2, and a decrease in sensitivity of amygdala GABAA
receptors to benzodiazepines (Anderson et al., 2007
). We have recently shown that the γ2 subunit of GABAA
receptors is a substrate for PKCε, and that phosphorylation by PKCε decreases the sensitivity of GABAA
receptors to benzodiazepines and ethanol (Qi et al., 2007
). Further studies will be necessary to determine if a PKCε-mediated alteration in the sensitivity of amygdala GABAA
receptors to ethanol regulates voluntary ethanol consumption.
Another possible mechanism by which PKCε could regulate alcohol intake may involve amygdala corticotropin-releasing factor (CRF). Amygdala CRF has been implicated in ethanol consumption, particularly in alcohol-dependent animals. Upon withdrawal from ethanol, CRF levels in the amygdala are elevated (Merlo Pich et al., 1995
). These changes are thought to drive ethanol consumption because CRF antagonists can reverse the increase in ethanol intake observed in ethanol-withdrawn rats (Valdez et al., 2002
). Recently, we have shown that PKCε regulates levels of amygdala CRF (Lesscher et al., 2008
mice show a 50% reduction in CRF peptide and mRNA in the amygdala. Furthermore, activation of PKCε increases CRF levels in primary amygdala neurons. Therefore it is possible that PKCε signaling in the amygdala controls voluntary ethanol consumption through regulation of amygdala CRF. Ongoing experiments with CRF1 receptor antagonists, which reduce ethanol consumption in rats (Funk et al., 2006b
; Marinelli et al., 2007
), should address this hypothesis. However, in rats, CRF antagonists, given systemically or infused into the amygdala, reduce ethanol consumption only in alcohol-dependent animals withdrawn from chronic intermittent ethanol exposure. These antagonists are without effect on voluntary ethanol consumption in non-dependent rats. Under the conditions of our limited access paradigm, mice drank moderate levels of alcohol and achieved blood alcohol levels (~50 mg/dl) that are not expected to result in dependence. Therefore it is likely that there are other PKCε-dependent mechanisms at work in this model that are CRF-independent and are yet to be elucidated.
Identification of downstream targets of PKCε in the amygdala that control voluntary ethanol consumption is an important challenge for future studies and will provide further insight in the neurobiological processes that increase ethanol consumption after repeated exposure.