Final Activity Report Summary - PKCEPSILON AND ALCOHOL (Amygdala CRF in reduced anxiety and alcohol consumption in PKCepsilon null mice) Alcoholism is a chronic disorder characterised by high rates of relapse after periods of abstinence. Increasing evidence suggests that stress and anxiety induced by withdrawal from drugs of abuse contributes to vulnerability to relapse. PKCepsilon -/- mice show reduced alcohol self-administration and reduced anxiety-like behaviour (Hodge et al., 1999, 2002). PKCepsilon -/- mice also show reduced corticotrophin releasing factor (CRF) levels in the amygdala. This neuropeptide has been implicated in anxiety, including withdrawal-induced anxiety, and in alcohol consumption. Therefore, in this project, we have studied the role of amygdala PKCepsilon in these behaviours. PKCepsilon directly regulates CRF in amygdala neurons. We cultured primary amygdala neurons and show that PKCepsilon activation results in increased CRF levels. A novel tool, RNA interference, was used to selectively reduce PKCepsilon in these amygdala neurons. After PKCepsilon knockdown, PKC activators no longer increased CRF levels in cultured amygdala neurons. To determine whether the reduction in amygdala CRF is important for the behaviour of PKCepsilon -/- mice, exogenous CRF was infused into the amygdala of PKCepsilon -/- and wild type mice. Injection of exogenous CRF into the amygdala increased anxiety-like behavior of PKCepsilon -/- mice to wild-type levels, suggesting that indeed reduced CRF levels contribute to the reduced anxiety-like behaviour observed in PKCepsilon -/- mice. We next used RNA interference to reduce PKCepsilon levels in the amygdala. To achieve this, lentiviral vectors were infused into the amygdala to locally express shRNAs designed against PKCepsilon. This treatment reduced PKCepsilon levels in the amygdala by 60% compared to a control virus. Bilateral knockdown of PKCepsilon in the amygdala decreased anxiety-like behavior in wild type mice. Finally, to explore the role of amygdala PKCepsilon in alcohol consumption we adapted a limited access alcohol consumption paradigm: a two-bottle choice task with access to water and 15 % alcohol in daily 2-h sessions. With this approach, mice drink high levels of alcohol and show a marked preference for alcohol (80 %). Local knockdown of PKCepsilon in the amygdala reduced alcohol consumption in this paradigm, particularly after multiple daily drinking sessions. Taken together, the results of this project suggest that amygdala PKCepsilon directly regulates CRF expression in the amygdala and controls anxiety and alcohol consumption in mice.