Neuropeptide Y Signaling in the Central Nucleus of Amygdala Regulates Alcohol-Drinking and Anxiety-Like Behaviors of Alcohol-Preferring Rats

Background: The neuropeptide Y (NPY) system of the central nucleus of amygdala (CeA) has been shown to be involved in anxiety and alcoholism. In this study, we investigated the molecular mechanisms by which NPY in the CeA regulates anxiety and alcohol drinking behaviors using alcohol-preferring (P) rats as an animal model. Methods: Alcohol-preferring rats were bilaterally cannulated targeting the CeA and infused with artificial cerebrospinal fluid (aCSF) or NPY. Alcohol drinking and anxiety-like behaviors were assessed by the 2-bottle free-choice paradigm and light/dark box (LDB) exploration test, respectively. The levels of NPY and related signaling proteins were determined by the gold immunolabeling procedure. The mRNA levels of NPY were measured by in situ RT-PCR. Double-immunofluorescence labeling was performed to observe the colocalization of NPY and Ca2+/calmodulin-dependent protein kinase IV (CaMK IV). Results: We found that NPY infusion into the CeA produced anxiolytic effects, as measured by the LDB exploration test, and also decreased alcohol intake in P rats. NPY infusion into the CeA significantly increased levels of CaMK IV and phosphorylated cAMP responsive element-binding (pCREB) protein and increased mRNA and protein levels of NPY, but produced no changes in protein levels of CREB or the catalytic alpha-subunit of protein kinase A (PKA-C alpha) in the CeA. We also observed that alcohol intake produced anxiolytic effects in P rats in the LDB test and also increased NPY expression and protein levels of pCREB and PKA-C alpha without modulating protein levels of CREB or CaMK IV, in both the CeA and medial nucleus of amygdala. In addition, we found that CaMK IV-positive cells were co-localized with NPY in amygdaloid structures of P rats. Conclusions: These results suggest that NPY infusion may increase the expression of endogenous NPY in the CeA, which is most likely attributable to an increase in CaMK IV-dependent CREB phosphorylation and this molecular mechanism may be involved in regulating anxiety and alcohol drinking behaviors of P rats.