The decreased phosphorylation of cyclic adenosine monophosphate (cAMP) response element binding (CREB) protein in the central amygda acts as a molecular substrate for anxiety related to ethanol withdrawal in rats

Background: Several lines of evidence indicate a high comorbidity between anxiety and alcohol abuse. This study investigated the molecular mechanisms in the amygdaloid neurocircuitry, governing anxiety related to ethanol withdrawal and also the phenomenon of alcohol preference. Methods: Male Sprague Dawley(R) rats were treated with ethanol or control diet for 15 days, and ethanol-fed rats were withdrawn for 0 and 24 hr. Ethanol-withdrawn or control diet-fed rats were bilaterally infused into central or basolateral amygdala with artificial cerebrospinal fluid or protein kinase A (PKA) activator or inhibitor. These rats were used to measure anxiety levels by the elevated plus-maze test. Protein levels of various signaling molecules related to cyclic adenosine monophosphate (cAMP)-response element binding (CREB) protein signaling in amygdaloid structures were determined by gold immunolabeling procedure. The messenger RNA levels of neuropeptide Y were determined by in situ polymerase chain reaction procedure. Results: Ethanol withdrawal (24 hr) after chronic exposure (15 days) produced anxiety in rats as measured by elevated plus-maze test. Ethanol withdrawal but not treatment significantly decreased the phosphorylation of CREB protein and protein levels of Ca2+/calmodulin-dependent protein kinase TV without modulating the protein levels of total CREB and alpha-catalytic subunit of protein kinase A (PKA-Calpha) in the central and medial amygdala. However, these changes were not observed in the basolateral amygdala. We also investigated the effects of manipulation of the phosphorylation status of CREB in the central amygdala by infusion of the PKA activator (Sp-cAMPS) or inhibitor (Rp-cAMPS) on anxiety levels in rats during ethanol withdrawal. When Sp-cAMPS is specifically infused into the central amygdala, it dose-dependently normalizes the decrease in CREB phosphorylation and prevents the development of anxiety in rats during ethanol withdrawal. On the other hand, Rp-cAMPS infusions into the central or basolateral amygdala decrease CREB phosphorylation, but only infusion into the central amygdala provokes anxiety and increases alcohol preference in normal rats. We also found that alcohol preference provoked by decreased CREB phosphorylation is related to decreased expression of the neuropeptide Y gene in the central amygdala. Conclusions: These novel results suggest the possibility that decreased CREB phosphorylation in the central amygdala acts as a common molecular correlate for anxiety and alcohol-drinking behaviors and also is correlated with anxiety related to ethanol withdrawal.