Alcohol Withdrawal Increases Protein Kinase A Activity in the Rat Inferior Colliculus

Abstract

Background Cyclic AMP-dependent protein kinase (PKA) signaling is a key target for the action of alcohol and may therefore play a role in the pathophysiology of alcohol withdrawal seizures (AWSs). Here, we investigated the role of PKA activity with respect to increased seizure susceptibility in rats that were subjected to alcohol withdrawal. Methods Adult male Sprague-Dawley rats received three daily doses of ethanol (or vehicle) for four consecutive days. Rats were then tested for susceptibility to acoustically evoked AWSs 3, 24, and 48 hours after the last alcohol dose. In separate experiments, the inferior colliculus (IC) was collected at these same time points from rats subjected to alcohol withdrawal and control rats following alcohol withdrawal. PKA activity, catalytic Cα (PKACα) protein, regulatory RIIα (PKARIIα) protein, and RIIβ (PKARIIβ) protein were measured in the IC. Lastly, in situ pharmacological studies were performed to evaluate whether inhibiting PKA activity in the IC suppressed AWSs. Results In the ethanol-treated group, AWSs were observed at the 24-hour time point, but not at the 3-hour or 48-hour time points. In the IC, PKA activity was significantly higher both 3 hours (i.e., before AWS susceptibility) and 24 hours after the last alcohol dose (when AWS susceptibility peaked) than in control rats. Consistent with these findings, protein levels of the PKACα subunit were significantly increased in the IC both 3 hours and 24 hours after the last alcohol dose. Lastly, in situ inhibition of PKA activity within the IC suppressed AWSs. Conclusions The increase in PKA activity and PKACα protein expression in the IC preceded the occurrence of AWSs, and inhibiting PKA activity within the IC suppressed acoustically evoked AWSs. Together, these findings suggest that altered PKA activity plays a key role in the pathogenesis of AWSs.

Targeted Inhibition of Upregulated Sodium-Calcium Exchanger in Rat Inferior Colliculus Suppresses Alcohol Withdrawal Seizures

“…Four SD rats from the cohort subjected to ethanol withdrawal were tested at 3, 24, and 48 hours following ethanol withdrawal to monitor acoustically evoked AWS susceptibility. Accordingly, 100% and 75% of tested SD rats (n=4) exhibited WRSs and GTCSs 24 hours following ethanol withdrawal; no AWS susceptibility was observed at 3 and 48 hours following ethanol withdrawal, consistent with earlier reports [24,25,43,45,48].…”

“…SN-6 and KB-R7943 were dissolved in dimethyl sulfonic acid (0.1%) and phosphate-buffered saline (pH 7.4) using sonication (80 kHz, 100% power). The solutions containing SN-6 or KB-R7943 were ltered before intra-IC microinjections at 5 μg/hemisphere; this dose was chosen based on our published reports [24,43].…”

“…In our model, blood ethanol concentrations were elevated 3-hours after the last dose of ethanol when no acoustically evoked seizure susceptibility was observed but returned to control levels 24-and 48-hours later when the seizure susceptibility peaked and resolved, respectively [24][25][26]43,45,48].…”

“…Acoustically evoked seizure testing Acoustically evoked seizure testing following alcohol withdrawal was performed as previously described [24,43]. Brie y, animals were placed in an acoustic chamber.…”

Alcohol withdrawal upregulates mRNA encoding for Ca V 2.1-α1 subunit in the rat inferior colliculus

“…The lack of change in the protein expression of Ca V 2.1-α1 subunits suggests that metabolic factors such phosphorylation may contribute to increase Ca V 2.1 P-type currents in IC neurons following alcohol withdrawal. Consistent with this hypothesis, we recently reported elevated protein kinase A (PKA) expression and function in IC neurons during the development of AWS susceptibility (Akifiresoye, Miranda, Lovinger, & N’Gouemo, 2016). Whether increased PKA phosphorylation affects Ca V 2.1-α1 subunits in IC neurons remains unknown.…”