Alcohol Withdrawal-Induced Seizure Susceptibility is Associated with an Upregulation of CaV1.3 Channels in the Rat Inferior Colliculus

Abstract

Background: We previously reported increased current density through L-type voltage-gated Ca(2+) (CaV1) channels in inferior colliculus (IC) neurons during alcohol withdrawal. However, the molecular correlate of this increased CaV1 current is currently unknown.

Methods: Rats received three daily doses of ethanol every 8 hours for 4 consecutive days; control rats received vehicle. The IC was dissected at various time intervals following alcohol withdrawal, and the mRNA and protein levels of the CaV1.3 and CaV1.2 α1 subunits were measured. In separate experiments, rats were tested for their susceptibility to alcohol withdrawal-induced seizures (AWS) 3, 24, and 48 hours after alcohol withdrawal.

Results: In the alcohol-treated group, AWS were observed 24 hours after withdrawal; no seizures were observed at 3 or 48 hours. No seizures were observed at any time in the control-treated rats. Compared to control-treated rats, the mRNA level of the CaV1.3 α1 subunit was increased 1.4-fold, 1.9-fold, and 1.3-fold at 3, 24, and 48 hours, respectively. In contrast, the mRNA level of the CaV1.2 α1 subunit increased 1.5-fold and 1.4-fold at 24 and 48 hours, respectively. At 24 hours, Western blot analyses revealed that the levels of the CaV1.3 and CaV1.2 α1 subunits increased by 52% and 32%, respectively, 24 hours after alcohol withdrawal. In contrast, the CaV1.2 and CaV1.3 α1 subunits were not altered at either 3 or 48 hours during alcohol withdrawal.

Conclusions: Expression of the CaV1.3 α1 subunit increased in parallel with AWS development, suggesting that altered L-type CaV1.3 channel expression is an important feature of AWS pathogenesis.

Keywords: Cacna1c mRNA; Cacna1d mRNA; Cav1.2 α1 subunit; Cav1.3 α1 subunit; alcohol withdrawal seizures.

Figure 1.

Prevalence of acoustically-evoked generalized seizures following ethanol withdrawal. These seizures consisted of wild running seizures (WRSs) or bouncing generalized tonic-clonic seizures (clonus [C]). At 24 hours after ethanol withdrawal, 100% and 60% of animals (n = 10) exhibited WRSs and clonus, respectively. No seizures were observed at 3 (n = 6) or 48 hours (n = 6) following ethanol withdrawal, and no seizures also were observed in the control group (n = 6, *p < 0.001 versus control; chi-square test).

Figure 2.

Ethanol withdrawal upregulates Cacna1d and Cacna1c mRNA expression in the inferior colliculus (IC). The levels of Cacna1d (Ca_V1.3 α1 subunit; panel A) and Cacna1c (Ca_V1.2 α1 subunit; panel B) mRNA were measured in the IC; the data are expressed as the fold change relative to the control group. (A) The level of Cacna1d mRNA was significantly higher in the IC 24 hours after alcohol withdrawal compared to the control group and the other alcohol-treated groups. (B) The level of Cacna1c Ca_V1.2 mRNA was significantly higher in the IC 24 and 48 hours after alcohol withdrawal compared to the control group. The level of Cacna1c Ca_V1.2 mRNA also was significantly elevated 24 hours after alcohol withdrawal compared to the 3-hour group. The data are shown as mean ± standard error of the mean. n = 6 rats per group. *p < 0.05 versus control (ANOVA followed by a Bonferroni test or Student-Newman-Keuls).

Figure 3.

Ethanol withdrawal increases the protein levels of Ca_V1.3 α1 subunits in the inferior colliculus (IC). Shown in insets are representative immunoblots of the (A<B) GAPDH, (A) Ca_V1.3 and (B) Ca_V1.2 α1 subunits measured from control IC samples and IC samples obtained at the indicated times after ethanol withdrawal. The bar graphs summarize the relative protein levels of Ca_V1.3 α1 and Ca_V1.2 subunits in the IC, expressed as a percentage of the control group. The density of the 260kDa immunoreactive band (i.e. the Ca_V1.3 α1 subunit; panel A) increased significantly in the IC 24 hours after ethanol withdrawal. The density of the 210kDa immunoreactive bands (i.e. the Ca_V1.2 α1 subunit; panel B) did not change significantly following ethanol withdrawal. The summary data are shown as the mean ± standard error of the mean. n = 8 rats per group. *p < 0.05 versus control (ANOVA followed by a Bonferroni test or Student-Newman-Keuls).