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

Abstract

The inferior colliculus (IC) is critical in initiating acoustically evoked alcohol withdrawal-induced seizures (AWSs). Recently, we reported that systemic inhibition of Ca²⁺ entry via the reverse mode activity of the Na⁺/Ca²⁺ exchanger (NCX_rev) suppressed AWSs, suggesting remodeling of NCX expression and function, at least in the IC, the site of AWS initiation. Here, we probe putative changes in protein expression in the IC of NCX isoforms, including NCX type 1 (NCX1), 2 (NCX2), and 3 (NCX3). We also evaluated the efficacy of targeted inhibition of NCX1_rev and NCX3_rev activity in the IC on the occurrence and severity of AWSs using SN-6 and KB-R943, respectively. We used our well-characterized alcohol intoxication/withdrawal model associated with enhanced AWS susceptibility. IC tissues from the alcohol-treated group were collected 3 h (before the onset of AWS susceptibility), 24 h (when AWS susceptibility is maximal), and 48 h (when AWS susceptibility is resolved) following alcohol withdrawal; in comparison, IC tissues from the control-treated group were collected at 24 h after the last gavage. Analysis shows that NCX1 protein levels were markedly higher 3 and 24 h following alcohol withdrawal. However, NCX3 protein levels were only higher 3 h following alcohol withdrawal. The analysis also reveals that bilateral microinjections of SN-6 (but not KB-R7943) within the IC markedly suppressed the occurrence and severity of AWSs. Together, these findings indicate that NCX1 is a novel molecular target that may play an essential role in the pathogenesis and pathophysiology of AWSs.

Keywords: Calcium signaling; Generalized tonic–clonic seizures; KB-R7943; Protein expression; SN-6.

Figure 1.. Experimental design

For molecular study, twenty-four rats subjected to ethanol withdrawal and not tested for seizures were randomly used at the 3^rd, 24^th, and 48^th hour time points. Control-treated rats were used at the 24^th time point after ethanol withdrawal. For pharmacological studies, twenty-four rats subjected to ethanol withdrawal and exhibiting seizure susceptibility were randomly assigned to three groups control-treated, SN-6, and KB-R7943. Seizure-resistant rats were used to evaluate the potential proconvulsant effect of inhibiting NCX reverse activity within the central nucleus of the IC. Twelve naïve Sprague-Dawley rats were used for the assessment of gross behaviors. These rats were assigned in three groups, including vehicle-treated (n=4), SN-6 (n=4), and KB-R7943 (n=4).

Figure 2.. Ethanol withdrawal increases the protein levels of NCX1 and NCX3 in the inferior colliculus before the onset of seizure susceptibility

Shown in insets are representative immunoblots of NCX1 (panel A), NCX2 (panel B), and NCX3 (panel C) measured from the control-treated samples collected 24 hours after vehicle administration and samples obtained at the indicated times after ethanol withdrawal. The bar graphs summarize the relative protein levels of NCX1, NCX2, and NCX3 in the IC after ethanol withdrawal, expressed as a percentage of the control-treated group. The density of the 120-kDa immunoreactive band (i.e., NCX1; panel A) increased significantly in the IC at the 3^rd and 24^th hour after ethanol withdrawal compared to the control-treated group. The density of the 103-kDa immunoreactive bands (i.e., NCX2; panel B) did not change significantly after ethanol withdrawal compared to the control-treated group. The density of the 106-kDa immunoreactive band (i.e., NCX3; panel C) increased significantly in the IC at the 3^rd hours after ethanol withdrawal compared to the control-treated group, and the ethanol withdrawal 24^th and 48^th hour time-points. The summary data are shown as the mean ± S.E.M. (8 rats per group). *P < 0.05 versus control (ANOVA followed by a Bonferroni correction).

Figure 3.

Representative sites of focal microinjections within inferior colliculi. All identified sites of microinjections were in the central nucleus of the IC, according to the atlas of Paxinos and Watson [44]. Vehicle (filled triangles), SN-6 (filled circles), and KB-R7943 (filled diamonds).

Figure 4.

Effects of focal microinjections of SN-6 and KB-R7943 within the central nucleus of the IC on the incidence, latency, duration, and severity of ethanol withdrawal seizures The effects of focal IC microinjections of SN-6 (5μg/2.5μl/hemisphere) and KB-R7843 (5μg/2.5μl/hemisphere), inhibitors of NCX1rev and NCX3rev activity, respectively, were evaluated at various post-treatment time points in animals exhibiting AWS susceptibility. A. Lower incidence of WRSs was observed at the 2^nd and 4^th hour posttreatment time points. No notable change in WRSs incidence was observed following focal IC microinjections of KBR-7943. B. Focal microinjection of SN-6 within the IC markedly reduced the incidence of GTCs at all tested posttreatment time points. Focal microinjection KB-R7943 within the IC also lowered the incidence of GTCS but at the 1^st, 2^nd, and 3^rd hour posttreatment time points. C. Focal microinjections of SN-6 within the IC significantly delayed the onset of seizures at the 2^nd and 4^th hour posttreatment time points. Focal IC microinjections of KB-R7943 did not substantially alter the seizure onset. D. Focal microinjections of SN-6 (but not KB-R7943) within the IC significantly reduced the seizure duration at all tested posttreatment time points. E. Focal microinjections of SN-6 (but no KB-7943) within the IC reduced and suppressed the seizure severity by the 1^st hour posttreatment time point. The summary data are mean %±S.E.M of WRSs and GTCSs, mean±S.E.M for seizure latency and duration, and median±S.E.M for seizure severity. *P < 0.05, **P < 0.01 versus vehicle-treated group Fisher exact test. *P < 0.05, **P < 0.01 versus vehicle-treated group Kruskal Wallis test for seizure severity or two-way ANOVA followed by Bonferroni correction for seizure latency and severity.