We used 70 male Sprague-Dawley (SD) rats (250-300 g; Taconic, Germantown, New York, Unites States) for the experiments. These animals were housed in standard polycarbonate cages with chow and water ad libitum and maintained in a temperature- and humidity-controlled room on a 12/12-hour light/dark cycle. Efforts were made to minimize the number of animals used in these experiments and their discomfort. All experimental procedures were approved by the Institution Animal Care and Use Committee (Protocol MED-20-03) and performed following the National Institutes of Health Guide for the Care and Use of Laboratory Animals .
To probe the role of NCX in the AWS pathophysiology, we used SN-6 (2-[[4-[(4-nitrophenyl)methoxy]phenyl]methyl]-4-thiazoli dinecarboxylic acid ethyl ester, R&D Systems, Minneapolis, Minnesota, United States) and KB-R7943 (2-[2-[4-(4-nitrobenzyloxy)phenyl]ethyl] isothioureamethanesulfonate, R&D Systems). SN-6 preferentially blocks NCX1rev activity, while KB-7943 inhibits NCX3rev more potently than its forward mode [40-42]. 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 filtered before intra-IC microinjections at 5 μg/hemisphere; this dose was chosen based on our published reports [24,43].
Cannula guide implantation
We used ketamine/xylazine (85/3 mg/kg, IP) to anesthetize the animals. Guide cannulae (21-gauge, Plastics One, Roanoke, VA, USA) were implanted bilaterally over the IC (9.15 mm posterior to bregma, 1.5 mm lateral to bregma), and the injection cannula was subsequently inserted vertically to 4.5 mm below the surface of the brain . A stylet was placed in each guide cannula to prevent clogging when not in use.
Ethanol administration procedure
Ethanol intoxication and withdrawal were performed as previously described [26,25,45-47]. Briefly, ethanol solution (30%, v/v, from a 95% stock solution, U.S.P., The Warner-Gram Company, Cockeysville, Maryland, Unites States) in Isomil (Abbotts’s lab, Chicago, Illinois, United States) was administered by intragastric intubation three times per day (at 8-hours intervals) for four days. The first dose of ethanol was 5 g/kg body weight, and subsequent doses were reduced and adjusted for each animal to achieve a moderate degree of intoxication determined based on a well-described intoxication scale [48,49]. The control-treated animals were maintained under similar conditions but received thrice daily the Isomil alone (without ethanol).
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 [24-26,43,45,48].
Acoustically evoked seizure testing
Acoustically evoked seizure testing following alcohol withdrawal was performed as previously described [24,43]. Briefly, animals were placed in an acoustic chamber. An auditory stimulus consisting of pure tones (100-105 decibels sound pressure level; Med Associated, St Albans, VT) was first presented until seizure activity was elicited or 60 seconds passed with no seizure activity. Animals that did not respond to tones were tested again 1-hour later using mixed sound at 110 decibels produced by an electrical bell. Acoustically evoked seizures following ethanol withdrawal consisted of wild running seizures (WRSs) that evolved into bouncing generalized tonic-clonic seizures (i.e., tonic-clonic seizures while the animal is lying on its belly, GTCSs) [24,50].
Western blot procedure
For Western blot analysis, animals subjected to ethanol withdrawal were not subjected to seizure testing because various degrees of seizure severity and duration can alter NCX protein expression . Control-treated SD rats (n=8) and ethanol-treated SD rats (n=8 per group) subjected to withdrawal (3-hour group, 24-hour group, and 48-hour group) were deeply anesthetized with Nembutal (100 mg/kg; i.p.), colliculi were surgically dissected and stored at –80°C until use. Tissue homogenates from each animal were lysed in 50 mM Tris-HCl (pH 7.4), 300 mM NaCl, 1% IGEPAL (Sigma-Aldrich, St. Louis, MO), 10% glycerol, 1 mM EDTA, and 1 mM Na3VO4 as described previously [25,42]. Briefly, nitrocellulose membranes (Bio-Rad, Hercules, CA) were incubated overnight at 4°C with primary rabbit antibodies (Alpha Diagnostic International, San Antonio, TX) against NCX1 (1:1000, Cat. #NCX12-A), NCX2 (1:1000; Cat. #NCX21-A), or NCX3 (1:1000, Cat. #NCX31-A). In addition, the membranes were incubated overnight with anti-glyceraldehyde 3-phosphate dehydrogenase (anti-GAPDH) antibody (1:10,000; Thermo Fisher Scientific, Waltham, Massachusetts, United States) at 4°C as an internal control. The membranes were probed with goat anti-rat IRDye800 (1:10,000; LI-COR Biosciences) and goat anti-rabbit IR-Dye680 (1:10,000; LI-COR Biosciences) for 1 hour at room temperature, then scanned using an Odyssey Fc imager (LI-COR Biosciences). All experiments were duplicated.
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].
Only animals exhibiting acoustically evoked AWSs (AWS-sensitive, n=24 out of 30 rats) were used for focal pharmacological studies to evaluate the antiseizure effects of NCXrev inhibitors. For microinjection procedures, stylets were removed from the implanted cannula guide tubes, and the vehicle, SN-6, or KB-R7943 was infused through an injection cannula (26 gauge, Plastics One) at a rate of 1 μl/min for 2.5 minutes; the injection cannula remained in place for additional 2.5 minutes. Acoustically evoked AWS-sensitive rats were closely monitored following intra-IC administration of vehicle (2.5 μl/hemisphere, n=8), SN-6 (5 μg/2.5 μl/hemisphere, n=8) or KB-R7943 (5 μg/2.5 μl/hemisphere, n=8) and tested for AWS susceptibility at 0.5, 1, 2, and 4 hours post-microinjections. Animals that do not show acoustically evoked AWSs (AWS-resistant) were used to assess the potential proconvulsant effects of SN-6 (5 μg/2.5 μl/hemisphere, n=3) or KB-R7943 (5 μg/2.5 μl/hemisphere, n=3). In another set of experiments, naive SD rats received intra-IC microinjections of SN- 6 (5 μg/2.5 μl/hemisphere, n=4) or KB-R7943 (5 μg/2.5 μl/hemisphere, n=4) and were subjected to 24 hours monitoring for potential abnormal behaviors. At the end of the pharmacological experiments, the animals received bilateral infusions of Fast green (0.25 μl/hemisphere, Electron Microscopy Science, Hatfield, PA, USA) at the microinjection sites. Animals were then euthanized with Nembutal or Euthasol (100 mg/kg i.p.), and coronal sections of the IC were obtained to verify the locations of microinjections microscopically.
The investigators were blinded to group allocation during experiments and data analysis. Origin 2022 software (Origin Northampton, Massachusetts, United States) was used for statistical analyses and graphs. For Western blot, we used densitometry to measure protein levels of NCX1, NCX2, and NCX3 in ethanol-treated samples relative to control-treated samples using LI-COR Image Studio Software; data from each sample was normalized to GAPDH. One-way ANOVA followed by a Bonferroni post hoc correction was performed to evaluate differences in protein expression levels. Data were first subjected to a normality test (Kolmogorov–Smirnov) and tests for homogeneity of variance (Levene’s test and Brown–Forsythe’s test) before ANOVA. For in vivo pharmacological studies and seizure testing, animals that did not display seizures, following intra-IC microinjection of SN-6 or KB-R7943, within the 60-s acoustic stimulation period were protected from seizure activity. For each group, the incidences of WRS and GTCSs were recorded and analyzed using Fisher’s Exact test. The seizure latency and seizure duration were analyzed using two-way ANOVA followed by Bonferroni post hoc correction. Finally, the seizure severity score was analyzed using the Kruskal-Wallis test. The summary data are presented as fold-change±S.E.M. for protein expression, mean±S.E.M. for seizure latency and seizure duration, median seizure score±mean average deviation for seizure severity, and the percentage (%) for the incidence of WRSs and GTCSs. Differences between groups were considered significant at P<0.05.