Postnatal Day 7 Ethanol Exposure is Associated with Apoptosis-Related Changes in GAD67-Positive Neurons and Candidate CaMKII/GSK-3β/BAX Signaling in the Mouse Spinal Dorsal Horn

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Background: Prenatal alcohol exposure can cause neurodevelopmental injury, but the cellular events linking altered inhibitory signalling to apoptotic changes in the developing spinal dorsal horn remain incompletely understood. Methods: C57BL/6J mice received two subcutaneous ethanol injections (2.5 g/kg each, 2 h apart) on postnatal day 7 (PD7). Dorsal horn injury and GABAergic changes were assessed by histology, immunostaining, and whole-cell patch-clamp recording. Intracellular Ca2+ signalling, CaMKII and GSK-3β regulation, mitochondrial apoptosis-related markers, oxidative stress, and cell viability were examined using calcium imaging, flow cytometry, Western blotting, and co-immunoprecipitation. Tetraethylammonium chloride (TEAC) and Bay K8644 were used to explore the contribution of membrane excitability and voltage-gated Ca2+ entry. Results: Ethanol exposure was associated with altered dorsal horn neuronal morphology, increased cleaved caspase-3, and prominent involvement of GAD67-positive neurons. It was also associated with increased GABA staining and mIPSC frequency, without a clear change in mIPSC amplitude; reduced intracellular Ca2+ signals; lower CaMKII Thr286 phosphorylation and CaMKII-GSK-3β association; decreased inhibitory GSK-3β Ser9 phosphorylation; and mitochondrial apoptosis-related changes, increased ROS, reduced cell viability, and caspase-3 activation. TEAC attenuated several ethanol-associated molecular and cellular injury markers, whereas Bay K8644 moderated selected Ca2+- and mitochondrial/oxidative stress-related outcomes. Conclusion: Acute PD7 ethanol exposure was associated with neuronal injury in the developing mouse spinal dorsal horn and with coordinated changes in GABAergic signalling, Ca2+-CaMKII regulation, GSK-3β activity, and apoptosis-related markers. These findings support a candidate membrane-potential/Ca2+-sensitive GABAergic and Ca2+-CaMKII-GSK-3β/BAX signalling framework, but do not establish a fixed causal sequence or behavioural consequence.

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