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
Source: PubMed Central Open Access, NCBI / U.S. National Library of Medicine
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 mode
Abstract
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.