Co-Expression of Glutamic Acid Decarboxylase Isoform 67, Membrane Nicotinic Acetylcholine Receptors,and Connexin 36 in Ischemia-Resistant Hippocampal Interneurons

As is known, hippocampal pyramidal neurons are highly sensitive to cerebral ischemia, while some other hippocampal neurons (particularly, interneurons) survive and keep their functional activity under these conditions for a longer time. We studied interneurons of the rat hippocampal organotypic culture after 30-min-long oxygen-glucose deprivation (OGD) using immunohistochemical approaches. Four and 24 h after OGD, the somata of interneurons with no signs of degeneration (revealed by propidium iodide, PI, staining) were immunopositive to antibodies against glutamic acid decarboxylase isoform 67 (GAD67) and to an extracellular domain of a7 nicotinic acetylcholine receptor (nAChR) but negative with respect to choline acetyltransferase (ChAT). GAD67/nAChR-positive interneurons were abundant within all layers of the hippocampal CA1-CA4 zones and also in the dentate gyrus. Co-localized GAD67/nAChR immunopositivity was also observed on numerous punctuate terminals close to the somata of pyramidal neurons stained by PI. After OGD followed by incubation with a blocker of gap junctions, carbenoxolone, only single PI-stained units were revealed in the pyramidal layer. In experiments with connexin 36 cyan fluorescent protein (Cx36-CFP) on gene-reporter mice, we have found that the combination of GAD67/nAChR immunopositivity and ChAT negativity in the hippocampus is specific for the interneuronal somata expressing Cx36-CFP, a component of electrotonic gap contacts in the neuronal networks. Our results indicate that OGD-resistant hippocampal interneurons display co-localization of GAD67, a7 nAChR, and Cx36-CFP. By these neurochemical features, OGD-resistant neurons can be classified as inhibitory GABA-ergic acetylcholine-sensitive interneurons able to couple electrotonically with other hippocampal units through Cx36-CFP-containing gap junctions. The existence of hippocampal interneurons coexpressing the above factors shows that further investigations towards elucidation of cooperative endogenic mechanisms responsible for cerebral neuroresistance are expedient.