Expression of glutamic acid decarboxylase and identification of GABAergic cells in the ischemic rat dentate gyrus.

We have investigated the glutamic acid dexcarboxylase (GAD) mRNA and protein isoforms as markers for ischemic loss of GABAergic neurons in the dentate hilus. Stereological counts of these neurons were performed in rats surviving 8 days after 10 min of transient forebrain ischemia, and in control rats (sham-operated and naïve). GAD65 and GAD67 were detected by both in situ hybridization and immunocytochemistry. No differences (three-way ANOVA, P > 0.05) were found between treatments (ischemia, sham-operated or naïve) when cell counts of identical GAD isoforms were compared at the same level (mRNA or protein). However, irrespective of treatments, the number of neurons expressing GAD65 mRNA was significantly higher than the number of neurons expressing GAD65 protein, and the number of neurons expressing GAD67 mRNA was significantly lower than the number of neurons expressing GAD67 protein. In parallel, we investigated the colocalization of the cell death marker Fluorojade B (FJB) with somatostatin- or GAD67-immunoreactivity in hilus of control and ischemic rats. Although the majority of FJB positive cells also contained somatostatin, a small number of GAD67 immunoreactive neurons contained FJB, suggesting that a small number of GABAergic neurons die after ischemia. In conclusion, this study provides direct evidence that a small proportion of GABAergic hilar neurons succumbs to ischemia. Stereological counts of neurons identified from their expression of either GAD isoform of mRNA or protein revealed a high inter-animal variation at any detection level in both naïve, sham-operated and ischemic rats. Therefore, counts of GABAergic neurons should be carefully interpreted in accordance with the marker used for identification.