Localization of cells preferentially expressing GAD67 with negligible GAD65 transcripts in the rat hippocampus. A double in situ hybridization study

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Two major forms of glutamic acid decarboxylase (GAD) are present in the mammalian brain, a 65-kDa isoform (GAD65) and a 67-kDa isoform (GAD67), and it is usually assumed that all GABAergic neurons contain both. The two forms have not yet been colocalized to the same neurons, because the GAD65 protein is found almost exclusively in axon terminals, while GAD67 is found predominantly in the cell body. Using double in situ hybridization (DISH) with both radioactive [35S] and non-radioactive (digoxigenin, DIG) probes, the distributions of GAD65 and GAD67 mRNA have been simultaneously examined in the rat hippocampus. The results suggest that [35S] radioprobes are slightly more sensitive than DIG probes, and that the reversal of labels is necessary in DISH studies to determine whether a neuronal subtype which expresses only one isoform of GAD may be present. The data indicate that the majority of cells (90%) showing labeling were labeled for both GAD65 and GAD67 mRNA. In sectors CA1 and CA3 approximately 5–10% of the cells positive for GAD67 showed little or no detectable GAD65 mRNA. In the hilus, however, GAD65 levels were higher, and all cells seem to express both GAD65 and GAD67 mRNA. Taken together, these results support the view that most GABAergic neurons in the hippocampus express both GAD65 and GAD67. However, it appears that some interneurons in the CA subfields differ from “classic” GABAergic interneurons by preferentially expressing the 67-kDa isoform of GAD under baseline conditions, with GAD65 mRNA levels very low or absent.