Localization of cells preferentially expressing GAD(67) with negligible GAD(65) transcripts in the rat hippocampus. A double in situ hybridization study

Two major forms of glutamic acid decarboxylase (GAD) are present in the mam malian brain, a 65-kDa isoform (GAD(65)) and a 67-kDa isoform (GAD(67)), an d it is usually assumed that all GABAergic neurons contain both. The two fo rms have not yet been colocalized to the same neurons, because the GAD(65) protein is found almost exclusively in axon terminals, while GAD(67) is fou nd predominantly in the cell body. Using double in situ hybridization (DISH ) with both radioactive [S-35] and non-radioactive (digoxigenin, DIG) probe s, the distributions of GAD(65) and GAD(67) mRNA have been simultaneously e xamined in the rat hippocampus. The results suggest that [S-35] radioprobes are slightly more sensitive than DIG probes, and that the reversal of labe ls is necessary in DISH studies to determine whether a neuronal subtype whi ch expresses only one isoform of GAD may be present. The data indicate that the majority of cells (90%) showing labeling were labeled for both GAD(65) and GAD(67) mRNA. In sectors CA1 and CA3 approximately 5-10% of the cells positive for GAD(67) showed little or no detectable GAD(65) mRNA, In the hi lus, however, GAD(65) levels were higher, and all cells seem to express bot h GAD(65) and GAD(67) mRNA. Taken together, these results support the view that most GABAergic neurons in the hippocampus express both GAD(65) and GAD (67). 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 GAD(65) mRNA levels very low or absent. (C) 1999 Elsevier Science B.V. All rights reserved.