COMPARATIVE LOCALIZATION OF MESSENGER-RNAS ENCODING 2 FORMS OF GLUTAMIC-ACID DECARBOXYLASE WITH NONRADIOACTIVE INSITU HYBRIDIZATION METHODS

Nonradioactive in situ hybridization methods with digoxigenin-labeled cRNA probes were used to localize two glutamic acid decarboxylase (GAD ) mRNAs in rat brain. These mRNAs encode two forms of GAD that both sy nthesize GABA but differ in a number of characteristics including thei r molecular size (65 and 67 kDa). For each GAD mRNA, discrete neuronal labeling with high cellular resolution and low background staining wa s obtained in most populations of known GABA neurons. In addition, the current methods revealed differences in the intensity of labeling amo ng neurons for each GAD mRNA, suggesting that the relative concentrati ons of each GAD mRNA may be higher in some groups of GABA neurons than in others. Most major classes of GABA neurons were labeled for each G AD mRNA. In some groups of GABA neurons, the labeling for the two mRNA s was virtually identical, as in the reticular nucleus of the thalamus . In other groups of neurons, although there was substantial labeling for each GAD mRNA, labeling for one of the mRNAs was noticeably strong er than for the other. In most brain regions, such as the cerebellar c ortex, labeling for GAD67 mRNA was stronger than for GAD65 mRNA, but t here were a few brain regions in which labeling for GAD65 mRNA was mor e pronounced, and these included some regions of the hypothalamus. Fin ally, some groups of GABA neurons were predominantly labeled for one o f the GAD mRNAs and showed little or no detectable labeling for the ot her GAD mRNA, as, for example, in neurons of the tuberomammillary nucl eus of the hypothalamus where labeling for GAD67 mRNA was very strong but no labeling for GAD65 mRNA was evident. The findings suggest that most classes of GABA neurons in the central nervous system (CNS) conta in mRNAs for at least two forms of GAD, and thus, have dual enzyme sys tems for the synthesis of GABA. Higher levels of one or the other GAD mRNA in certain groups of GABA neurons may be related to differences i n the functional properties of these neurons and their means of regula ting GABA synthesis.