THE JOURNAL OF COMPARATIVE NEUROLOGY zyx 331:339-362 (1993) Comparative Localization of mRNAs Encoding Two Forms of Glutamic Acid Decarboxylase With Nonradioactive In Situ Hybridization Methods MONIQUE ESCLAPEZ, NIRANJALA J.K. TILLAKARATNE, ALLAN J. TOBIN, AND CAROLYN R. HOUSER Brain Research (M.E., A.J.T., C.R.H.) and Molecular Biology Institutes (A.J.T.), Departments ofAnatomy and Cell Biology (M.E.,C.R.H.) and Biology (N.J.K.T.,A.J.T.), University of California, Los Angeles, and Neurology and Research Services, VA Medical Center, West Los Angeles, Wadsworth Division (C.R.H.), Los Angeles, California 90024 ABSTRACT 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 synthesize GABA but differ in a number of characteristics including their molecular size (65 and 67 kDa). For each GAD mRNA, discrete neuronal labeling with high cellular resolution and low background staining was obtained in most populations of known GABA neurons. In addition, the current methods revealed differences in the intensity of labeling among neurons for each GAD mRNA, suggesting that the relative concentrations 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 GAD mRNA. In some groups of GABA neurons, the labeling for the two mRNAs 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 stronger than for the other. In most brain regions, such as the cerebellar cortex, labeling for GAD67 mRNA was stronger than for GAD65 mRNA, but there were a few brain regions in which labeling for GAD65 mRNA was more pronounced, and these included some regions of the hypothalamus. Finally, some groups of GABA neurons were predominantly labeled for one of the GAD mRNAs and showed little or no detectable labeling for the other GAD mRNA, as, for example, in neurons of the tuberomammillary nucleus 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) contain mRNAs for at least two forms of GAD, and thus, have dual enzyme systems 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 in the functional properties of these neurons and their means of regulating GABA synthesis. CI 1993 Wiley-Liss, Inc. Key words: GABA, digoxigenin, glutamate decarboxylase, RNA probes, rat brain Neurons that use GABA as a neurotransmitter have generated a great deal of interest, not only because of their functional importance in many regions of the central nervous system (CNS), but also because of their fascinating diversity. Even within the same brain region, GABA neu- rons vary widely in their morphology (Freund et al., '83; Houser et al., '83; DeFelipe et al., zyxwvutsr '86), the specificity of their connections (Somogyi et al., '85; Freund and Antal, '88; Soriano and Frotscher, '89; Freund and Meskenaite, '921, their content of neuropeptides and calcium-binding proteins (Hendry et al., '84, '89; Schmechel et al., '84; Somogyi et al., '84; Kosaka et al., '85, '87; Lin et al., '86; Demeulemeester et al., '88; Katsumaru et al., '88; DeFelipe et al., '89; Nitsch et al., '90) and their functional effects Accepted January 25,1993. Address reprint requests to Dr. M. Esclapez, 73-364 CHS, Brain Research Institute, UCLA, Los Angeles, zyxwv CA 90024. zyxw O 1993 WILEY-LISS, INC.