Figure 8 Labeling patterns in Weaver cerebellum with GAD, CaBP, and proenkephalin hybridization probes. Dark-field photomicrographs of sections of Weaver cerebellum hybridized with antisense GAD RNA (A), antisense CaBP RNA (B), antisense proenkephalin RNA (C), or sense CaBP RNA(control, D). A, B, and C: Arrows point to labeled objects in the cortical layer. D: Arrows point to locations equivalent to those marked in A, B, and C. Bar=0.5 mm.

CA3 region of the ventral hippocampus. This leads to a long-term increase in seizure susceptibility that we at first thought might be mediated by a decreased ability to produce GABA. Somewhat surprisingly, we discovered that the GABAergic interneurons of the hilus, in fact, had increased levels of GAD mRNA. We are currently investigating the other changes that accompany the reorganization of the hippocampus that follows such injections. These preliminary data, however, show that some GABAergic neurons are able to change the level of GAD mRNA in response to experimental manipulation. We can document these changes by quantitative in situ hybridization.

Summary

This work led to a number of surprises, some of which were frankly more pleasant to us than others. The most pleasant, of course, was the discovery of the enzymatic activity of the fusion protein. This allowed the unambiguous identification of the cDNA as encoding glutamate decarboxylase.

A second, less pleasant, surprise was the large size of the human GAD gene. Not only did this mean that studies of the GAD gene in humans would be more work, but it also made the detection of point mutations more challenging. The difference in the size of the rat GAD gene was also a surprise, one whose basis we do not now know.

Third, another disappointment, was that GAD maps to a region of chromosome 2 that is more or less a genetic wasteland. Despite the expectation that changes in GAD could lead to a variety of neurological and neuropsychiatric disease, so far we have found no genetic disease that cosegregates with GAD. We have no evidence for any point mutations in GAD associated with any of the diseases that we examined. As far as GAD is concerned, our focus seems to have changed from considering GAD a candidate gene to considering a variety of disorders as candidate diseases.

Figure 9 (see facing page) Labeling patterns in reeler cerebellum with GAD, calbindin, and proenkephalin hybridization probes. Dark-field photomicrographs of sections of reeler cerebellum hybridized with antisense GAD RNA (A), antisense calbindin RNA (B), antisense proenkephalin RNA (C), or sense calbindin RNA (control, D). A: Arrows point to labeled objects at the boundary between the molecular and granular layers (c=central cell mass, d=deep nucleus in A and B). B: Arrow at left points to a labeled object in the granular layer. Arrows at right point to labeled objects at the boundary between the molecular and granular layers. C: Arrows at left point to labeled objects in the deep nucleus. Arrow at right points to labeled object in the granular layer. D: c=central cell mass, d=deep nucleus, g=granular layer. Bar=0.5 mm.

Fourth, another surprise, was the expression of GAD in the gonads in a set of RNAs whose sizes differ from those of the mature 3.7 kb mRNA. We have some evidence in human GAD of differential splicing of GAD transcripts, and we hope to elucidate the structural basis of the altered RNAs in the testes and ovaries.

Our use of GAD cDNA as a tool to understand the commitment to a neuronal set of differentiation suggests that Purkinje cells and Golgi II cells of the cerebellum are committed to a GABAergic pathway early in ontogeny. These cells develop with apparently little sensitivity to their altered environments.

Finally, we examined a model that increases seizure susceptibility. While we expected to observe a decrease in overall GAD expression in GABAergic hippocampal neurons, instead we found a twofold increase in a specific subset of hilar interneurons. We think this represents a compensatory reaction to the cell death induced by the kainate injection.

Our current work is now addressing the regulatory signals responsible for specific expression of GAD and is attempting to use the GAD cDNA to produce neuronal lines in which we can study the mechanism of GABA secretion. Such overproducing GABA lines would also be useful as sources of excess GABA in grafting experiments that might provide local continuing sources of GABA.

Acknowledgments

This paper summarizes some previous and some ongoing work in my laboratory. It has been supported by grants from NINCDS (NS 22256, NS 20356) and by a program project grant to A.V. Delgado-Escueta (NS 21908). The people whose work I have discussed are Mark Erlander, Sophie Feldblum, Gretchen Frantz, Daniel Kaufman, Yutaka Kobayashi, Niranjala Tillakaratne, Teresa Wood, and Carol Wuenschell. I am grateful to them and to Karen McKeown for their contributions to this paper.

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