Elsevier

Molecular Brain Research

Volume 71, Issue 2, 25 August 1999, Pages 201-209
Molecular Brain Research

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

https://doi.org/10.1016/S0169-328X(99)00185-0Get rights and content

Abstract

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.

Introduction

The synthesis of GABA from glutamate in the brain is dependent upon the enzyme glutamate decarboxylase (GAD). Two distinct isoforms of GAD exist: a 67-kDa form (GAD67) and a 65-kDa form (GAD65), both of which are capable of synthesizing GABA [13]. Each form has been conserved during phylogenesis, with rat and human forms showing approximately 98% and 94% homology for GAD67 and GAD65, respectively. Within a given species, however, there is much less conservation. In rat, for example, the identity between GAD65 and GAD67 is only 65% [13]. While it is generally believed that most GABAergic neurons express both forms of GAD, this hypothesis has never been systematically tested. In the tuberomammillary nucleus of the hypothalamus, a subpopulation of neurons has been shown to express high levels of GAD67 with little or no GAD65 being detectable [18]. This observation raises the possibility that a unique subpopulation of GABAergic cells which preferentially expresses GAD67 or GAD65 may exist in other regions of the brain.

Immunocytochemical colocalizations of GAD67 and GAD65 are confounded by the fact that the two isoforms show different subcellular localizations. For example, immunoreactivity (IR) for GAD65 is predominantly found in axon terminals 17, 23, 25, while immunoreactive product for GAD67, though also detected in axon terminals, shows a predilection for cellular bodies [22]. This differential cellular distribution makes it virtually impossible to use a double immunohistochemical approach to determine if both forms are constitutively expressed in all or only some GABAergic cells. The differential distribution of GAD65 and GAD67 IR within neurons, however, suggests the possibility that there may be functional differences for the two forms, and provides support for the idea that some neurons may only synthesize one isoform of GAD without the other.

While previous in situ hybridization studies of the distribution of GAD mRNAs 18, 19have demonstrated that both isoforms are usually found in the same neuronal populations, single-labeling in situ hybridization cannot reliably determine if individual cells are expressing both forms. In many populations of neurons, GAD67 mRNA levels appear to be higher than those of GAD65 [19]. Such a difference could occur if all of the cells in these populations are expressing higher levels of GAD67 than of GAD65. Alternatively, there could be a neuronal subpopulation which primarily or perhaps exclusively expresses GAD67 in these regions.

A distinction between these possibilities could potentially be made by using a simultaneous localization of mRNA for the two GAD isoforms. Double in situ hybridization (DISH) allows for colocalization of separate mRNAs within a given cell. In the study described below, DISH has been used to examine the distribution of GAD65 and GAD67 mRNA in the hippocampus of the adult rat.

Section snippets

Tissue preparation

Three-month old male (n=4) and female (n=4) rats were decapitated under ketamine:xylazine anesthesia (IP; 3 ml/kg). Brains were immediately removed from calvaria, snap frozen in −18°C isopentane, and stored at −80°C until sectioning (time from injection to freezing <5 min). Brains were sliced at 16 μm thickness and mounted (4 brains/slide) on Superfrost Plus slides (Fisher Scientific, Pittsburgh, PA). Slides were fixed in 4% paraformaldehyde in phosphate buffer (pH 7.4; 15 min) and then

Results

To determine if cRNA probes were binding to only their respective forms of GAD, or if one or both probes could bind to both forms of GAD mRNA in situ, both single and double ISH were run simultaneously with the concentration of radioactive probes equivalent in each case (Fig. 1). In DISH experiments, the radioactive signal from each form of GAD did not decrease when compared to levels seen in single-labeling experiments. This suggests that the DIG-labeled probes were not competing with the [35S

Sensitivity of probes and single-labeled cells

In the current study, labeling of riboprobes with [35S] or DIG permitted the detection of the majority of GAD mRNA positive cells, although the radioprobes appear to be slightly more sensitive. In the hilus, both GAD65 and GAD67 mRNA radioprobes detected cells which were DIG negative, while DIG probes almost never detected cells which were not also positive for radioprobes. Thus, it appears that mRNAs for the two forms of GAD are not coregulated at the same levels in all cells, and that there

Acknowledgements

We express our gratitude to Drs. Alan Tobin and Niranjula Tillakaratne for the gift of rat GAD65 and GAD67 cDNA. This work was supported by grants from the National Institute of Mental Health (MH 00432, MH 42261, and MH 31154).

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