Glutamate decarboxylase (GAD65) gene expression is increased by dopamine receptor agonists in a subpopulation of rat striatal neurons

Abstract

The mRNA levels encoding for the two isoforms of glutamate decarboxylase (GAD65 and GAD67) were measured in the adult rat striatum following systemic administration of dopamine receptor agonists. Double-labeling in situ hybridization histochemistry was used to measure GAD65 or GAD67 mRNA levels in neurons labeled or not with a preproenkephalin (PPE) cRNA probe. Chronic treatment with the D1/D2 dopamine receptor agonist apomorphine or with the D1 dopamine receptor agonist SKF-38393 induced an increase in GAD65 but not GAD67 mRNA levels in different sectors of the striatum. These effects were abolished by pre-administration of the D1 dopamine receptor antagonist SCH-23390. On double-labeled sections, GAD65 mRNA labeling was distributed in neurons labeled and unlabeled with the PPE cRNA probe. About half of all neuronal profiles labeled with the GAD65 cRNA probe were also labeled with the PPE cRNA probe. Quantification of labeling at cellular level demonstrated a significant increase of GAD65 mRNA levels in PPE-unlabeled neurons. On the other hand, no significant changes of GAD65 mRNA levels were detected in PPE-labeled neurons. Our results demonstrate a differential effect of dopamine receptor agonists on striatal GAD65 and GAD67 gene expression. In particular, we show that GAD65 mRNA levels are selectively increased in presumed striato-nigral neurons following treatments with dopamine receptor agonists. These data provide evidence that the GAD65 isoform is preferentially involved in the regulation of GABAergic neurotransmission in striato-nigral neurons.

Introduction

Striatal projection neurons use γ-aminobutyric acid (GABA) as their neurotransmitter and contain the GABA-synthesizing enzyme, glutamate decarboxylase (GAD) 6, 31, 44, 47, 51. The enzyme GAD exists at least as two isoforms (GAD65 and GAD67) of distinct molecular weight that are encoded by different genes 12, 29. Anatomical studies have shown that the mRNAs encoding for the two GADs are co-localized in many neurons of the basal ganglia including the striatum 13, 43. Striatal GABAergic projection neurons in the rat can be subdivided into two distinct subpopulations. The striato-pallidal neurons preferentially express the peptide enkephalin and the D2 dopamine receptor and the striato-nigral/entopeduncular neurons preferentially express the peptides substance P and dynorphin and the D1 dopamine receptor 18, 19, 21, 24, 25, 35, 36, 65.

There is extensive evidence that dopamine receptors are involved in the regulation of striatal GABAergic neurons. For instance, dopamine or D1 receptor agonists are able to increase striatal GABA release as measured in vitro on brain slices 4, 17, 60or in vivo by push-pull cannula or microdialysis techniques 23, 64. The stimulatory effect of D1 dopamine receptors on striatal GABA release appears to involve GABAergic neurons of the striato-nigral/entopeduncular pathway since application of SKF-38393 in the striatum increases concomitantly the release of GABA in the striatum, the substantia nigra and the entopeduncular nucleus 16, 64. On the other hand, D2 dopamine receptors appear to exert an inhibition 4, 23, 50or have no effect [64]on striatal GABA release.

The regulation of striatal GABAergic neurons by dopamine receptors can also be evidenced through measurements of GAD activity or GAD gene expression. For instance, rats with 6-OHDA lesions of dopamine neurons exhibit increased GAD activity [52], GAD67 mRNA levels and GAD immunoreactivity 40, 52, 54, 59. Similar increases are observed following blockade of D2 dopamine receptors in adult rats 8, 32, 40, 49, 54which suggests that dopamine through D2 receptors is involved in a tonic inhibition of striatal GAD activity and GAD67 gene expression. Striatal GAD65 gene expression, on the other hand, is not altered following D2 dopamine receptor blockade or lesion of dopamine neurons in adult rats 32, 54. However, a recent study has shown that the administration of D1 dopamine receptor agonists to adult rats induces an increase in striatal GAD65 but not GAD67 mRNA levels [32]. In addition to demonstrating a differential regulation of the two GAD isoforms by dopamine receptor subtypes, this result suggests that the GAD65 isoform is preferentially up-regulated in response to D1 receptor stimulation in striato-nigral neurons of the adult rat. Such a possibility would provide further evidence for the still debated stimulatory role of D1 dopamine receptors on GABAergic striato-nigral neurons [26].

The goal of the present study was first to provide further evidence that chronic administration of dopamine D1 receptor agonists to adult rats induce a selective increase in GAD65 mRNA levels and second to determine whether this increase occurs in presumed striato-pallidal, striato-nigral or both subpopulations of neurons. The effects of the dopamine receptor agonists apomorphine or SKF-38393 on the levels of the GAD65 mRNA were measured in striatal neurons labeled or not with a preproenkephalin (PPE) cRNA probe which was used as a preferential marker of striato-pallidal neurons.