Elsevier

Neuroscience

Volume 78, Issue 1, 27 February 1997, Pages 87-97
Neuroscience

Differential distribution of glutamate decarboxylase-65 and glutamate decarboxylase-67 messenger RNAs in the entopeduncular nucleus of the rat

https://doi.org/10.1016/S0306-4522(96)00593-3Get rights and content

Abstract

The entopeduncular nucleus is one of the major output nuclei of the basal ganglia, with topographically organized projections to both motor and limbic structures. Neurons of the entopeduncular nucleus use GABA as the principal transmitter, and glutamate decarboxylase (the GABA synthetic enzyme) is widely distributed throughout the region. Previous studies have shown that glutamate decarboxylase exists in two forms (glutamate decarboxylase-65 and glutamate decarboxylase-67), and that the messenger RNAs for these different enzymes are widely distributed in rat brain. The purpose of the present experiment was to describe the distribution of glutamate decarboxylase-65 and glutamic decarboxylase-67 messenger RNAs throughout the entopeduncular nucleus using recently developed oligodeoxynucleotide probes and in situ hybridization histochemical methods. In agreement with previous studies, northern analysis of rat brain poly(A)+ messenger RNA preparations showed that the glutamate decarboxylase-65 and glutamate decarboxylase-67 probes used in the present study hybridized to messenger RNAs of approximately 5.7 and 3.7 kb, respectively. Film autoradiographic analysis revealed large region-dependent, isoform-specific differences in the levels of expression of the two messenger RNAs, with glutamate decarboxylase-65 messenger RNA predominating in rostral and medial regions of the entopeduncular nucleus and glutamate decarboxylase-67 messenger RNA most abundant in the caudal region. Cellular analysis showed that these region-dependent differences in labelling were due to differences in the relative amounts of glutamate decarboxylase-65 and glutamate decarboxylase-67 messenger RNAs expressed per cell rather than the number of cells expressing each form of glutamate decarboxylase messenger RNA. The differences in the distribution of glutamate decarboxylase-65 and glutamate decarboxylase-67 messenger RNAs are closely related to the organization of limbic and motor circuits of the entopeduncular nucleus, suggesting that GABAergic transmission through the limbic pathway is regulated predominantly by glutamate decarboxylase-65, whereas glutamate decarboxylase-67 is of principal importance in the motor pathway.

These data provide additional evidence that the neurons of the limbic and motor subregions of the entopeduncular nucleus are neurochemically distinct.

Section snippets

Animals

Female rats (200–250 g) of an outbred Sprague–Dawley strain (ALAB, Sollentuna, Sweden) were used for these experiments. The animals were housed at approximately 22°C on a fixed light cycle (12 h light–12 h dark), with food and water available at all times. Animals were rendered unconscious by CO2 inhalation to minimize discomfort and decapitated. Brains were rapidly removed, frozen with powdered dry ice and either used immediately (northern analysis) or stored for up to one week at −70°C (in situ

Specificity of glutamate decarboxylase-65 and glutamate decarboxylase-67 oligodeoxyribonucleotide probes

Northern analysis performed on poly(A)+-enriched mRNA preparations from whole rat brain showed that the GAD65 and GAD67 oligodeoxynucleotide probes used in the present study labelled mRNAs of approximately 5.7 and 3.7 kb (Fig. 1).

Film autoradiograms demonstrating GAD65 and GAD67 mRNA labelling in brain sections collected at several levels of the striatum and pallidal complex are shown in Fig. 2. Both GAD mRNA probes labelled brain regions known to contain GABAergic neurons with high resolution

Discussion

In the present study, recently developed oligodeoxynucleotide probes[19]were used to describe the distribution of GAD65 and GAD67 mRNAs in the EP. Analysis of film autoradiograms comparing the distribution of the two isoforms of the GAD enzyme throughout the entire extent of the EP demonstrated a clear difference in the distribution of GAD65 and GAD67 mRNAs. More specifically, GAD65 mRNA labelling was most abundant in the rostral two-thirds of the EP, with lower but detectable levels in the

Conclusion

The results of the present study provide an additional neurochemical marker for discriminating the limbic and motor pathways through one of the major output nuclei of the basal ganglia, the EP. In doing so, these data, along with previously published reports, provide further evidence for a possible means whereby neurotransmission through motor and limbic pathways of the basal ganglia are integrated via a habenular–raphé–striatal circuit involving serotonin.

Acknowledgements

This work was supported by grant no. 8318 from the Swedish Medical Research Council.

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