QUANTITATIVE ULTRASTRUCTURAL-LOCALIZATION OF GLUTAMATE-DEHYDROGENASE IN THE RAT CEREBELLAR CORTEX (VOL 62, PG 1133, 1994)

Glutamate dehydrogenase is one of the main enzymes involved in the for mation and metabolism of the neurotransmitter glutamate. In the presen t study we investigated the enzyme ultrastructurally in the cerebellar cortex, a region rich in well defined glutamatergic neurons; by pre-e mbedding immunocytochemical staining (peroxidase-antiperoxidase), as w ell as by post-embedding immunogold labelling employing a new system f or quantitation and for specificity testing under the conditions of th e immunocytochemical procedure. A new antiserum against immunologicall y purified bovine liver glutamate dehydrogenase or antibodies isolated from this by affinity chromatography were used in rats fixed by perfu sion with aldehydes. The pre-embedding method displayed peroxidase rea ction preferentially in mitochondria of astroglial cells (including th e Bergmann glia). Mitochondria of neuronal tissue elements were usuall y free of peroxidase-reaction product. Extra-mitochondrial staining wa s not observed. The post-embedding immunogold method was employed to o vercome penetration problems and allow semiquantitative analysis of lo calization and specificity. The highest densities of gold particles we re found over the mitochondria in astroglial cell elements (including the Bergmann glia). Mitochondria in cell bodies of Bergmann glia had a lower particle density than those in astrocytic processes. In the lat ter, analysis of frequency distribution revealed no evidence of a popu lation of mitochondria lacking glutamate dehydrogenase, but suggested the presence of populations with different levels of immunoreactivity. Comparison with the labelling of embedded bovine liver glutamate dehy drogenase indicated that the enzyme constitutes a high proportion (10% ) of the total matrix protein of these mitochondria. A weaker but sign ificant labelling was found in oligodendrocytes of the white matter. T he labelling of mitochondria in neuronal elements including glutamater gic messy fibre terminals was of the order of 15% of that in astroglia l mitochondria. No difference was detected between glutamatergic neuro ns (messy and parallel fibres, granular cells) and non-glutamatergic n eurons (Purkinje cells). The particle density over non-mitochondrial a reas was very close to background over empty resin. The results, obtai ned with different methods of tissue and antibody preparation, agree t o show that the present form of glutamate dehydrogenase is restricted to mitochondria and preferentially localized in astrocytes.