Immunocytochemical localization of glutamate decar☐ylase in rat cerebellum with a new antiserum

Abstract

An antiserum prepared in sheep against rat brain glutamic acid decar☐ylase, the biosynthetic enzyme for γ-aminobutyric acid, has been employed to localize, at the light and electronmicroscopic levels, neurons containing glutamate decar☐ylase in the cerebellum of adult rats using the unlabeled antibody enzyme method of S ternberger (1979). With high dilutions of the primary antiserum (1:4000 and higher) glutamate decar☐ylase-immunoreactivity was localized in synaptic terminals of basket, stellate and Golgi cells and Purkinje cell axon recurrent collaterals in the cerebellar cortex, as well as in axosomatic and axodendritic synaptic terminals in the deep cerebellar nuclei, all of which contain pleomorphic synaptic vesicles. The reaction product was present in the axoplasm and at the cytoplasmic aspect of the plasmamembrane, the membrane of synaptic vesicles and axoplasmic reticulum, the outer mitochondrial membrane and also the microtubules. Lower dilutions of antiserum (1:1500) revealed light, specific immunoreactivity in somata of Purkinje, basket, stellate and Golgi-cells of the cerebellar cortex. We obtained specimens in which the vast majority of the members of these cells were stained. Staining of stellate cells was the least intense of all positive neurons in the cerebellar cortex. Very light labeling of some small cell bodies of the deep cerebellar nuclei was also observed. Granule cells, parallel fibers, mossy fibers and climbing fibers in the cerebellar cortex, the large neurons of the cerebellar nuclei, and glial cells were negative. When the antiserum was absorbed against acetone-dried rat liver powder, the same staining pattern was obtained, however, dilutions of 1:500 were necessary to obtain equally strong staining of neuronal cell bodies. These data indicate the specificity of the new anti-serum to rat brain glutamate decar☐ylase. They confirm and complement earlier immunocytochemical work with an antiserum to mouse brain glutamate decar☐ylase. The new antiserum not only shows that nearly all inhibitory axons in the cerebellum are glutamate decar☐ylase positive but also detects immunoreactivity in neuronal cell bodies in the cerebellar cortex without administration of blockers of axoplasmic transport.