Cytochrome oxidase immunohistochemistry in rat brain and dorsal root ganglia: Visualization of enzyme in neuronal perikarya and in parvalbumin-positive neurons

Abstract

Histochemical detection of cytochrome oxidase activity has been widely used to deduce patterns of neuronal electrical activity in the CNS. Here we investigated the utility of cytochrome oxidase localization by immunohistochemistry and compared immunostaining with histochemical staining patterns in dorsal root ganglia of the rat. In addition, a limited survey of cytochrome oxidase immunostaining density within what are thought to be highly active parvalbumin-immunoreactive neurons was conducted. The immunohistochemical approach produced granular cytoplasmic immunolabelling in neuronal cell bodies and allowed identification of individual labelled cells in all brain regions including those within dense immunoreactive networks of neuropil. Neuronal somata exhibited a wide range of staining densities which were particularly evident in the hippocampus and dorsal root ganglia. The distribution of neurons intensely immunoreactive for cytochrome oxidase within various structures was consistent with previous histochemical descriptions of enzyme activity. Densitometric measurements of immunohistochemical reaction product in individual neurons of hippocampus, substantia nigra, cerebellum and dorsal root ganglia showed that the rate of product deposition was linear with time under conditions chosen for comparisons of staining density. Quantitative analysis of cytochrome oxidase immunohistochemical and histochemical staining densities within the same cells in adjacent sections of dorsal root ganglion gave a correlation coefficient of r = 0.75 (P < 0.001). In sections processed immunohistochemically for both cytochrome oxidase and parvalbumin, most but not all parvalbumin-containing cells displayed dense cytochrome oxidase immunolabelling. Conversely, many examples were found of neurons that were densely stained for cytochrome oxidase, but lacked parvalbumin. Immunohistochemistry for cytochrome oxidase reveals the enzyme in neuronal cell bodies with a clarity not usually seen with the histochemical method. Combination of this immunohistochemical approach with simultaneous immunolabelling of other neuronal markers, as shown here in the case of parvalbumin, is expected to assist the elucidation of patterns of activity in neurochemically identified cell types and anatomically defined neural systems.