Immunocytochemical localization of cyclic GMP: Light and electron microscope evidence for involvement of neuroglia

Abstract

Cyclic CGMP microscopy immunoreactivity in the rat''s cerebellum was studied with light and EM by the indirect fluorescence method and the peroxidase-antiperoxidase method. Labeled cells included neuroglial cells in the cerebellar cortex, white matter and deep nuclei; some stellate and basket cells in the cortex; and some large neurons in the deep nuclei. No evidence was found for sagittal microzonation in the cGMP distribution. In the labeled cells, cGMP immunoreactive sites were localized to surface membranes, organelles and the cytoplasmic matrix. Specificity was indicated by the same pattern of labeling after treatment with cGMP immunoglobulin that had been adsorbed with cyclic(c)AMP and by the failure to label after treatment with normal rabbit sera or with cGMP immunoglobulin that had been adsorbed with 1 mM cGMP. Cerebella treated with cAMP antisera showed immunoreactivity in Purkinje cells, granule cells and Golgi cells in addition to neuroglia in cortex and deep nuclei. Sequential norepinephrine and glutamate superfusions generally intensified cGMP immunoreactivity, not only in neuroglial cells but also in the background. Under these conditions some Purkinje cells and some granule cells were also labeled. Increased cGMP immunoreactivity was also obtained by treatment with harmaline, GABA and aminooxyacetic acid, muscimol or apomorphine in order of decreasing effectiveness. Serotonin and colchicine produced no detectable increase of cGMP immunoreactivity above normal, and diazepam and sodium pentobarbital decreased it. In these experiments, diethyl ether was preferable to sodium pentobarbital for anesthesia on account of the depressive action of the latter on cGMP immunoreactivity. Drugs that increase cerebellar activity enhance cGMP levels, whereas those that decrease cerebellar activity decrease cGMP levels. It is unclear whether these fluctuations in cGMP levels are a direct consequence of neurotransmitter function or are sequelae to other related events. Some neurons and many neuroglial cells are the major sites of cGMP in the cerebellum.