Proc. Natl. Acad. Sci. USA Vol. 76, No. 3, pp, 1485-1488, March 1979 Neurobiology Immunocytochemical localization of cyclic GMP: Light and electron microscope evidence for involvement of neuroglia (cerebellum/cyclic AMP/y-aminobutyric acid/harmaline/diazepam) V. CHAN-PALAY* AND S. L. PALAYt Departments of *Neurobiology and tAnatomy, Harvard Medical School, Boston, Massachusetts 02115 Contributed by Sanford L. Palay, December 7, 1978 ABSTRACT Guanosine 3',5'-cyclic monophosphate (cGMP) presents cytopharmacological evidence from immunocyto- immunoreactivity in the rat's cerebellum was studied with light chemistry for the cellular and subcellular location of cGMP in and electron microscopy by the indirect fluorescence method some cerebellar neurons and particularly in neuroglial cells. and the peroxidase-antiperoxidase method. Labeled cells in- cluded neuroglial cells in the cerebellar cortex, white matter, and deep nuclei; some stellate and basket cells in the cortex; and MATERIALS AND METHODS some large neurons in the deep nuclei. No evidence was found Adult 200-300 g Sprague-Dawley rats, the indirect immu- for sagittal microzonation in the cGMP distribution. In the la- nofluorescence method (11), and the peroxidase-antiperoxidase beled cells, cGMP immunoreactive sites were localized to sur- face membranes, organelles, and the cytoplasmic matrix. (PAP) method (12) for light and electron microscopy were used. Specificity was indicated by the same pattern of labeling after Rabbit antisera were raised against the 2'-O-succinyl derivative treatment with cGMP immunoglobulin that had been adsorbed of cGMP or cAMP conjugated to limpet hemocyanin (13). More with adenosine 3',5'-cyclic monophosphate (cAMP) and by the than 50% of 2'-O-succinyl-cGMP ([125I]iodotyrosine methyl failure to label after treatment with normal rabbit sera or with ester derivative) or 2'-O-succinyl-cAMP ([1251]iodotyrosine cGMP immunoglobulin that had been adsorbed with 1 mM methyl ester derivative) (<0.01 pmol) was bound at a serum cGMP. Cerebella treated with cAMP antisera, however, showed immunoreactivity in Purkinje cells, granule cells, and Golgi cells dilution of 1:2000. Specificity was determined by radioim- in addition to neuroglia in cortex and deep nuclei. Sequential munoassay. The antisera raised against cGMP gave 20-30% norepinephrine and glutamate superfusions generally intensi- displacement of bound '25I-labeled cGMP with 5 fmol of fied cGMP immunoreactivity, not only in neuroglial cells but acetylated cGMP. Acetylated cAMP did not produce significant also in the background. Under these conditions some Purkinje displacement up to 1000 fmol. Corresponding experiments with cells and some granule cells were also labeled. Increased cGMP antisera raised against cAMP produced the equivalent appro- immunoreactivity was also obtained by treatment with har- maline, y-aminobutyric acidand aminooxyacetic acid, musci- priate result, indicating its specificity. The immunoglobulin mol, y-aminobutyric acid, or apomorphine in order of de- (IgG) fractions of these antisera were used. The basic cyto- creasing effectiveness. Serotonin and colchicine produced no chemical methods have been described (14). Cryostat sections detectable increase of cGMP immunoreactivity above normal, (10,um thick) of cerebella from formaldehyde-perfused brains and diazepam and sodium pentobarbital decreased it. In these and unfixed cerebella frozen in liquid nitrogen 1-2 min after experiments, diethyl ether was preferable to sodium pento- decapitation were treated with the IgG and stained by the barbital for anesthesia on account of the depressive action of the latter on cGMP immunoreactivity. Thus, drugs that increase immunofluorescence method, using a fluorescein isothiocya- cerebellar activity enhance cGMP levels, whereas those that nate-conjugated goat IgG. Vibratome sections (20-30 Am thick, decrease cerebellar activity decrease cGMP levels. However, cut in cold 0.05 M Tris-HCI buffer, pH 7.6) of cerebella from it is not clear whether these fluctuations in cGMP levels are a perfused brains were treated sequentially with cGMP IgG (18 direct consequence of neurotransmitter function or are sequelae hr at 40C in a humid environment, dilution 1:100 in 0.5% Triton to other related events. The present study suggests that some X-100/0.05 M Tris-HCI buffer, pH 7.6), goat-anti-rabbit IgG, neurons and many neuroglial cells are the major sites of cGMP PAP antiserum, and diaminobenzidine with hydrogen peroxide, in the cerebellum. prior to postfixation in 2% osmium tetroxide, dehydration with The mammalian cerebellum contains unusually high concen- methanol, and embedding in epoxy resin. These sections were trations of endogenous guanosine 3',5'-cyclic monophosphate then examined with the light microscope and subsequently (cGMP) (1). Experiments on brain slices, cultures, and in vitro thin-sectioned in serial order for electron microscopy. No preparations suggest that cGMP may be associated with a va- counterstains were used for electron microscopy. Two forms riety of possible neurotransmitter substances in mechanisms that of anesthesia were employed and their results were compared: are not understood. Selective changes of cerebellar cGMP in- 2% sodium pentobarbital (0.1 ml/100 g of body weight) injected dependent of adenosine 3',5'-cyclic monophosphate (cAMP) intraperitoneally and diethyl ether by inhalation. Two fixatives can be produced by a number of agents and conditions such as were used in the perfusions, either 4% (wt/vol) formaldehyde alcohol (2), excitatory and inhibitory transmitter substances (3, in Na phosphate buffer, pH 7.4, or 4% formaldehyde and 0.25% 4), decapitation (5), and benzodiazepines (6). Considerable glutaraldehyde in phosphate buffer, pH 7.4, both at 4°C; each speculation marks the interpretation of the correlations among was preceded by a wash with cold Ca2+_free Tyrode's buf- cGMP levels, putative transmitter substances, and their possible fer. localization in cerebellar neurons, particularly Purkinje cells Nine separate sets of experiments were performed, on groups (refs. 7 and 8; reviewed in ref. 9, p. 229). The presence of cGMP of three rats each, using drugs to alter cGMP levels detectable in Purkinje cells has not been demonstrated (10). This study by immunocytochemistry. All animals were perfused with fixative at the stated intervals after drug administration: (i) The publication costs of this article were defrayed in part by page charge payment. This article must therefore by hereby marked "ad- Abbreviations: cGMP, guanosine 3',5'-cyclic monophosphate; cAMP, vertisement" in accordance with 18 U. S. C. §1734 solely to indicate adenosine 3',5'-cyclic monophosphate, PAP, peroxidase-antiperoxidase; this fact. GABA, y-aminobutyric acid. 1485 1486 Neurobiology: Chan-Palay and Palay Proc. Natl. Acad. Sci. USA 76 (1979) Diazepam, 25 mg/kg of body weight, intraperitoneal, 30 min in all experiments using a scale of 1+ to 4+ for increasing prior to perfusion; (ii) muscimol, 10 mg/kg of body weight, amounts and intensities of reactions. Electron microscopy was intravenous, 30 min prior to perfusion; (iii) apomorphine, 5 conducted on serial sections of six specimens obtained from mg/kg of body weight, intraperitoneal, 5 min prior to perfu- normal, untreated, anesthetized animals. The sections were sion; (iv) harmaline, 40 mg/kg of body weight, intraperitoneal, treated with cGMP IgG, and cGMP IgG adsorbed with cGMP 30 min prior to perfusion; (v) y-aminobutyric acid (GABA) 10 was used for controls. ,uM, 2-,l intracerebellar injections, 5-7 min prior to perfusion; (vi) GABA, 10 AM, 2-,A intracerebellar injections, 5-7 min prior RESULTS to perfusion with aminooxyacetic acid, 10 mg/250 g of body cGMP immunoreactivity was greater: (i) in the cerebella from weight, intraperitoneal, 30 min prior; (vii) norepinephrine, 300 animals anesthetized with ether compared to sodium pento- JAM, and sodium L-glutamate, 3 mM in sterile saline at 37°C, barbital; (ii) in the more sensitive PAP method compared to superfused in sequence for 10 min each by using a push-pull immunofluorescence; (iii) in tissues fixed in formaldehyde cannula and agar superfusion chamber over the cerebellar without glutaraldehyde compared to unfixed frozen material. cortical surface; (viii) serotonin, 0.1 ,uM (250,l), intraventric- The presence of glutaraldehyde in the primary fixative en- ular infusion over 3 hr with monoamine oxidase inhibition hanced morphological preservation for electron microscopy (clorgyline, 10 mg/100 g of body weight); (ix) colchicine (3 but reduced immunoreactivity. Colchicine treatment did not ,ug/,ul), intracerebellar injection, 2 ,ul per electrode track, and enhance cGMP immunoreactivity. In the molecular layer, some 25 ,ul intraventricularly 24 hr prior to perfusion. stellate and basket somata (approximately 30%) were cGMP Parasagittal and transverse sections from three cerebellar immunoreactive. The Golgi epithelial neuroglial somata sur- regions (vermis, hemisphere, and paravermis including deep rounding Purkinje cells and their radial fibers (15) displayed nuclei) in each animal were processed. Tissues from the separate the most intense immunoreactivity. Purkinje cell somata re- experiments and controls were handled at the same time in mained unreactive with all modes of fixation and drug ma- order to reduce technical differences. Controls for specificity nipulations attempted except for the superfusion with norepi- included: cAMP antisera; cyclic GMP IgG adsorbed overnight nephrine