Proc. Natl. Acad. Sci. USA Vol. 76, No. 4, pp. 2067-2071, April 1979 Neurobiology Immunocytochemical localization of y-aminobutyric acid transaminase at cellular and ultrastructural levels (cerebellum/synapses/harmaline/muscimol) V. CHAN-PALAY*, J.-Y. WUt, AND S. L. PALAYt Departments of *Neurobiology and tAnatomy, Harvard Medical School, Boston, Massachusetts 02115; and tDepartment of Cell Biology, Baylor College of Medicine, Houston, Texas 77030 Contributed by Sanford L. Palay, January 3, 1979

ABSTRACT y-Aminobutyric acid transaminase (GABA- equilibrium in dilute buffer, deuterium oxide, and guanidine Tase; 4-aminobutyrate:2-oxaglutarate aminotransferase, EC hydrochloride solution, and polyacrylamide gel electrophoresis 2.6.1.19) immunoreactivity in the rat's cerebellum was studied as described (4-6). Antisera to GABA-Tase were produced in by light and electron microscopy with indirect immunofluo- rescence and peroxidase-antiperoxidase methods. Evidence is rabbits by weekly infrascapular injections of 30 .tg of enzyme presented for neuronal and neuroglial compartments of in complete Freund's adjuvant; serum was collected after the GABA-Tase. Labeled neurons included stellate, basket, Purkinje, fourth injection. GABA-Tase antisera were characterized by and Golgi cells of the cortex and a few large neurons in the deep immunodiffusion microcomplement fixation and immuno- nuclei. Labeled neuroglia included those surrounding Purkinje electrophoresis as described (6-9). GABA-Tase antisera were cells, their radial fibers in the molecular layer, and astrocytes used at dilutions of 1:100 or 1:200, and normal rabbit preim- in the granular layer and deep nuclei. No evidence for sagittal microzonation was found. At the ultrastructural level, GABA- mune sera served as the controls for cytochemical specificity Tase immunoreactive sites were localized to cell surface in light and electron microscope studies. membranes, intracellular organelles, and the cytoplasmic ma- Cryostat sections (10 Atm thick) of cerebella from formal- trix. GABA-Tase immunoreactivity at synapses could be local- dehyde-perfused brains and unfixed cerebella frozen in liquid ized precisely to pre- and postsynaptic membranes in y-ami- nitrogen or Freon 60-120 sec after decapitation were treated nobutyric acid (GABA)containing as well as non-GABA-con- taining neurons. Specific label was absent from tissues treated with the antisera and stained by the immunofluorescence with normal rabbit preimmune sera. GABA-Tase labeling was method using a fluorescein isothiocyanate-conjugated goat more intense in tissues from animals anesthetized with ether immunoglobulin. Vibratome sections (10-30 Am thick, cut in than with barbiturates and after formaldehyde fixation without cold 0.05 M Tris buffer) of cerebella from perfused brains were glutaraldehyde. Increased GABA-Tase immunoreactivity was treated sequentially with antisera (18 hr, 4°C in a humid en- observed on treatment with colchicine, GABA with oxamic acid, vironment, diluted in 0.5% Triton X-100/0.05 M Tris buffer), GABA, harmaline, norepinephrine and glutamate, or diazepam (in order of decreasing effectiveness). Serotonin produced no goat-anti-rabbit immunoglobulin, PAP antiserum, and 0.022% detectable change, and apomorphine and muscimol decreased diaminobenzidine with 0.3% hydrogen peroxide. The sections the immunoreactivity. were then fixed in 2% osmium tetroxide, dehydrated in methanol, and embedded in epoxy resin. These sections were There is considerable interest in the identification of cellular examined with the light microscope and/or subsequently sites of the biosynthesis and metabolism of y-aminobutyric acid thin-sectioned in serial order for electron microscopy (12, 13). (GABA), a major inhibitory transmitter in vertebrate and in- No counterstains were used for electron microscopy. Two forms vertebrate nervous systems. Localization of GABA transaminase of anesthesia were employed: 2% sodium pentobarbital (0.1 (GABA-Tase; 4-aminobutyrate:2-oxaglutarate aminotrans- ml/100 g body weight) and diethyl ether. Two fixatives were ferase, EC 2.6.1.19), an enzyme involved in GABA degradation, used in the perfusions, preceded by cold Ca2+-free Tyrode's has been attempted by histochemical stains (1, 2) and by light buffer at 40C: 4% formaldehyde in phosphate buffer (pH 7.4) microscope immunocytochemistry (3). Immunocytochemical or 4% formaldehyde with 0.25% glutaraldehyde in phosphate methods have been made possible by the successful purification buffer (pH 7.4). and characterization of GABA-Tase from mouse brain (4-6) Nine separate sets of experiments were performed, on groups and the use of purified GABA-Tase for production of specific of three rats each, using drugs to alter GABA-Tase levels de- antibodies (6-9). The present investigation attempts to localize tectable by immunocytochemical methods. All animals were GABA-Tase ultrastructurally with the aid of GABA-Tase an- perfused with fixative at the stated intervals after drug ad- tisera and to examine changes in GABA-Tase immunoreactivity ministration: (i) diazepam, 25 mg/kg body weight, intraperi- induced by administration of pharmacological agents and toneal, 30 min prior to perfusion; (ii) muscimol, 10 mg/kg body neurotransmitter substances. weight intravenous, 30 min prior to perfusion; (iii) apomor- phine, 5 mg/kg body weight intraperitoneal, 5 min prior to MATERIALS AND METHODS perfusion; (iv) harmaline, 40 mg/kg body weight intraperito- Adult Sprague-Dawley rats (200-300 g), the indirect immu- neal, 30 min prior to perfusion; (v) GABA, 10 ,AM (in 2 ,u) in- nofluorescence method (10), and the peroxidase-antiperoxidase tracerebellar, 5-7 min prior to perfusion; (vi) GABA, 10MM (in (PAP) method (11) for light and electron microscopy were used. 2 ,ul) intracerebellar, 5-7 min prior to perfusion concurrently GABA-Tase was purified from mouse brain and its purity was with oxamic acid, 10 mg/250 g body weight, intraperitoneal, established by gel electrophoresis, high-speed sedimentation 30 min prior; (vii) norepinephrine, 0.3 mM, and L-sodium glutamate, 3 mM, in sterile saline at 37°C superfused for 10 The publication costs of this article were defrayed in part by page min, each sequentially, by using a push-pull cannula; (viii) se- charge payment. This article must therefore be hereby marked "ad- vertisement" in accordance with 18 U. S. C. §1734 solely to indicate Abbreviations: GABA, y-aminobutyric acid; GABA-Tase, GABA this fact. transaminase; PAP, peroxidase-antiperoxidase. 2067 Downloaded by guest on September 26, 2021 2068 Neurobiology: Chan-Palay et al. Proc. Natl. Acad. Sci. USA 76 (1979)

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i FIG. 1. (Legend appears at the bottom of the next page.) Downloaded by guest on September 26, 2021 Neurobiology: Chan-Palay et al. Proc. Natl. Acad. Sci. USA 76 (1979) 2069 rotonin, 0.1 AtM (250 ,ul) intraventricular infusion over 3 hr with apomorphine and muscimol decreased it (Fig. 1 a-c and e-h). a monoamine oxidase inhibitor (clorgiline, 10 mg/100 gibo4dy No specific staining was obtained in control tissues treated with weight); (ix) colchicine, 3 gg/,.l intracerebellar injections of preimmune rabbit serum (Fig. ld). 2 Al per electrode track and 25 Al intraventricularly, 24 hr prior Electron microscopy confirmed the light microscope results. to perfusion. Labeled neurons included Golgi cells, basket and stellate cells, Sections in the sagittal and transverse planes from three and Purkinje cell somata and their dendrites. Neuroglial cells cerebellar regions (vermis, hemisphere, and paravermis in- between Purkinje cells were also labeled, as were their processes cluding deep nuclei) of each animal were processed. Tissues surrounding neural elements in the molecular layer, other cells from the separate experiments and controls were handled at in the granular layer, and blood vessels (Figs. ii and 2 a-d). In the same time in order to reduce technical differences. The the labeled neuron or neuroglial cell, two components were resulting material was studied with the light microscope, and recognized-membranous and cytoplasmic. The membranous neurons and neuroglia with GABA-Tase immunoreactivity reactive material was detectable on the plasma membranes, on were identified in the cerebellar cortex, white matter, and deep outer nuclear membranes, on outer mitochondrial membranes, cerebellar nuclei. The distribution and intensity of immuno- on outer membranes of the granular and smooth endoplasmic reactivity were determined for specimens in all experiments reticulum, and on microtubules and neurofilaments. The by using a scale of 1+ to 4+ for increasing accumulation of cytoplasmic matrix appeared as a dark reactive flocculent reaction product. These were compared in order to determine material between the cellular organelles. Where the label oc- effects of prolonged manipulation on levels of detectable im- curred only in neuroglial cells, the unlabeled neuronal elements munoreactivity. Electron microscopy was carried out on serial were encircled by reactive glial processes (Fig. li, for example), sections of seven specimens obtained from normal anesthetized around Purkinje cells (Fig. 2a), stellate cells, Purkinje cell animals and treated with GABA-Tase antisera. dendrites (Fig. 2b), and mossy fibers (Fig. 2d). At the synaptic interface, the presynaptic and postsynaptic membranes alone RESULTS could be specifically labeled (Fig. 2c). GABA-Tase immunoreactivity was greater: (i) in cerebella from animals anesthetized with ether than with barbiturates; (ii) in DISCUSSION tissues treated by the PAP method than by the immunofluo- Evidence is provided for the presence of GABA-Tase in the rescence method; (iii) in tissues fixed in formaldehyde without neuronal and neuroglial compartments of the cerebellum. In glutaraldehyde than in unfixed frozen material (the presence the neuronal pool, cells that have been observed with GABA- of glutaraldehyde in the primary fixative enhanced morpho- Tase immunoreactivity in their cytoplasm and membranes are logical preservation for electron microscopy but decreased cerebellar GABA neurons previously shown to have glutamic immunoreactivity); and (iv) in tissues obtained after colchicine acid decarboxylase immunoreactivity (3) and [3H]GABA up- administration. take (16, 17). GABA-Tase immunoreactivity was also observed In the molecular layer, numerous stellate and basket cell on postsynaptic membranes alone of axons belonging to non- somata (greater than 80%) were immunoreactive. The neuro- GABA-containing neurons such as granule cells. This indicates glial somata surrounding Purkinje cells and their radial fibers that GABA-Tase is a major cytoplasmic and membrane-related (14) displayed the most intense reaction. Purkinje cell somata degradative enzyme in GABA-synthesizing neurons but is, in were unreactive with both methods of anesthesia when gluta- addition, selectively bound to the postsynaptic membrane at raldehyde was present in the fixative but appeared as single GABA synapses on non-GABA-containing neurons. Neuroglial immunoreactive cells or as groups of up to 10 or 12 immuno- cells, particularly those enveloping GABA-containing neurons, reactive cells when formaldehyde was used alone or after col- have significant amounts of cytoplasmic and membrane-bound chicine treatment, respectively. These results indicate that the GABA-Tase. Although many Purkinje cells have GABA-Tase content of GABA-Tase may differ from one Purkinje cell to immunorpactivity, some have none. All Purkinje cells and their another. In the granular layer, Golgi neurons were always in- dendrites, however, are ensheathed by GABA-Tase immuno- tensely reactive, as were neuroglial cells, but granule cells and reactive neuroglial processes. The presence of GABA-Tase in axons were not. In the deep nuclei, some large neurons and GABA neurons, neuroglia, and non-GABA neurons associated neuroglial cells were reactive and this reactivity was most in- with GABA synapses indicates the participation of these cells tense in large and small neurons after harmaline treatment. The in important mechanisms for terminating transmitter ac- total amount of immunoreactivity was considerably lower in tion-the uptake and degradation of GABA. the deep nuclei than in the cortex. Labeled structures were The present studies indicate that an increase in GABA-Tase randomly scattered in the cerebellum, and no sagittal micro- immunoreactivity can be induced by application of GABA and zonation (15) in GABA-Tase distribution was seen. Changes in intensified with the use of oxamic acid, and by glutamate. These the intensity of GABA-Tase immunoreactivity were detectable effects may be explained by increases in enzyme substrate on administration of various drugs. Increased reactivity was (GABA), in coenzyme-ligand interactions (oxamic acid) and obtained by treatment with colchicine, GABA and oxamic acid, in enzyme product interactions (glutamate). Colchicine, a drug GABA, harmaline, norepinephrine and glutamate, or diazepam that blocks axoplasmic transport of proteins, also increases de- in order of decreasing effectiveness. Serotonin produced no monstrable GABA-Tase. The finding that muscimol, a potent detectable increase in GABA-Tase reactivity above normal, and GABA agonist, does not increase GABA-Tase levels suggests that

FIG. 1 (on preceding page). Light micrographs (a-h) of cerebellar cortex with immunoreactive neuronal and neuroglial elements visualized by GABA-Tase antisera using the PAP method. (a) Tissue from normal ether-anesthetized rat (control preparation), showing GABA-Tase immunoreactivity in groups of Purkinje cells (crossed arrows) separated by individual or several unlabeled cells (arrows). (X120.) (b) Immuno- reactive Purkinje cells (PC, crossed arrows) and radial neuroglial fibers. (X480). (c) Unlabeled Purkinje cell (PC) and primary dendrites surrounded by intensely immunoreactive neuroglial cells (arrows) and their processes. Labeled basket cells (B) are also present. (X480.) (d) Control tissue treated with normal rabbit preimmune sera. (X120.) (e and f) Treatment with apomorphine decreases immunoreactivity below normal in Purkinje cell (PC) and glial compartments. (X120 and X480.) (g and h) Treatment with harmaline increases GABA-Tase immunoreactivity in the neuroglia around Purkinje cells (PC) and in the granular layer. (X120 and X480.) (i) Electron micrograph of two GABA-Tase immunoreactive neuroglial cells (GI) and processes near granule cells (gr c) of the cerebellar cortex. (No counterstains; X16,800.) Downloaded by guest on September 26, 2021 2070 Neurobiology: Chan-Palay et al. Prroc. Natl. Acad. Sci. USA 76 (1979)

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binding sites for GABA-Tase and transmitter receptor sites are 5. Schousboe, A., Wu, J.-Y. & Roberts, E. (1974) J. Neurochem. 23, separate. With the exception of muscimol and apomorop~ii 11,89-1 195. the selective changes elicited in levels of GABA-Tase reactivity 6. Wu, J.-Y. (1976) in GABA in Nervous System Function, eds. by pharmacological treatments indicate that drugs and neu- Roberts, E., Chase, T. N. & Tower, D. B. (Raven, New York), pp. rotransmitter substances that increase the activity of cerebellar 7-55. 7. Saito, K., Schousboe, A., Wu, J.-Y. & Roberts, E. (1974) Brain Res. GABA neurons directly (GABA, norepinephrine, glutamate) 65,287-296. or indirectly (diazepam, harmaline) increase immunologically 8. Wong, E., Schousboe, A., Saito, K., Wu, J.-Y. & Roberts, E. (1974) detectable levels of the enzyme. These studies indicate that the Brain Res. 68, 133-139. ability to localize the precise sites of a degradative enzyme such 9. Saito, K. (1976) in GABA in Nervous System Function, eds. as GABA-Tase provides a powerful means for investigation of Roberts, E., Chase, T. N. & Tower, D. B. (Raven, New York), pp. neurotransmitter mechanisms at cellular and subcellular 103-111. levels. 10. Coons, A. G. (1958) in General Cytochemical Methods, ed. Danielli, J. F. (Academic, New York), pp. 399-422. We thank Mr. H. Cook for photographic assistance. This work was 11. Sternberger, L. (1974) in Immunocytochemistry, Foundations supported in part by U.S. Public Health Service Grant NS 10536, NS of Immunology Series, eds. Osler, A. & Weiss, L. (Prentice Hall, 13224, the Huntington Chorea Foundation, the Parkinson's Disease New Jersey), p. 171. Project of Massachusetts General Hospital (Boston), a Louise Harkness 12. Chan-Palay, V. & Palay, S. L. (1977) Proc. Natl. Acad. Sci. USA Ingalls Fellowship in Research on Parkinson's Disease, and an Alfred 74,4050-4054. P. Sloan Foundation Fellowship (V.C-P.). 13. Chan-Palay, V. & Palay, S. L. (1979) Proc. Natl. Acad. Sci. USA, 76, 1485-1488. 1. Van Gelder, N. M. (1965) J. Neurochem. 12, 231-237. 14. Palay, S. L. & Chan-Palay, V. (1974) Cerebellar Cortex, Cytology 2. Hyde, J. C. (1978) in Amino Acids as Chemical Transmitters, and Organization (Springer, Berlin). ed. Fonnum, F. (Plenum, New York), 49-53. 15. Chan-Palay, V., Palay, S. L., Brown, J. T. & Van Itallie, C. (1977) 3. Barber, R. & Saito, K. (1976) in GABA in Nervous System Exp. Brain Res. 30,561-576. Function, eds. Roberts, E., Chase, T. N. & Tower, D. B. (Raven, 16. Ljungdahl, A., Seiger, A., Hokfelt, T. & Olson, L. (1973) Brain New York), 113-132. Res. 61, 379-384. 4. Schousboe, A., Wu, J.-Y. & Roberts, E. (1973) Biochemistry 12, 17. Chan-Palay, V. (1977) Cerebellar Dentate Nucleus, Organiza- 2868-2873. tion, Cytology, and Transmitters (Springer, Berlin).

FIG. 2 (on preceding page). Electron micrographs of GABA-Tase immunoreactive cells in the cerebellar cortex from normal untreated animals anesthetized with ether and visualized by the PAP method without counterstains. (a) A neuroglial cell (GL) with unlabeled nucleus and immu- noreactive surface membranes, organelles, and cytoplasmic matrix surround an unlabeled Purkinje cell soma (PC) and other unlabeled neuronal elements. (X24,600.) (b) Immunoreactive neuroglial processes (GI) in the molecular layer surround unlabeled basket cell soma (B) and Purkinje dendrites and thorns (Pcd). (X11,700.) (c) GABA-Tase immunoreactive pre- and postsynaptic membranes (arrow and crossed arrow) at the synapse between a thorn (t) and axon (Ax), surrounded by GABA-Tase labeled glial (GI) process. (X25,400.) (d) GABA-Tase labeled glial cell (GI) with unlabeled nucleus and processes containing mitochondria with labeled outer membranes surround a nonreactive mossy fiber rosette (MF). (X9750.) Downloaded by guest on September 26, 2021