14 LLIN.~S, R., BLOEDEL, J. R., AND HILLMAN, D. E., Functional characterization of neuronal circuitry of frog cerebellar cortex, J. Neurophysiol., 32 (1969) 847-870. 15 LLIN,/~S, R., BLOEDEL,J. R., AND ROBERTS, W., Antidromic invasion of Purkinje cells in frog cerebellum, J. Neurophysiol., 32 (1969) 881-892. 16 LLIN~S, R., NICHOLSON,C., FREEMAN, J. A., AND HILLMAN, D. E., Dendritic spikes and their inhibition in alligator Purkinje cells, Science, 160 (1968) 1132-1135.
17 N1CHOLSON,C., AND LLIN~S,R., Inhibition of Purkinje cells in the cerebellum of elasmobranch fishes, Brain Researeh, 12 (1969) 477481. 18 RAM6NYCAJAL,S., Histologie du Syst~me Nerveux de l'Homme et des Vertdbrds, 2 vols., Maloine, Paris, 1911, pp. 72-79. 19 SOVELO,C., Ultrastructural aspects of the cerebellar cortex of the frog. In R. LLIN~S(Ed.), Neurobiology of Cerebellar Evolution and Development, Amer. Med. Ass., Chicago, Ill., 1969, pp. 327 371. 20 SOTELO,C., Stellate cells and their synapses on Purkinje cells in the cerebellum of the frog, Brain Research, 17 (1970) 510-514. (Accepted June 1lth, 1970) Brain Research, 22 (1970) 386-391
Cellular localization of labeled gamma-aminobutyric acid (3H-GABA) in rat cerebellar cortex: an autoradiographic study Brain slices incubated in physiological buffer solutions take up and accumulate gamma-aminobutyric acid (GABA) (for ref. see ref. 10), which is considered to be an inhibitory transmitter substance in mammalian central nervous system (see e.g. refs. 2, 12, 16). It has been suggested that the uptake of GABA from the extracellular space may serve as an important mechanism for inactivation of this transmitter 10 similar to that operating in monoamine neurons (see ref. 9). However, in contrast to monoamines there is comparatively little known about the cellular localization both of exogenously administered as well as endogenous G A B A (cf. refs. 6, 16). In the present paper some preliminary results will be reported obtained by autoradiographic studies on slices of rat cerebellar cortex incubated with 3H-GABA. Thin slices of rat cerebellar cortex were incubated with 3H-GABA (specific activity 2 Ci/mM, New England Nucl. Corp., Boston, Mass.) (4 • 10-6M) in a Dextran (Pharmacia, Uppsala, Sweden) containing Tyrode buffer solution for 45 rain (ref. 8). After rinsing for 20 rain the slices were either frozen in liquid propane cooled by liquid nitrogen, freeze-dried, reacted with paraformaldehyde and embedded in Araldite 7 or fixed in 5 ~ glutaraldehyde, dehydrated and embedded in Epon. 1-5/~m thick sections, cut on an LKB ultrotome, were covered with Ilford L4 emulsion (Ilford Ltd., Essex, Great Britain) with the help of a loop techniquea3, a4 and exposed for 2-6 weeks. The autoradiographs were examined in a Leitz Orthoplan light microscope equipped with an Ultropak system (E. Leitz GmbH, Wetzlar, W. Germany). Our preliminary results demonstrate an increased number of grains diffusely over the cerebellar cortex as compared to background aclivity over parts of the sections containing no tissue. Furthermore, strong and well-defined accumulations of grains Brain Research, 22 (1970) 391-396
SHORT COMMt NIt ATIONS
Fig. I. All figures (I 7) are autoradiographs from slices of rat cerebellar cortex incuba~,ed with :~H-GABA. Strong accumulations o f grains (single arrow) are seen in the molecular layer (tool), mainly in its superficial part. One accumulation (double arrowsl is localized deeper. Under most of these accumulations cell bodies can be identified. 450. Fig. 2. Accumulation of grains over a cell body and possibly over" a cell process (single arrowl m the superficial zone of the molecular layer. 840. Fig. 3. In addition to a few large accumulations (arrow heads) over cell bodies smaller accumulations are seen in the superficial zone of the molecular layer (mol), :, 500. Fig. 4. Large accumulations of grains (arrow heads) at rather regular intervals are seen i n 1he granular layer (granl very closely to the Purkinje cell layer. :, 575.
Brain Research, 22 (1970) 391 396
were found over several structures as described below. Principally, both diffuse and well-defined activity was higher in sections from the superficial layer of the slices. No obvious differences were found between freeze-dried and glutaraldehyde fixed tissue. (1) Strong accumulations of grains were seen over cell bodies in the molecular layer, mainly in its outer zone (Figs. 1-3). Occasionally, grains seemed to cover a process emanating from such a cell (Fig. 2). In this area mainly stellate cells, oligodendroglial cells and, around vessels, also pericytes are found (for original work and references on cerebellar anatomy used in this study, see e.g. refs. 3, 5; for glial components, see especially ref. 17). Thus, the accumulations of grains may be related to at least one of these structures. Similarly, accumulations of grains described under (2)-(5) may correspond to one (or more) of the structures mentioned under the respective paragraph. (2) Small accumulations (diameter, mostly about 1-3 #m) were found in the outer zone of the molecular layer (Fig. 3). In this area axon terminals of stellate cells and of parallel fibers and end feet of Bergmann glia are localized. (3) Large, comparatively weak accumulations were observed at regular intervals at the border between the granular and Purkinje cell layer (Figs. 4-6). They were localized in the immediate vicinity of the Purkinje cell bodies, sometimes almost investing its basal (towards the granular layer) part (Figs. 5, 6). Sometimes these accumulations seemed to have 'processes' extending towards the molecular layer (Fig. 6). Toluidine blue staining of the sections revealed that generally these accumulations were not localized over cell bodies. In this area mainly Golgi cells, Bergmann glia and axon terminals of the basket cells and Purkinje cell collaterals are found. (4) Occasionally accumulations of grains were found over cell bodies in the molecular layer near the Purkinje cell layer (Fig. 6). Here mainly basket cells, Bergmann glia and oligodendroglial cells may be found. (5) Small accumulations with a varying diameter were found over the neuropile in the granular layer (Figs. 6, 7). In this area so called cerebel[ar glomeruli, i.e. presynaptic nerve terminals of mossy fibers contacting dendrites of the granular cells, and also glial processes may be found. (6) Accumulations are also found over the walls of blood vessels, especially over small cell bodies. They may represent pericytes when localized around vessels within the brain tissue. Also over the leptomeninges (the pia mater and the arachnoid) and cell bodies associated with them accumulations are found. (7) It may be emphasized that only a low activity was found over Purkinje and granular cell bodies. The present results demonstrate specific and well-localized accumulations of grains in the rat cerebellar cortex, and thus extend previous results on the distribution of 3H-GABA obtained by whole-body autoradiography 6. These accumulations may correspond to sites where 3H-GABA have been taken up and retained in unchanged form since, according to Iversen and Neal t°, GABA metabolites formed during incubation procedure are only found in the incubation medium, and thus do not seem to be retained in the tissue slices. It should, however, be pointed out that higher conBrain Research, 22 (1970) 391-396
Fig. 5. Large accumulations (arrow heads) are seen some of which seem to 'embrace" Purkinje cell bodies (P), Note extensive *processes" (arrows) around one Purkinje cell body. > 700. Fig. 6. Large accumulations of grains (arrow heads) near Purkinje cell bodies (P). Two strong accumulations (Ca and Cz) overlying cell bodies are seen in the molecular layer (tool) near the Purkinje cells. Note small accumulations (arrows) in the granular layer (gran) >< 600. Fig, 7. Many small, strong accumulations are seen in the granular layer (gran), mostly localized over the neuropile between the granular cells, ~" 700.
Brain Research, 22 (1970) 391-396
centrations of G A B A have been used in the present study. Therefore, the possible role of metabolites is at present investigated using aminooxyacetic acid (AOAA), a potent inhibitor of G A B A transaminase is, the main enzyme responsible for the breakdown of GABA. Previous morphological, biochemical and physiological experiments have been interpreted to indicate that in cerebellum G A B A may be present in neuron systems with an inhibitory function, i.e. Purkinje cells, basket cells, stellate cells and Golgi type II cells (for ref. and discussion see ref. 16). Furthermore, uptake and subcellular studies seem to indicate that at least a large part of exogenous G A B A is taken up into neuronal elements and indeed seems to mix with endogenous G A B A pools, possibly localized to n e u r o n s s,1°,u,15. A recent study with 3H-GABA by Ehinger 4 also seems to indicate accumulations of grains over neurons in rabbit retina. The identification of the structures accumulating G A B A in the present study is hampered mainly by the low resolution power of the light microscope and by distortions of the tissue caused by the incubation procedure. It is of interest, however, that Purkinje cell bodies have only a very low activity. It may be added that Csillik and K n y i M r 1 have recently failed to demonstrate accumulation in Purkinje cells of p4C]thiosemicarbazide which is thought to indicate localization of glutamate decarboxylase, the G A B A synthesizing enzyme. Indeed, at the present time, it cannot be established with certainty that any of the cell bodies, over which grains are found, are neurons, although for example those described under (2) and (4) may represent stellate and basket cells, respectively. The possible significance of the present data will be discussed in detail in a full paper. Furthermore, to obtain a better comprehension of the cellular localization of exogenous G A B A in rat cerebellum we are now performing autoradiographic studies at the ultrastructural level. We would like to thank Prof. Gunnar Grant and Dr. Constantino Sotelo for a valuable discussion of the present findings. This work has been supported by a grant from the Swedish Medical Research Council (B71-14X-2887-02A) and by grants from Therese och Johan Anderssons Minne, OIlie och Elof Ericssons Stiftelse and Magnus Bergvalls Stiftelse. Department of Histology, Karolinska Institutet, Stockholm (Sweden)
TOMAS HOKFELT AKE LJUNGDAHL
] CSILLiK, B., AND KNYIHAR, E., Distribution of 14C-thiosemicarbazide in the rat brain: an attempt to localize sites of 7-aminobutyric acid production, Nature (Lond.), 225 (1970) 562-563.
2 CtrRTJS, D. R., Pharmacology and neurochemistry of mammalian central inhibitory processes. In C. yON EULER,S. SKOGLUNDAND U. SODE~tBERG(Eds.), Structure and Function oflnhibitory Neuronal Mechanisms, Vol. 10, Pergamon Press, Oxford, 1968, pp. 429455. 3 ECCLES,J. C., ITO, M., ANDSZENTAGOTHAI,J., The Cerebellum as a Neuronal Machine, Springer, Berlin, 1967, 335 pp. 4 EHINGER,B., Autoradiographic identification of rabbit neurons that take up GABA, Experientia (Basel), in press. 5 Fox, C. A., ANDSNIDER,R. S. (Eds.), The Cerebellum, Progress in Brain Research, VoL 25, Elsevier, Amsterdam, 1967, 355 pp. Brain Research, 22 (1970) 391-396
6 HESPE, W., ROBERTS, E., AND PRINS, H., Autoradiographic investigation of the distribution of [14C]GABA in tissues of normal and aminooxyacetic acid-treated mice, Brain Research, 14 (1969) 663 -671. 7 HOKFEL1, T., A modification of the histochemical fluorescence method for the demo~stration of catecholamines and 5-hydroxytryptamine, using Araldite as embedding mediura~, Histochem. Cytochem., 13 (1965) 518-519. 8 H(~KFELT, T., JONSSON, G., AND LJUNGDAHL, A., Regional uptake and subcellular localization of [3H]gamma-aminobutyric acid (GABA) in rat brain slices, L(fe Sci., 9 (1970) 203 212. 9 IVERSEN, L. L., The Uptake and Storage of Noradrenaline in Sympathetic Nerves, Cambridge University Press, Cambridge, 1963, 253 pp. 10 IVERSEN, L. L., AND NEAL, M. J., The uptake of [~H]GABA by slices of rat cerebral cortex, J. Neurochem., 15 (1968) 1141 1149. I 1 IWRSEN, L. L., AND SNYDER, S , Synaptosomes: different populations storing catecholamines and gamma-aminobutyric acid in homogenates of rat brain, Nature (Lond.), 220 (1968) 796-798. 12 KRNJEW6, K., AND SCHWARTZ, S., The inhibitory transmitter in cerebral cortex. In C. YoN EUL~R, S. SKOGLUNDAND U. S/3DERBERG(Eds.), Structure and Function of Inhibitory Neuronal Mechanisms, Vol. 10, Pergamon Press, Oxford, 1968, pp. 419-427. 13 MILLER, O. L., Jr., STONe, G. E., AND PRY:SCOTT, D. M., Autoradiography of soluble materials, J. Cell Biol., 23 (1964) 654-658. 14 NACIATA,T., AND NAWA, T., A modification of dry-mounting technique for radioautography of water-soluble compounds, Hi3tochemie, 7 (I 966) 370-371. 15 NEAI_, M..I., AND IVERSEN, L. I.., Subcellular distribution of endogenous and [:3H]7-aminobutyric acid in rat cerebral cortex, J. Neurochem., 16 (I 969) 1245-1252. 16 ROBERTS, E., Some biochemical-physiological correlations in studies of 7-aminobutyric acid. In ('. VON If_ULI'.R, S. SKOGLUND AND U. S/3DERBERG reds.), Structure and Function of Inhibitory Neuronal Mechanisms, Vol. I0, Pergamon Press, Oxford, 1968, pp. 401-418. 17 SOTELO, C., Cerebellar neuroglia: morphological and histochemical aspects. In C. A. Fox AND R. S. SNmV:R (Eds.), The Cerebellum, Progress in Brain Research, Vol. 25, Elsevier. Amsterdam 1967, pp. 226 250. 18 WaLLACH. D. P., Studies on the GABA pathway. The inhibition of 7-aminobutyric acid-¢tketoglutaric acid transaminase in vitro and in vivo by U7524 (amino-oxyacetic acid), Biochem. Pharmacol., 5 (1961) 323-331. (Accepted June 22nd, 1970)
Brain Research, 22 (1970) 391-396