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Autoradiographic localisation of [3H]GABA and [3H]glutamate over satellite glial cells
Brain Research, 66 (1974) 275-288
275
© Elsevier ScientificPublishing Company, Amsterdam - Printed in The Netherlands
A U T O R A D I O G R A P H I C LOCALISATION OF [3H]GABA AND [3H]GLUTAMATE OVER SATELLITE GLIAL CELLS
F. SCHON AND J. S. KELLY
MRC Neurochemicat Pharmacology Unit, Department of Pharmacology, Medical School, Hills Road, Cambridge CB2 2QD (Great Britain) (Accepted July 25th, 1973)
SUMMARY
The uptake of the putative amino acids transmitter substances GABA, glycine and glutamate was studied autoradiographically in adult rat sensory ganglia following in vitro incubation. As these ganglia are devoid of any synaptic innervation, they offer a convenient preparation for examining glial cell uptake. GABA was exclusively localised within the satellite glial ceils surrounding unlabelled sensory neuronal cell bodies. GABA was also accumulated within glial cells surrounding unmyelinated axons and in the Schwann cell bodies of the large myelinated fibres in the dorsal root ganglia. Glutamate was also exclusively taken up into satellite glial cell bodies. The lack of accumulation in sensory neuronal cell bodies was surprising in view of the suggested role of glutamate as the possible mammalian sensory transmitter substance. The uptake of glycine, leucine and alanine was much less than that for GABA or glutamate. All 3 amino acids were localised equally within neuronal and satellite glial cell bodies presumably by the neutral amino acid uptake system present in all cells.
INTRODUCTION
Homogenates and small slices of the mammalian central nervous system actively accumulate exogenous 7-aminobutyric acid (GABA) when incubated in the presence of low concentrations of the amino acid, and electron microscopic autoradiography has shown that this uptake is primarily localised within nerve terminal regions 4,2°. However, on larger slices and in vivo the evidence suggests that this uptake is not exclusively into neurones but also involves glial cells. In the cerebellum the Bergmann glial cells which surround Purkinje cell bodies accumulate exogenous GABA 17,18
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as do Schwann cells in the corpus callosum17. GABA uptake in vitro in the retina is also predominantly into Mfiller glial cells2s. A high affinity GABA uptake system has been reported in the rat superior cervical ganglia5, where it is known that the preganglionic fibres utilize acetylcholine and the postganglionic cell bodies noradrenaline as transmitter substances6,7,zL The present study was, therefore, undertaken to localise the uptake of GABA and certain other amino acids into adult rat sensory ganglion by autoradiographic procedures on the basis that (1) such ganglia are devoid of any morphologically identifiable synapses, (2) they contain a single population of neurones, the sensory neuronal cell bodies, which probably do not utilise GABA as their transmitter substance, and (3) they contain large numbers of easily identifiable satellite glial cells surrounding each of the sensory neurones~L An uptake of glutamic acid into the sensory ganglion was also examined since the distribution of glutamic acid within the dorsal roots and spinal cord13,24 has led to the suggestion that glutamic acid is the transmitter synthesised by the sensory ganglion cells. A high affinity, stereospecific uptake system for L-glutamic acid has been described in homogenates and small slices of rat spinal cord and cerebral cortexz,3,27. METHODS
Incubation of ganglia with radioactive amino acids Adult albino Wistar rats (150-300 g) were killed by decapitation and their spinal columns rapidly dissected in ice cold Krebs-phosphate medium pH 7.4. Individual sensory ganglia were desheathed and preincubated for 10 min in 0.5 ml of oxygenated Krebs-phosphate medium at 37 °C23. Tritiated amino acid was then added (final concentration ---- 50 #Ci/ml; range = 3-25/zM) and the ganglia incubated for a further 20 min with shaking after which they were recovered, blotted on Whatman No. 1 filter paper, washed 5 times in fresh cold buffer and fixed for 1-2 h by immersion in a 5 ~ glutaraldehyde in Krebs-phosphate buffer20.
Tissue/medium ratios In biochemical experiments, ganglia were incubated with a final amino acid concentration of 1/~Ci/ml (range 60-500/zM). After washing in cold Krebs the ganglia were immersed in 1 ml of Soluene Tm 100 (Packard) for 8 h at room temperature. The radioactivity was estimated by liquid scintillation spectrometry after the addition of 2 ml of ethoxyethanol and 10 ml of phosphor (0.4~ butyl-PBD in toluene). The radioactivity was also estimated in an aliquot of the stock tritiated amino acid solutions. The uptake into the ganglia was expressed in terms of tissue/medium ratio defined as the disint./min in 1 g of tissue/the disint./min in 1 ml of incubation medium.
Preparation of ganglia for autoradiography After fixation the ganglia were rinsed thoroughly in 0.1 M sodium phosphate buffer pH 7.4 and immersed for 1 h in 1 ~ osmium tetroxide solution in the same buffer, dehydrated and embedded in an Epoxy resin. For light microscopic autoradiog-
[3H]GABA
IN SENSORY GANGLIA
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raphy thick (2 #m) sections were cut, mounted on glass slides, dipped in Ilford L4 emulsion and stored at 4 °C for 1-12 weeks in light-tight boxes. Slides were developed for 3 min in a Kodak DK170 developer at 20 °C, fixed for 5 min in 25 ~ (w/v) sodium thiosulphate, washed, dried and mounted for light microscopic examination. For electron microscopic autoradiography ultrathin sections (approx. 75 nm) were cut on an LKB ultratome Mark 3, mounted on celloidin-coated slides, stained for 5 min in lead citrate, carbon coated, dipped in Ilford L4 emulsion and then stored for 1-12 weeks. The autoradiographs were developed for 2 min in a Kodak D19B developer and fixed for 5 min in 25 ~ sodium thiosulphate solution. Finally the celloidin films were floated onto a water surface and mounted on 50 #m mesh copper grids. The thin sections were then examined using a Philips 300 electron microscope.
Radioactive materials [2,3-3H]GABA, specific activity : 10Ci/mmole, N.E.N. Chemicals, Dreieichenhain, Germany; [1-3H]glycine, specific activity : 2 Ci/mmole; L-[3H]alanine, specific activity : 34 Ci/mmole; L-[3H]leucine, specific activity : 15.2 Ci/mmole; OL-[3H]glutamic acid, specific activity : 2.8 Ci/mmole, all from the Radiochemical Centre, Amersham. Metabolism Incorporation of [3H]amino acids into protein. After incubation ganglia were homogenised in cold 0.4 N perchloric acid (PCA), the PCA homogenate centrifuged and the pellet resuspended and washed 3 times in cold PCA. Thereafter, the pellet was dissolved in 1 ml of Soluene (Packard Instrument Co.) overnight and counted. Chromatographic analysis of ganglion extracts following incubation with [3HIamino acids. Individual ganglia were incubated for 10 min with [3H]glutamic acid and for 20 min with [3H]glycine and [ZH]GABA; thereafter, they were washed in cold incubation medium and homogenised in 250 #1 of 70 % ethanol. The homogenate was centriguged for 10 min at 1000 × g and the supernatant run overnight on descending paper chromatograms with radioactive and amino acid markers in a butanolacetic acid-water (4:1:1, v/v/v) system 22. The chromatograms were stained with 0.25 % ninhydrin in acetone and cut up into 1 cm strips from origin to solvent front. The radioactivity in each strip was eluted into 2 ml of ethoxyethanol for 1 h and estimated by liquid scintillation counting. RESULTS
Tissue/medium ratios In preliminary studies on the uptake of the amino acids, GABA, glutamate and glycine in sensory ganglion in vitro it was found that the amount of [3H]glycine taken up after 20 min was fairly low, whilst there was a substantial uptake of both [3H]glutamate and [3H]GABA. This difference in the amount of radioactivity accumulated in ganglion incubated with the 3 different amino acids is expressed in Table I as tissue/medium ratios (radioactivity in 1 mg of tissue divided by the radioactivity in
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TABLE I THE UPTAKE AND METABOLISM OF CERTAIN AMINO ACIDS IN RAT SENSORY GANGLIA FOLLOWING
in vitro
INCUBATION
Amino acid
Time (min)
T/M ratio
Average length of autoradiographie exposure in weeks
% Unmetabolised amino acid after incubation 2 determinations
GABA DL-Glu Gly
20 10 20
4.7 2.1 1.6
1 2 5
84.2 85.2 80.3
1 #1 of incubation medium). Longer exposure periods were, therefore, necessary in order to obtain autoradiographs of ganglia incubated with [3H]glycine comparable to those with [3H]GABA. The average exposure period used to produce autoradiographs of similar intensity is presented in the third column of Table I. The amount of radioactive amino acid incorporated into protein during the incubation period must have been extremely small since all but 1 ~ of the radioactivity was extractable with PCA. The final column of Table I shows the percentage of radioactivity extracted from the ganglia which was located on chromatograms as a single peak, coincident with the RF of authentic GABA. When the radioactivity was measured in whole ganglia fixed with glutaraldehyde after incubation with [3H]GABA, approximately 70 ~ of the activity was retained in the tissue.
Light microscopic localisation of [3H]GABA The sensory ganglia of adult rats are 1-2 mm in diameter and surrounded by a thick connective tissue sheath beneath which lie the large sensory neuronal cell bodies (40-150 #m in diameter). Each neurone is completely surrounded by tightly apposed satellite glial cells. Autoradiographs of desheathed ganglia incubated with 5 × 10-6 M GABA (50/zCi/ml) were prepared and developed after a 1 week exposure period. An abundance of silver grains was seen throughout the ganglia when cut in transverse section (Fig. 1). The grains were concentrated exclusively over the satellite glial cell bodies surrounding the sensory neurones which were themselves almost devoid of labelling. In addition the fibre tract running through the ganglia to form the dorsal root was relatively free of silver grains. In preliminary experiments using ganglia incubated with intact connective sheaths, the greatest density of silver grains was observed over satellite cells in the outer regions of the ganglia. The presence of the intact sheath thus appeared to reduce amino acid penetration into the ganglia. The sheath itself showed negligible autoradiographic activity, as seen in Fig. 2a. Again the satellite glial cells and their processes were intensely labelled. The same autoradiographic distribution of [3H]GABA was observed in sensory ganglia from neonatal rats as well as in both adult and neonatal cat ganglia. The pres-
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Fig. 1. A dark field autoradiograph showing photomontage through a whole sensory ganglia incubated for 20 rain with 10-6 M [3H]GABA. Radioactivity is distributed throughout the ganglia, localised exclusively in the satellite glial cells stlrrounding the large unlabelled sensory neuronal cell bodies. The sensory fibre tract (indicated by a white arrowhead) shows only sparse labelling. The epoxy resin above the ganglia is almost totally devoid of background silver grains. Exposure time, 3 weeks; calibration bar, 100/,m.
ence of the drug amino-oxyacetic acid (AOAA) 29 at a concentration of 10/zM during the preincubation and the incubation period did not alter the silver grain distribution, but greatly increased the intensity of the autoradiograph. Recently the ganglion has been shown to contain detectable levels of GABA-glutamate transaminase activity (Gottesfeld et al., in preparation).
Electron microscopic localisation of [3HJGABA Electron microscopic autoradiography confirmed the glial localisation of GABA. The sensory neuronal cell bodies are completely enwrapped by narrow sheaths of glial cytoplasm which appear in transverse section as long thin projections. An intense accumulation of silver grains was observed throughout these satellite glial cell processes (Fig. 3). The axonal processes leaving the small neuronal cell bodies are unmyelinated as are the initial segments of the axons of the large cell bodies 14. These axons are also enclosed by tightly apposing glial cells which accumulate GABA (Fig. 4).
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Fig. 2. a: dark field autoradiography of rat sensory ganglia incubated with 10 6 M [aH]GABA. Heavy accumulations of silver grains over satellite glial ceils (indicated by arrowheads in b) were observed. The neuronal cell bodies (N) and the connective tissue (CT) were relatively free of silver grains. Exposure time, 1 week; calibration bar, 20 pm. In Figs. 2 and 4-6 the phase contrast micrograph contains the lettering referred to in the description of the upper dark field autoradiograph. b: phase contrast of the same area.
Fig. 3. a: electron microscopic autoradiograph of rat sensory ganglia. Relatively few silver grains were found over the sensory neuronal cytoplasm (NC) in the lower half of the picture and the extracellular space (ECS)in the upper portion. The silver grains clearly overlie the satellite glial cell process (indicated by arrowheads). Calibration bar, 2 #m. b: The axon (A) seen in transverse section is devoid of radioactivity. Large numbers of silver grains are observed over the nucleus (GN) and the cytoplasm of the glial cell completely surrounding the axon. Calibration bar, 2 #m.
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Fig. 4. a and b: dark field autoradiograph and corresponding phase contrast field of rat sensory ganglia incubated with 1.75 × 10-a MDL-[3H]glutamic acid. Silver grains are predominantly located over satellite glial cells (indicated by arrowheads). The connective tissue sheath (CT) and neuronal cell bodies (N) were relatively free of labelling. Exposure time, 4 weeks; calibration bar, 20/~m.
Light microscopic localisation of glutamic acid T h e a u t o r a d i o g r a p h i c l o c a l i s a t i o n o f o g - [ a H ] g l u t a m i c acid was investigated after i n c u b a t i n g ganglia for a 10 min p e r i o d with the r a d i o a c t i v e a m i n o acid at a conc e n t r a t i o n o f 10 -6 M. A s h o r t i n c u b a t i o n time was used in a n a t t e m p t to minimize m e t a b o l i s m o f this a m i n o acid. The d i s t r i b u t i o n o f silver grains was identical to t h a t o b s e r v e d for G A B A . The satellite glial cells were heavily labelled whilst the n e u r o n a l cell b o d i e s a n d the connective tissue sheath were u n l a b e l l e d (Fig. 4).
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Fig. 5. a and b: dark field autoradiograph of rat sensory ganglia incubated with 10 a M [aH]glycine. Silver grains were distributed equally over the neuronal (N) and the satellite glial cell bodies (arrowheads). Relatively few silver grains were found over the sensory fibre tract (ST). Exposure time, 6 weeks; calibration bar, 20 t~m.
Light microscopic localisation of glycine, alanine and leucine W h e n ganglia were i n c u b a t e d with tritiated glycine (25 × 10 -6 M ) , alanine (1.5 × 10 -6 M ) a n d leucine (3.3 × 10 -6 M ) , u n d e r exactly the same c o n d i t i o n s as described f o r G A B A , a n d the a u t o r a d i o g r a p h s exposed f o r 5 weeks (cf T a b l e 1), the silver grains arising f r o m these 3 a m i n o acids were localised equally over the n e u r o n a l cell b o d i e s a n d satellite glial celis. Figs. 5 a n d 6 show this d i s t r i b u t i o n in the ganglia
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Fig. 6. a and b: dark field autoradiograph and cgrresponding phase contrast field of rat sensory ganglia incubated with 1.5 >: 10 a M alanine. Silver grains are predominantly located over the neuronal and satellite gliaI cell bodies (arrowheads). The connective tissue sheath (CT) and the myelinated fibres (MF) are relatively free of labelling. Exposure time, 6 weeks; calibration bar, 20/~m.
i n c u b a t e d with [ZH]glycine a n d a l a n i n e respectively. The connective tissue sheath a n d the m y e l i n a t e d fibre t r a c t were a g a i n sparsely labelled.
GABA localisation in the dorsal root fibre tract T h e presence o f [ Z H ] G A B A u p t a k e in v a r i o u s r a t p e r i p h e r a l nerve fibre tracts in the r a t has been r e p o r t e d b y B o w e r y a n d B r o w n 5. T o investigate the localisation o f this u p t a k e , s h o r t lengths o f the d o r s a l r o o t fibre t r a c t were dissected, i n c u b a t e d
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with [3H]GABA in vitro, and prepared for light microscope level autoradiography. The silver grains were localised over Schwann cell bodies. The myelin sheaths and the interior of the axons were devoid of silver grains. An identical distribution of silver grains was found following in vitro incubation of ventral roots. Dorsal root section: effect on G A B A uptake
Transection of the efferent dorsal root caused no significant change in [3H]GABA uptake into the sensory ganglia when the uptake was investigated over a 10 min period at 25 °C. Short lengths of the afferent and efferent sensory trunks were dissected from rats lesioned 1 month earlier incubated with GABA and prepared for light microscopic autoradiography. Although uptake into Schwann cells in the afferent trunk was unaffected, uptake into the efferent trunk proximal to the transection was greatly reduced presumably as a result of the extensive degeneration of myelinated axons and supporting Schwann cells observed histologically. Cells which invaded the tract following such transections did not accumulate [ZH]GABA under the conditions of the experiment. DISCUSSION
In sensory ganglia, the sole cellular component which accumulates exogenous GABA are the satellite glial cells as revealed by both light and electron microscopic autoradiography. This provides a convenient system for comparing the properties of the predominantly neuronal uptake of GABA in brain slices and homogenates z 1,23 with glial uptake in the sensory ganglia in vitro. Such a study is reported in a companion paper 3a. The autoradiographic distribution and the biochemically measured uptake of GABA was unchanged in ganglia isolated from the spinal cord by rhizotomy l month earlier. This is in good agreement with the morphological observation that such deafferentation causes no marked cellular changes in either the sensory neurones or their satellite glial cells 1~. The increased number of silver grains observed over satellite glial cells following incubation in the presence of AOAA, confirms a similar observation on glial cells in the corpus callosum by H6kfelt and Ljungdah117 who suggested that glial cells may contain large amounts of the enzyme GABA-transaminase. Histochemical studies have revealed large amounts of the enzyme in extraneuronal structures such as the pia mater and ependymal cells36. Glycine, alanine and leucine all showed the same autoradiographic distribution being localised over both satellite glial cells and neuronal cell bodies. Since the amount of the 3 amino acids taken up was much less than the amount of GABA or glutamate accumulated, it is probable that they are accumulated by a low affinity neutral amino acid uptake system present in all brain tissues 1. The localisation of DE-[3H]glutamate in rat sensory ganglion like that of GABA, was exclusively in satellite glial cells. This extraneuronal localisation of glutamate is in clear agreement with other recent studies which showed that in the retina it is localised in Mfiller glial cells 12, in rat cerebellar slices in the Bergmann glial cells 19
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and at the insect neuromuscular junction, where it is implicated as the neurotransmitter substance, it was localised over Schwann cells az. A high affinity uptake system of glutamate has also been reported in frog sciatic nerves 3v. It is somewhat surprising that the sensory neuronal cell bodies themselves do not accumulate glutamic acid as it has been cited repeatedly as the putative mammalian sensory transmitter 24. The situation could, of course, resemble that observed in the cerebellum where the Purkinje cells which utilize GABA as their synaptic transmitter is will only accumulate exogenous GABA into their cell bodies under special circumstances. For instance Purkinje cells can be labelled by adding [aH]GABA to neurones growing either in tissue culture~"5,a4 orin explants of cerebellum attached to the iris of a recipient rat's eye (Ljungdahl et al., to be published). Presumably in both these situations the glial cells no longer form an effective barrier to the entry of GABA into the vicinity of the neurone. In the lobster neuromuscular junction no neuronal uptake is observeda°; all the GABA released is actively transported into Schwann cells surrounding the inhibitory synaptic endings. A similar situation was reported in the insect, where neuronally released glutamate is taken up exclusively into extraneuronal sites 3z. The role played by the glial as opposed to the neuronal uptake system in the mammalian central nervous system is not obvious. It has been suggested that the extraneuronal uptake may serve to prevent neurotransmitter substances in the blood reaching and activating receptors on neurones in the central nervous system a6. However, intraarterially administered GABA depolarises neurones of both sensory and superior cervical ganglia9,10 which would appear to preclude such an explanation. A more attractive hypothesis would be that specialised glial cells exist in close association with all neurones to ensure more rapid or efficient removal of neuronally released GABA. On the other hand, presynaptic inhibition in the dorsal horn of the spinal cord is blocked by both picrotoxin 11 and bicuculline 26 and mimicked by iontophoretically applied GABA s. GABA containing interneurones may therefore synapse with the preterminal regions of the primary sensory axons. The presence on the sensory neurones of GABA receptors and of glial cells capable of accumulating GABA may therefore be related to this role of GABA on the primary afferent terminals. The capacity to accumulate exogenous GABA may alternatively be present in all glial cells 16 but only play a physiological role in CNS regions where GABA has a neurotransmitter function. The presence of extraneuronal GABA uptake in ventral root fibres, in superior cervical ganglia and in preganglionic sympathetic nerve trunks supports such a view.
ACKNOWLEDGEMENTS
The authors wish to thank Dr. L. L. Iversen and the members of his laboratory for their help throughout this work and Mr. D. C. Chapman for skilled technical assistance. F. S. is an M R C Scholar at the Physiological Laboratory, Cambridge, Great Britain.
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REFERENCES 1 ABADOM,P. N., AND SCHOLEFIELD, P. G., Amino acid transport in brain cortex slices. II. Competition between amino acids, Canad. J. Biochem., 40 (1962) 1591-1602. 2 BALCAR, V. J., AND JOHNSTON, G. A. R., The structural specificity of the high affinity uptake of L-glutamate and L-aspartate by rat brain slices, J. Neurochem., 19 (1972) 2657-2666. 3 BALCAR, V.J., AND JOHNSTON, G. A. R., High affinity uptake of transmitters; Studies on the uptake of L-aspartate, GABA, L-glutamate and glycine in cat spinal cord, J. Neurochem., 20 (1973) 529-539. 4 BLOOM,F. E., AND IVERSEN,L. L., Localizing 3H-GABA in nerve terminals of rat cerebral cortex by electron microscopic autoradiography, Nature (Lond.), 229 (1971) 628-630. 5 BOWERY,N. G., AND BROWN, D. A., ~-Aminobutyric acid uptake by sympathetic ganglia, Nature New Biol., 238 (1972) 89-91. 6 BROWN, G. L., AND FELDBERG,W., The action of potassium on the superior cervical ganglion of the cat, J. Physiol. (Lond.), 86 (1936) 290-305. 7 CANNON, W. B., AND LISSAK,K., Evidence for adrenaline in adrenergic neurones, Amer. J. Physiol., 125 (1939) 765-777. 8 CURTIS, D. R., AND RYALL, R. W., Pharmacological studies upon spinal presynaptic fibres, Exp. Brain Res., 1 (1966) 195-204. 9 DE GROAT, W. C., GABA-depolarization of a sensory ganglion; antagonism by picrotoxin and bicuculline, Brain Research, 38 (1972) 429-432. 10 DE GROAT, W. C., LALLEY, P. M., AND SAUM, W. R., Depolarization of dorsal root ganglia in the cat by GABA and related amino acids; antagonism by picrotoxin and bicuculline, Brain Research, 44 (1972) 273-277. 11 ECCLES, J.C., SCHMIDT, R . F . , AND WILLIS, W.D., Pharmacological studies on presynaptic inhibition, J. Physiol. (Lond.), 168 (1963) 500-530. 12 EHIN6ER, B., AND FALCK, B., Autoradiography of some suspected neurotransmitter substances: GABA, glycine, glutamic acid, histamine, dopamine and L-DOPA, Brain Research, 33 (1971) 157-172. 13 GRAHAM,L. T., SHANK, R. P., WERMAN,R., AND APRISON, M. H., Distribution of some synaptic transmitter suspects in cat spinal cord, J. Neuroehem., 14 (1967) 465-472. 14 HA, H., Axonal bifurcation in the dorsal root ganglion of the cat: A light and electron microscopic study, J. comp. Neurol., 140 (1970) 227-240. 15 HARE, W. K., AND HINSEY, J. C., Reactions of dorsal root ganglion cells to section of peripheral and central processes, J. eomp. Neurol., 73 (1940) 489-502. 16 HENN, F. A., AND HAMBER6ER, A., Glial cell function: Uptake of transmitter substances, Proc. nat. Acad. Sei. (Wash.), 68 (1971) 2686-2690. 17 HOKFELT, T., AND LJUNGDAHL,/~k., Uptake of [3H]noradrenaline and [3H]7-aminobutyric acid in isolated tissues of rat: an autoradiographic and fluorescence microscopic study. In O. ER~NKO (Ed.), Histochemistry of Nervous Transmission, Progress in Brain Research, Vol. 34, Elsevier, Amsterdam, 1971, pp. 87-102. 18 HOKFELT, T., AND LJUNGDAHL,/~., Autoradiographic identification of cerebral and cerebellar cortical neurons accumulating labelled gamma-aminobutyric acid (3H-GABA), Exp. Brain Res., 14 (1972) 354-362. 19 HtSKFELT,T., AND LJUNGDAHL,/~., Applications of cytochemical techniques to the study of suspected transmitter substances in the nervous system. In E. COSTA,L. L. IVERSENAND R. PAOLETTI (Eds.), Advances in Biochemical Psychopharmacology, Vol. 6, Raven Press, New York, 1972, pp. 1-37. 20 IVERSEN,L. L., AND BLOOM, F. E., Studies of the uptake of [aH]GABA and [3H]glycine in slices and homogenates of rat brain and spinal cord by electron microscopic autoradiography, Brain Research, 41 (1972) 131-143. 21 IVERSEN,L. L., AND JOHNSTON,G. A. R., GABA uptake in rat central nervous system: comparison of uptake in slices and homogenates and the effects of some inhibitors, J. Neurochem., 18 (1971) 1939-1950. 22 IVERSEN, L.L., AND KRAVITZ, E.A., The metabolism of ~-aminobutyric acid (GABA) in the lobster nervous system - - uptake of GABA in nerve muscle preparations, J. Neurochem., 15 (1968) 609-620. 23 ][VERSEN,L. L., AND NEAL, M. J., The uptake of [aH]-GABA by slices of rat cerebral cortex, J. Neurochem., 15 (1968) 1141-1149.
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24 JOHNSON,J. L., Glutamic acid as a synaptic transmitter in the nervous system. A review, Brabt Research, 37 (1972) 1-19. i 25 LASHER, R. S., AND ZAOON, I. S., The effect of potassium on neuronal differentiation in culturesl of dissociated newborn rat cerebellum, Brain Research, 41 (1972) 482-488. [ 26 LEVY, R. A., REPKIN, A. H., AND ANDERSON, E. G., The effect of bicuculline on primary afferent terminal excitability, Brain Research, 32 (1971) 261-265. 27 LOGAN, W. J., AND SNYDER, S. H., High affinity uptake systems for glycine, glutamic and aspartic acids in synaptosomes of rat central nervous tissues, Brain Research, 42 (1972) 413-431. 28 NEAL, M.J., AND IVERSEN, L.L., Autoradiographic localization of 3H-GABA in rat retina, Nature New Biol., 235 (1972) 217-218. 29 NEAL, M.J., AND STARR, M. S., Effects of inhibitors of y-aminobutyrate aminotransferase o n the accumulation of 3H-y-aminobutyric acid by the retina, Brit. J. Pharmacol., 47 (1973) 543-555. i 30 ORKAND, P. M., AND KRAWTZ, E. A., Localization of the sites of y-aminobutyric acid (GABA) I uptake in lobster nerve-muscle preparations, J. Cell Biol., 49 (1971) 75-89. 31 PANESSI,E., BIANCHI, R., CALLIGARIS,B., VENTURA, R., AND WEIBEL, E. R., Quantitative relationships between nerve and satellite cells in spinal ganglia. An electron microscopical study. I . Mammals, Brain Research, 46 (1972) 215-234. 32 SALPETER, M. M., AND FAEDER, I. R., The role of sheath cells in glutamate uptake by insect nerve-muscle preparations. In O. ER~NK6 (Ed.), Histoehemistry of Nervous Transmission, Progress in Brain Research, Vol. 34, Elsevier, Amsterdam, 1971, pp. 103-114. 33 SCHON, F., AND KELLY, J. S., The characterisation of [3H]GABA uptake into the satellite glial cells of rat sensory ganglia, Brain Research, 66 (1974) 289-300. 34 SOTELO, C., PR~VAT, A., AND DRrAN, M. J., Localisation of [3H]GABA in tissue culture of rat cerebellum using electron microscopy radioautography, Brain Research, 45 (1972) 302-308. 35 VON EOLER, U. S., AND PURKHOLD, A., Effect of sympathetic denervation on the noradrenaline and adrenaline content of the spleen, kidney and salivary glands in the sheep, Acta physiol, stand., 24 (1951) 212-217. 36 VAN GELDER, N. M., A comparison of ~-aminobutyric acid metabolism in rabbit and mouse nervous tissue, J. Neurochem., 12 (1965) 239-244. 37 WHEELER, D. D., AND BOYARSKY, L. L., Influx of glutamic acid in peripheral nerve characteristics of influx, J. Nettroehem., 15 (1968) 1019-1031.
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© Elsevier ScientificPublishing Company, Amsterdam - Printed in The Netherlands
A U T O R A D I O G R A P H I C LOCALISATION OF [3H]GABA AND [3H]GLUTAMATE OVER SATELLITE GLIAL CELLS
F. SCHON AND J. S. KELLY
MRC Neurochemicat Pharmacology Unit, Department of Pharmacology, Medical School, Hills Road, Cambridge CB2 2QD (Great Britain) (Accepted July 25th, 1973)
SUMMARY
The uptake of the putative amino acids transmitter substances GABA, glycine and glutamate was studied autoradiographically in adult rat sensory ganglia following in vitro incubation. As these ganglia are devoid of any synaptic innervation, they offer a convenient preparation for examining glial cell uptake. GABA was exclusively localised within the satellite glial ceils surrounding unlabelled sensory neuronal cell bodies. GABA was also accumulated within glial cells surrounding unmyelinated axons and in the Schwann cell bodies of the large myelinated fibres in the dorsal root ganglia. Glutamate was also exclusively taken up into satellite glial cell bodies. The lack of accumulation in sensory neuronal cell bodies was surprising in view of the suggested role of glutamate as the possible mammalian sensory transmitter substance. The uptake of glycine, leucine and alanine was much less than that for GABA or glutamate. All 3 amino acids were localised equally within neuronal and satellite glial cell bodies presumably by the neutral amino acid uptake system present in all cells.
INTRODUCTION
Homogenates and small slices of the mammalian central nervous system actively accumulate exogenous 7-aminobutyric acid (GABA) when incubated in the presence of low concentrations of the amino acid, and electron microscopic autoradiography has shown that this uptake is primarily localised within nerve terminal regions 4,2°. However, on larger slices and in vivo the evidence suggests that this uptake is not exclusively into neurones but also involves glial cells. In the cerebellum the Bergmann glial cells which surround Purkinje cell bodies accumulate exogenous GABA 17,18
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as do Schwann cells in the corpus callosum17. GABA uptake in vitro in the retina is also predominantly into Mfiller glial cells2s. A high affinity GABA uptake system has been reported in the rat superior cervical ganglia5, where it is known that the preganglionic fibres utilize acetylcholine and the postganglionic cell bodies noradrenaline as transmitter substances6,7,zL The present study was, therefore, undertaken to localise the uptake of GABA and certain other amino acids into adult rat sensory ganglion by autoradiographic procedures on the basis that (1) such ganglia are devoid of any morphologically identifiable synapses, (2) they contain a single population of neurones, the sensory neuronal cell bodies, which probably do not utilise GABA as their transmitter substance, and (3) they contain large numbers of easily identifiable satellite glial cells surrounding each of the sensory neurones~L An uptake of glutamic acid into the sensory ganglion was also examined since the distribution of glutamic acid within the dorsal roots and spinal cord13,24 has led to the suggestion that glutamic acid is the transmitter synthesised by the sensory ganglion cells. A high affinity, stereospecific uptake system for L-glutamic acid has been described in homogenates and small slices of rat spinal cord and cerebral cortexz,3,27. METHODS
Incubation of ganglia with radioactive amino acids Adult albino Wistar rats (150-300 g) were killed by decapitation and their spinal columns rapidly dissected in ice cold Krebs-phosphate medium pH 7.4. Individual sensory ganglia were desheathed and preincubated for 10 min in 0.5 ml of oxygenated Krebs-phosphate medium at 37 °C23. Tritiated amino acid was then added (final concentration ---- 50 #Ci/ml; range = 3-25/zM) and the ganglia incubated for a further 20 min with shaking after which they were recovered, blotted on Whatman No. 1 filter paper, washed 5 times in fresh cold buffer and fixed for 1-2 h by immersion in a 5 ~ glutaraldehyde in Krebs-phosphate buffer20.
Tissue/medium ratios In biochemical experiments, ganglia were incubated with a final amino acid concentration of 1/~Ci/ml (range 60-500/zM). After washing in cold Krebs the ganglia were immersed in 1 ml of Soluene Tm 100 (Packard) for 8 h at room temperature. The radioactivity was estimated by liquid scintillation spectrometry after the addition of 2 ml of ethoxyethanol and 10 ml of phosphor (0.4~ butyl-PBD in toluene). The radioactivity was also estimated in an aliquot of the stock tritiated amino acid solutions. The uptake into the ganglia was expressed in terms of tissue/medium ratio defined as the disint./min in 1 g of tissue/the disint./min in 1 ml of incubation medium.
Preparation of ganglia for autoradiography After fixation the ganglia were rinsed thoroughly in 0.1 M sodium phosphate buffer pH 7.4 and immersed for 1 h in 1 ~ osmium tetroxide solution in the same buffer, dehydrated and embedded in an Epoxy resin. For light microscopic autoradiog-
[3H]GABA
IN SENSORY GANGLIA
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raphy thick (2 #m) sections were cut, mounted on glass slides, dipped in Ilford L4 emulsion and stored at 4 °C for 1-12 weeks in light-tight boxes. Slides were developed for 3 min in a Kodak DK170 developer at 20 °C, fixed for 5 min in 25 ~ (w/v) sodium thiosulphate, washed, dried and mounted for light microscopic examination. For electron microscopic autoradiography ultrathin sections (approx. 75 nm) were cut on an LKB ultratome Mark 3, mounted on celloidin-coated slides, stained for 5 min in lead citrate, carbon coated, dipped in Ilford L4 emulsion and then stored for 1-12 weeks. The autoradiographs were developed for 2 min in a Kodak D19B developer and fixed for 5 min in 25 ~ sodium thiosulphate solution. Finally the celloidin films were floated onto a water surface and mounted on 50 #m mesh copper grids. The thin sections were then examined using a Philips 300 electron microscope.
Radioactive materials [2,3-3H]GABA, specific activity : 10Ci/mmole, N.E.N. Chemicals, Dreieichenhain, Germany; [1-3H]glycine, specific activity : 2 Ci/mmole; L-[3H]alanine, specific activity : 34 Ci/mmole; L-[3H]leucine, specific activity : 15.2 Ci/mmole; OL-[3H]glutamic acid, specific activity : 2.8 Ci/mmole, all from the Radiochemical Centre, Amersham. Metabolism Incorporation of [3H]amino acids into protein. After incubation ganglia were homogenised in cold 0.4 N perchloric acid (PCA), the PCA homogenate centrifuged and the pellet resuspended and washed 3 times in cold PCA. Thereafter, the pellet was dissolved in 1 ml of Soluene (Packard Instrument Co.) overnight and counted. Chromatographic analysis of ganglion extracts following incubation with [3HIamino acids. Individual ganglia were incubated for 10 min with [3H]glutamic acid and for 20 min with [3H]glycine and [ZH]GABA; thereafter, they were washed in cold incubation medium and homogenised in 250 #1 of 70 % ethanol. The homogenate was centriguged for 10 min at 1000 × g and the supernatant run overnight on descending paper chromatograms with radioactive and amino acid markers in a butanolacetic acid-water (4:1:1, v/v/v) system 22. The chromatograms were stained with 0.25 % ninhydrin in acetone and cut up into 1 cm strips from origin to solvent front. The radioactivity in each strip was eluted into 2 ml of ethoxyethanol for 1 h and estimated by liquid scintillation counting. RESULTS
Tissue/medium ratios In preliminary studies on the uptake of the amino acids, GABA, glutamate and glycine in sensory ganglion in vitro it was found that the amount of [3H]glycine taken up after 20 min was fairly low, whilst there was a substantial uptake of both [3H]glutamate and [3H]GABA. This difference in the amount of radioactivity accumulated in ganglion incubated with the 3 different amino acids is expressed in Table I as tissue/medium ratios (radioactivity in 1 mg of tissue divided by the radioactivity in
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TABLE I THE UPTAKE AND METABOLISM OF CERTAIN AMINO ACIDS IN RAT SENSORY GANGLIA FOLLOWING
in vitro
INCUBATION
Amino acid
Time (min)
T/M ratio
Average length of autoradiographie exposure in weeks
% Unmetabolised amino acid after incubation 2 determinations
GABA DL-Glu Gly
20 10 20
4.7 2.1 1.6
1 2 5
84.2 85.2 80.3
1 #1 of incubation medium). Longer exposure periods were, therefore, necessary in order to obtain autoradiographs of ganglia incubated with [3H]glycine comparable to those with [3H]GABA. The average exposure period used to produce autoradiographs of similar intensity is presented in the third column of Table I. The amount of radioactive amino acid incorporated into protein during the incubation period must have been extremely small since all but 1 ~ of the radioactivity was extractable with PCA. The final column of Table I shows the percentage of radioactivity extracted from the ganglia which was located on chromatograms as a single peak, coincident with the RF of authentic GABA. When the radioactivity was measured in whole ganglia fixed with glutaraldehyde after incubation with [3H]GABA, approximately 70 ~ of the activity was retained in the tissue.
Light microscopic localisation of [3H]GABA The sensory ganglia of adult rats are 1-2 mm in diameter and surrounded by a thick connective tissue sheath beneath which lie the large sensory neuronal cell bodies (40-150 #m in diameter). Each neurone is completely surrounded by tightly apposed satellite glial cells. Autoradiographs of desheathed ganglia incubated with 5 × 10-6 M GABA (50/zCi/ml) were prepared and developed after a 1 week exposure period. An abundance of silver grains was seen throughout the ganglia when cut in transverse section (Fig. 1). The grains were concentrated exclusively over the satellite glial cell bodies surrounding the sensory neurones which were themselves almost devoid of labelling. In addition the fibre tract running through the ganglia to form the dorsal root was relatively free of silver grains. In preliminary experiments using ganglia incubated with intact connective sheaths, the greatest density of silver grains was observed over satellite cells in the outer regions of the ganglia. The presence of the intact sheath thus appeared to reduce amino acid penetration into the ganglia. The sheath itself showed negligible autoradiographic activity, as seen in Fig. 2a. Again the satellite glial cells and their processes were intensely labelled. The same autoradiographic distribution of [3H]GABA was observed in sensory ganglia from neonatal rats as well as in both adult and neonatal cat ganglia. The pres-
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Fig. 1. A dark field autoradiograph showing photomontage through a whole sensory ganglia incubated for 20 rain with 10-6 M [3H]GABA. Radioactivity is distributed throughout the ganglia, localised exclusively in the satellite glial cells stlrrounding the large unlabelled sensory neuronal cell bodies. The sensory fibre tract (indicated by a white arrowhead) shows only sparse labelling. The epoxy resin above the ganglia is almost totally devoid of background silver grains. Exposure time, 3 weeks; calibration bar, 100/,m.
ence of the drug amino-oxyacetic acid (AOAA) 29 at a concentration of 10/zM during the preincubation and the incubation period did not alter the silver grain distribution, but greatly increased the intensity of the autoradiograph. Recently the ganglion has been shown to contain detectable levels of GABA-glutamate transaminase activity (Gottesfeld et al., in preparation).
Electron microscopic localisation of [3HJGABA Electron microscopic autoradiography confirmed the glial localisation of GABA. The sensory neuronal cell bodies are completely enwrapped by narrow sheaths of glial cytoplasm which appear in transverse section as long thin projections. An intense accumulation of silver grains was observed throughout these satellite glial cell processes (Fig. 3). The axonal processes leaving the small neuronal cell bodies are unmyelinated as are the initial segments of the axons of the large cell bodies 14. These axons are also enclosed by tightly apposing glial cells which accumulate GABA (Fig. 4).
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Fig. 2. a: dark field autoradiography of rat sensory ganglia incubated with 10 6 M [aH]GABA. Heavy accumulations of silver grains over satellite glial ceils (indicated by arrowheads in b) were observed. The neuronal cell bodies (N) and the connective tissue (CT) were relatively free of silver grains. Exposure time, 1 week; calibration bar, 20 pm. In Figs. 2 and 4-6 the phase contrast micrograph contains the lettering referred to in the description of the upper dark field autoradiograph. b: phase contrast of the same area.
Fig. 3. a: electron microscopic autoradiograph of rat sensory ganglia. Relatively few silver grains were found over the sensory neuronal cytoplasm (NC) in the lower half of the picture and the extracellular space (ECS)in the upper portion. The silver grains clearly overlie the satellite glial cell process (indicated by arrowheads). Calibration bar, 2 #m. b: The axon (A) seen in transverse section is devoid of radioactivity. Large numbers of silver grains are observed over the nucleus (GN) and the cytoplasm of the glial cell completely surrounding the axon. Calibration bar, 2 #m.
[3H]GABA IN ~
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SENSORY GANGLIA
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Fig. 4. a and b: dark field autoradiograph and corresponding phase contrast field of rat sensory ganglia incubated with 1.75 × 10-a MDL-[3H]glutamic acid. Silver grains are predominantly located over satellite glial cells (indicated by arrowheads). The connective tissue sheath (CT) and neuronal cell bodies (N) were relatively free of labelling. Exposure time, 4 weeks; calibration bar, 20/~m.
Light microscopic localisation of glutamic acid T h e a u t o r a d i o g r a p h i c l o c a l i s a t i o n o f o g - [ a H ] g l u t a m i c acid was investigated after i n c u b a t i n g ganglia for a 10 min p e r i o d with the r a d i o a c t i v e a m i n o acid at a conc e n t r a t i o n o f 10 -6 M. A s h o r t i n c u b a t i o n time was used in a n a t t e m p t to minimize m e t a b o l i s m o f this a m i n o acid. The d i s t r i b u t i o n o f silver grains was identical to t h a t o b s e r v e d for G A B A . The satellite glial cells were heavily labelled whilst the n e u r o n a l cell b o d i e s a n d the connective tissue sheath were u n l a b e l l e d (Fig. 4).
[ a H ] G A B A IN SENSORY GANGLIA
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Fig. 5. a and b: dark field autoradiograph of rat sensory ganglia incubated with 10 a M [aH]glycine. Silver grains were distributed equally over the neuronal (N) and the satellite glial cell bodies (arrowheads). Relatively few silver grains were found over the sensory fibre tract (ST). Exposure time, 6 weeks; calibration bar, 20 t~m.
Light microscopic localisation of glycine, alanine and leucine W h e n ganglia were i n c u b a t e d with tritiated glycine (25 × 10 -6 M ) , alanine (1.5 × 10 -6 M ) a n d leucine (3.3 × 10 -6 M ) , u n d e r exactly the same c o n d i t i o n s as described f o r G A B A , a n d the a u t o r a d i o g r a p h s exposed f o r 5 weeks (cf T a b l e 1), the silver grains arising f r o m these 3 a m i n o acids were localised equally over the n e u r o n a l cell b o d i e s a n d satellite glial celis. Figs. 5 a n d 6 show this d i s t r i b u t i o n in the ganglia
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1~. SCHON AND J. S. KELLY
*?
r
Fig. 6. a and b: dark field autoradiograph and cgrresponding phase contrast field of rat sensory ganglia incubated with 1.5 >: 10 a M alanine. Silver grains are predominantly located over the neuronal and satellite gliaI cell bodies (arrowheads). The connective tissue sheath (CT) and the myelinated fibres (MF) are relatively free of labelling. Exposure time, 6 weeks; calibration bar, 20/~m.
i n c u b a t e d with [ZH]glycine a n d a l a n i n e respectively. The connective tissue sheath a n d the m y e l i n a t e d fibre t r a c t were a g a i n sparsely labelled.
GABA localisation in the dorsal root fibre tract T h e presence o f [ Z H ] G A B A u p t a k e in v a r i o u s r a t p e r i p h e r a l nerve fibre tracts in the r a t has been r e p o r t e d b y B o w e r y a n d B r o w n 5. T o investigate the localisation o f this u p t a k e , s h o r t lengths o f the d o r s a l r o o t fibre t r a c t were dissected, i n c u b a t e d
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with [3H]GABA in vitro, and prepared for light microscope level autoradiography. The silver grains were localised over Schwann cell bodies. The myelin sheaths and the interior of the axons were devoid of silver grains. An identical distribution of silver grains was found following in vitro incubation of ventral roots. Dorsal root section: effect on G A B A uptake
Transection of the efferent dorsal root caused no significant change in [3H]GABA uptake into the sensory ganglia when the uptake was investigated over a 10 min period at 25 °C. Short lengths of the afferent and efferent sensory trunks were dissected from rats lesioned 1 month earlier incubated with GABA and prepared for light microscopic autoradiography. Although uptake into Schwann cells in the afferent trunk was unaffected, uptake into the efferent trunk proximal to the transection was greatly reduced presumably as a result of the extensive degeneration of myelinated axons and supporting Schwann cells observed histologically. Cells which invaded the tract following such transections did not accumulate [ZH]GABA under the conditions of the experiment. DISCUSSION
In sensory ganglia, the sole cellular component which accumulates exogenous GABA are the satellite glial cells as revealed by both light and electron microscopic autoradiography. This provides a convenient system for comparing the properties of the predominantly neuronal uptake of GABA in brain slices and homogenates z 1,23 with glial uptake in the sensory ganglia in vitro. Such a study is reported in a companion paper 3a. The autoradiographic distribution and the biochemically measured uptake of GABA was unchanged in ganglia isolated from the spinal cord by rhizotomy l month earlier. This is in good agreement with the morphological observation that such deafferentation causes no marked cellular changes in either the sensory neurones or their satellite glial cells 1~. The increased number of silver grains observed over satellite glial cells following incubation in the presence of AOAA, confirms a similar observation on glial cells in the corpus callosum by H6kfelt and Ljungdah117 who suggested that glial cells may contain large amounts of the enzyme GABA-transaminase. Histochemical studies have revealed large amounts of the enzyme in extraneuronal structures such as the pia mater and ependymal cells36. Glycine, alanine and leucine all showed the same autoradiographic distribution being localised over both satellite glial cells and neuronal cell bodies. Since the amount of the 3 amino acids taken up was much less than the amount of GABA or glutamate accumulated, it is probable that they are accumulated by a low affinity neutral amino acid uptake system present in all brain tissues 1. The localisation of DE-[3H]glutamate in rat sensory ganglion like that of GABA, was exclusively in satellite glial cells. This extraneuronal localisation of glutamate is in clear agreement with other recent studies which showed that in the retina it is localised in Mfiller glial cells 12, in rat cerebellar slices in the Bergmann glial cells 19
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and at the insect neuromuscular junction, where it is implicated as the neurotransmitter substance, it was localised over Schwann cells az. A high affinity uptake system of glutamate has also been reported in frog sciatic nerves 3v. It is somewhat surprising that the sensory neuronal cell bodies themselves do not accumulate glutamic acid as it has been cited repeatedly as the putative mammalian sensory transmitter 24. The situation could, of course, resemble that observed in the cerebellum where the Purkinje cells which utilize GABA as their synaptic transmitter is will only accumulate exogenous GABA into their cell bodies under special circumstances. For instance Purkinje cells can be labelled by adding [aH]GABA to neurones growing either in tissue culture~"5,a4 orin explants of cerebellum attached to the iris of a recipient rat's eye (Ljungdahl et al., to be published). Presumably in both these situations the glial cells no longer form an effective barrier to the entry of GABA into the vicinity of the neurone. In the lobster neuromuscular junction no neuronal uptake is observeda°; all the GABA released is actively transported into Schwann cells surrounding the inhibitory synaptic endings. A similar situation was reported in the insect, where neuronally released glutamate is taken up exclusively into extraneuronal sites 3z. The role played by the glial as opposed to the neuronal uptake system in the mammalian central nervous system is not obvious. It has been suggested that the extraneuronal uptake may serve to prevent neurotransmitter substances in the blood reaching and activating receptors on neurones in the central nervous system a6. However, intraarterially administered GABA depolarises neurones of both sensory and superior cervical ganglia9,10 which would appear to preclude such an explanation. A more attractive hypothesis would be that specialised glial cells exist in close association with all neurones to ensure more rapid or efficient removal of neuronally released GABA. On the other hand, presynaptic inhibition in the dorsal horn of the spinal cord is blocked by both picrotoxin 11 and bicuculline 26 and mimicked by iontophoretically applied GABA s. GABA containing interneurones may therefore synapse with the preterminal regions of the primary sensory axons. The presence on the sensory neurones of GABA receptors and of glial cells capable of accumulating GABA may therefore be related to this role of GABA on the primary afferent terminals. The capacity to accumulate exogenous GABA may alternatively be present in all glial cells 16 but only play a physiological role in CNS regions where GABA has a neurotransmitter function. The presence of extraneuronal GABA uptake in ventral root fibres, in superior cervical ganglia and in preganglionic sympathetic nerve trunks supports such a view.
ACKNOWLEDGEMENTS
The authors wish to thank Dr. L. L. Iversen and the members of his laboratory for their help throughout this work and Mr. D. C. Chapman for skilled technical assistance. F. S. is an M R C Scholar at the Physiological Laboratory, Cambridge, Great Britain.
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24 JOHNSON,J. L., Glutamic acid as a synaptic transmitter in the nervous system. A review, Brabt Research, 37 (1972) 1-19. i 25 LASHER, R. S., AND ZAOON, I. S., The effect of potassium on neuronal differentiation in culturesl of dissociated newborn rat cerebellum, Brain Research, 41 (1972) 482-488. [ 26 LEVY, R. A., REPKIN, A. H., AND ANDERSON, E. G., The effect of bicuculline on primary afferent terminal excitability, Brain Research, 32 (1971) 261-265. 27 LOGAN, W. J., AND SNYDER, S. H., High affinity uptake systems for glycine, glutamic and aspartic acids in synaptosomes of rat central nervous tissues, Brain Research, 42 (1972) 413-431. 28 NEAL, M.J., AND IVERSEN, L.L., Autoradiographic localization of 3H-GABA in rat retina, Nature New Biol., 235 (1972) 217-218. 29 NEAL, M.J., AND STARR, M. S., Effects of inhibitors of y-aminobutyrate aminotransferase o n the accumulation of 3H-y-aminobutyric acid by the retina, Brit. J. Pharmacol., 47 (1973) 543-555. i 30 ORKAND, P. M., AND KRAWTZ, E. A., Localization of the sites of y-aminobutyric acid (GABA) I uptake in lobster nerve-muscle preparations, J. Cell Biol., 49 (1971) 75-89. 31 PANESSI,E., BIANCHI, R., CALLIGARIS,B., VENTURA, R., AND WEIBEL, E. R., Quantitative relationships between nerve and satellite cells in spinal ganglia. An electron microscopical study. I . Mammals, Brain Research, 46 (1972) 215-234. 32 SALPETER, M. M., AND FAEDER, I. R., The role of sheath cells in glutamate uptake by insect nerve-muscle preparations. In O. ER~NK6 (Ed.), Histoehemistry of Nervous Transmission, Progress in Brain Research, Vol. 34, Elsevier, Amsterdam, 1971, pp. 103-114. 33 SCHON, F., AND KELLY, J. S., The characterisation of [3H]GABA uptake into the satellite glial cells of rat sensory ganglia, Brain Research, 66 (1974) 289-300. 34 SOTELO, C., PR~VAT, A., AND DRrAN, M. J., Localisation of [3H]GABA in tissue culture of rat cerebellum using electron microscopy radioautography, Brain Research, 45 (1972) 302-308. 35 VON EOLER, U. S., AND PURKHOLD, A., Effect of sympathetic denervation on the noradrenaline and adrenaline content of the spleen, kidney and salivary glands in the sheep, Acta physiol, stand., 24 (1951) 212-217. 36 VAN GELDER, N. M., A comparison of ~-aminobutyric acid metabolism in rabbit and mouse nervous tissue, J. Neurochem., 12 (1965) 239-244. 37 WHEELER, D. D., AND BOYARSKY, L. L., Influx of glutamic acid in peripheral nerve characteristics of influx, J. Nettroehem., 15 (1968) 1019-1031.