Brain Research, 119 (1977) 189-198 © Elsevier/North-Holland Biomedical Press, Amsterdam - Printed in The Netherlands

P O T A S S I U M - S T I M U L A T E D y - A M I N O B U T Y R I C ACID N E U R O N S A N D GLIA

189

RELEASE F R O M

/~KE SELLSTROM and ANDERS HAMBERGER Institute of Neurobiology, University of GiJteborg, GiJteborg (Sweden)

(Accepted April 21st, 1976)

SUMMARY Potassium-stimulated [ZH]GABA release has been studied with fractions of glial cells, neuronal perikarya and synaptosomes using a superfusion technique. A monotonic increase in the [SH]GABA release was observed when external K + concentration was raised from 5 to 15 mM. However, there was no further increase in the [SH]GABA release when K + was raised above 15 mM. The KCl-stimulated release was saturable at the level of 60-100 ~ stimulation. Potassium-stimulated [SH]GABA release from glial cells was unchanged in the absence of calcium while release from synaptosomes and neuronal perikarya demonstrated Ca2+-dependence. The potassium-stimulated [aH]GABA release was enhanced when superfusion was performed in the presence of 10-5 M ouabain.

INTRODUCTION ?-Aminobutyric acid (GABA) is a widespread inhibitory neurotransmitter in mammalian cerebral cortex, cerebellum and spinal cord 8,15,zl,22,sz. The intra- and extracellular concentrations of GABA are regulated by synthesis via glutamate decarboxylase in GABAergic nerve terminalsl,S,9,~4,26, z~, high-affinity uptake in nerve terminals, synapse-associated glial cells and postsynaptic neuronal perikaryaS,XZ,~4,16,85 and subsequent catabolism via GABA transaminase in the aforementioned nervous tissue compartments31. The high-affinity uptake of GABA as a means of rapid termination of the inhibitory postsynaptic action has been characterized for nerve terminalsln,x0, ~3. In addition to presynaptic uptake, however, bulk-isolated glial cells and neuronal perikarya13,20, as well as glial cells in tissue culture~7, 9,s, have also been shown to possess high-affinity uptake. The ionic requirements for GABA uptake in synaptosomes, glial cells and neurons are very similar, i.e. absolutely dependent on external sodium, opti-

190 mally active at similar potassium concentrations, and inhibited by ouabain ~3,16,19, 23,29. There also appears to be cell-specificity, glia and synaptosomes showing optimal uptake at different calcium concentrations 29. Some questions have been raised about the existence of a bona fide net uptake of GABA under high-affinity conditions, since uptake under certain conditions is counteracted by an equal eiitux20, 30. Spontaneous efflux, as well as effiux of GABA stimulated by exogenous GABA, has to some extent been characterized for synaptosomes~O,82, glial cells in superior cervical or dorsal root ganglion 4,2~ and bulk-isolated glial cells and neuronal perikarya 3°. In addition to spontaneous efflux, however, neuronal and glial cell tissue models also show a stimulated release of GABA under conditions designed to depolarize the membrane, i.e., electrical pulses or high concentrations of potassium in the external medium4,5,7,1zAT, 18,25,34. We have studied both spontaneous efflux and potassium-stimulated release of labeled GABA from bulkisolated neurons, glia and synaptosomes by the use of a superfusion system which delivers well-defined potassium pulses to the isolated ceils. At issue is the physiological meaning of such a K+-stimulated release since glial cells, strategically located, could modulate neuronal excitability via release of GABA. MATERIALS AND METHODS

Materials Ficoll was obtained from Pharmacia Fine Chemicals, y-[2,3-3H]aminobutyric acid (GABA) from N E N Chemicals GmbH, and ethyleneglycol-bis(fl-aminoethylether)-N',N'-tetraacetic acid (EGTA), aminooxyacetic acid (AOAA), ouabain and GABA from Sigma Chemicals, St. Louis, Mo. Munktell No. F 1 filter paper (Grycksbo, Sweden) was employed. All other chemicals were reagent grade. Preparation of neuron- and glia-enriched fractions and synaptosomes Neuronal cell bodies and glial cells were prepared as described previously TM. Whole brains minus cerebellum from 6 to 8 white rabbits were sliced, using a mechanical chopper, and incubated in a balanced salt medium for 45 min at 37 °C. The suspended tissue was disrupted by passage through nylon mesh (1000 #m and 500/zm) attached to the ends of plastic syringes. The crude cell suspension was filtered through nylon meshes of decreasing pore sizes down to 50/~m. The final filtrate was centrifuged for 5 min at 150 × g, and the pellet-resultant mixed with Ficoll to a final concentration of 20 ~ . Glial and neuronal perikaryal fractions were obtained via centrifugation on a discontinuous sucrose-Ficoll gradient at 64,000 × g for 110 min. The cell fractions were collected, diluted and pelleted at 2000 × g. Purified synaptosomes were prepared by homogenization of rabbit cortex in 0.32 M sucrose, 10 m M Tris • HC1 (pH 7.4), centrifugation of the homogenate for 10 min at 900 × g to obtain the P1 supernatant, sedimentation of the crude mitochondrial fraction (PD by centrifugation at 10,000 × g for 20 min, and isolation of the synaptosomal band on a gradient made up of 7.5 ~o and 13 ~ Ficoll in 0.32 M sucrose. Density gradient centrifugation was carried out at 64,000 × g × 50 min 6. The interphase

191 between 7.5 ~ and 13 ~ Ficoll was collected, diluted with 0.32 M sucrose, and pelleted 10,000 x g × 30 min.

Incubation system for labeling of the intracellular GABA pool with fZHJGABA The different brain cell fractions and synaptosomes were incubated for 10 min in a medium containing 35 m M Tris • HCI (pH 7.4), 120 m M NaC1, 5 m M KCI, 2.5 m M MgCI~, 20 mMglucose, 1.5 mMCaC12, 5 × 10-SMAOAA and 10-7M [2,3-3H]GABA as previously described 3°. In superfusion experiments where AOAA was excluded, AOAA was also omitted in the prior incubation. Following the incubation, the fractions were sedimented onto a piece of filter paper by centrifugation for 5 min at 900 × g. The filter paper and its content was placed inside the superfusion chamber 30 to initiate release.

Superfusion Superfusion experiments were carried out as described previouslys°. Unless otherwise indicated in the Results, incubation and superfusion media were of the same composition. The cells were perfused with medium by delivering a supply of fluid to the volume in the open chamber, and 1.5 ml portions of medium/min resulting from the outflow were collected by means of a peristaltic pump. The cells were washed with perfusion medium for 20 min before introducing changes in the composition of the medium, in order to obtain a steady-state characterization of spontaneous efflux. Following spontaneous release, stimulating pulses of 0.5 or 1 min duration, consisting of high-potassium medium, were delivered via a peristaltic pump. The radioactivity of [3H]GABA released at appropriate intervals during the stimulation period was measured by counting the effluent in a Packard Liquid Scintillation counter using Triton-X 100 (1:2, v/v) in toluene-Permablend III (Packard 5.5 g/l) as scintillation solvent.

Calculations The release rate v(i), during the time period, i, is calculated according to the following: V(i) =

The amount of [3H]GABA in fraction i The total amount of [3H]GABA at time i

× 100.

The stimulated release rate Vs(i) is what we are measuring and the unstimulated release rate V(i) is obtained by extrapolation from the non-stimulated part of the efflux curve. Stimulatory effects of elevated KC1 in the perfusion medium, occurring from time a to time b, are expressed according to the following: b [Vs(i) - - V(i)] Stimulation =

i~a

× 100.

b W(i) i=a

192 Effects of Ca2+-withdrawal and ouabain on the K+-stimulated release are expressed as a percentage compared to the control. RESULTS

Labeling of the intracellular GABA-pool with [SH]GABA and spontaneous GABA effiux Cerebrocortical slices, as well as fractions enriched in nerve endings, neuronal perikarya and glial cells, actively accumulate G A B A from the incubation medium via high-affinity uptake systems. At a concentration of approximately 10-7M, high tissue-to-medium ratios of 100 are characteristically obtained for cortex slices 16, while for nerve endings, glial cells and neuronal perikarya the tissue-to-medium ratios are 150, 50 and 10 respectively 13. Glial-neuronal fractions and synaptosomes were incubated for 10 min under high-affinity conditions with [~H]GABA, and spontaneous efflux permitted to proceed by means of the continuous superfusion system. Radioactivity was released at a rate which has been characterized previously 3°. The G A B A release stimulated by exogenous G A B A has been shown to have Vma~ in the same range as G A B A high-affinity uptake20, z0,32. Potassium-stimulated release Potassium chloride at 15 m M delivered during a 1 min pulse stimulated [SH]G A B A release from isolated glia as well as neuronal perikarya and synaptosomes. Sodium chloride and choline chloride at the same concentration, in contrast, had no effect on the release of [SH]GABA. The increase due to K+-stimulated release (10 m M K ÷) compared to spontaneous efflux is shown in Table I. Giial and synaptosomal release showed about a 7 0 ~ increase compared to unstimulated control whereas release from neuronal perikarya showed a 40 ~ increase. CaZ+-requirements for K+-stimulated release These experiments were performed in superfusion medium containing 0.5 m M

TABLE I

Potassium-stimulated [ZH] GABA effluxfrom glial cells, neuronalperikarya and synaptosomes The potassium concentration was raised from 5 to 15 mM for 1 rain. The results are expressed as the difference between stimulated and unstimulated efflux, with efflux calculated in percentage of what is left in cells. Values are given as mean :k S.D.

Percentage release in stimulated efflux Percentage release in non-stimulated efflux Per cent of stimulation

Glial cells

Neurons

Synaptosomes

1.75 & 0.18 (6)

7.64 :k 0.63 (6)

1.76 -k 0.40 (6)

1.05 -k 0.09 67 ~

5.40 ~_ 0.36 41%

1.00 ± 0.15 76

193 TABLE II Effects of various media compositions on the potassium-stimulated [3HJGABA efflux from glial cells, neuronal perikarya and synaptosomes All experiments were performed on material which was exposed to the medium during perfusion for 15 min before the K + stimulation. Efflux rate was expressed by per cent of control. Values given represent mean ± S.D. from the number of experiments shown within brackets. Medium

Glial cells

Neurons

Synaptosomes

Control --Ca z+ + 0.5 MEGTA + 10-5 M Ouabain

100 106 4- 33 (7) 411 ± 38 (4)

100 15 4- 6 (7) 241 ± 21 (4)

100 44 ± 9 (5) 389 5= 23 (4)

E G T A with no additions of Ca 2+ ions. Such changes of the medium composition slightly affected the effluent rate. However, an altered spontaneous release rate would be corrected for by using the devised way of calculating the stimulations (see Methods). The potassium-stimulated [3H]GABA release from synaptosomes when Ca 2+ was withdrawn from the medium resulted in a release of [SH]GABA which was 44 ~o of the control value (Table II). Ca2+-withdrawal was even more effective in inhibiting release from neuronal perikarya (15 ~ of control value). In contrast, the K+-stimulated release from isolated glial cells was unaffected in spite of the fact that Ca z+ was withdrawn from the medium. Effects o f ouabain on K+-stimulated release The addition of 10-SM ouabain to the superfusion medium significantly increased spontaneous [aH]GABA release from isolated glia/neurons and synaptosomes al. In spite of that, ouabain also caused a prominent increase in the K+-stimulated release, with glia, neurons and synaptosomes being stimulated between 200 and 400% (Table II). Effects o f variation in K+-concentration on release o f [3H]GABA Maximal stimulation of [3H]GABA release from glia and neurons was obtained using a 1 min pulse of 15 m M potassium chloride; concentrations higher than 15 m M (45 and 75 m M ) caused no further release (see Fig. 1). The stimulated G A B A release from synaptosomes showed a moderate increase when the K + concentration was raised from 15 to 45 and 75 mM. The increment of stimulation for all K+-concentrations was in the range 90-100 ~ in the presence of AOAA. In the absence of AOAA, the stimulated efflux of G A B A showed a similar plateau level at all K+-concentrations, the stimulation being in the range of 30 ~ for neurons and synaptosomes and 50 ~ for glial cells. The effect of delivering shorter pulses of potassium chloride in lower concentrations was also investigated (Fig. 2). Pulses of 0.5 min duration, containing 7, 9 and 11 m M potassium, resulted in a continuous increase of [3H]GABA release compared to spontaneous efflux. In the experiments where K + was raised from 15 m M to

194

10

20

30 mM

40

50

60

70

K""

Fig. 1. The figure illustrates the potassium-induced stimulation of the GABA efflux rate (see Materials and Methods). Values represent means from 3 to 4 experiments with a S.D. of 5-15 %. Cells were preincubated in [3H]GABA and superfused either in the presence of AOAA (solid line) or in the absence of AOAA (broken line). Potassium pulses at the concentrations indicated were applied for one minute. Symbols: @, synaptosomes; I1[, glial cells; V, neurons.

75 m M c o n c e n t r a t i o n , sucrose was a d d e d to the superfusion m e d i u m to c o m p e n s a t e for tonicity changes. DISCUSSION Electrical s t i m u l a t i o n or high c o n c e n t r a t i o n s o f p o t a s s i u m in the external med i u m are widely used in vitro to simulate the reactions occurring in vivo in nervous tissue during n e u r o n a l activity. Since t r a n s m i t t e r release is a m a j o r event in the transmission o f the nervous impulse, efforts have been m a d e to establish a t r a n s m i t t e r role for G A B A by showing s t i m u l a t e d release in response to increased extracellular p o t a s sium. K + - s t i m u l a t e d release o f G A B A has been shown for cortical b r a i n slices 1~,17,a4, retina is a n d purified nerve terminals 5,7. However, in the use o f tissues w h i c h lack p r e s y n a p t i c terminals, such as s u p e r i o r cervical ganglion 4, a n d d o r s a l r o o t ganglion zS,

195 $0

40

3o

Z --

20

I-

U

t0

5

6

7

8

9

10

11

mM K +

Fig. 2. Cells incubated in [aH]GABA in the presence of AOAA were exposed to 0.5 min pulses of increasing concentrations of potassium, during superfusion. Values represent means of 3--4 experiments, S.D. approximately 1 0 % Symbols: Q, synaptosomes; , , glial cells; V , neurons.

it has also been possible to demonstrate K÷-stimulated release of GABA. Autoradiography indicated that it was the glial elements which took up labeled GABA by a highaffinity uptake process, hence being responsible for the release phenomenon. Highaffinity uptake of labeled GABA followed by spontaneous effiux has also been demonstrated for glial cells in tissue culture 27 and bulk-isolated glial cells 80. The possibility that glial cells may be involved in the modulation of neuronal excitability via control of the levels of neuroactive amino acids in the extracellular milieu of neurons has been discussedla,27, 3°. When the endogenous GABA stores of bulk-isolated glial cells, purified synaptosomes and neuronal perikarya are labeled with [3H]GABA under high-affinity uptake conditions, a spontaneous efflux of the label occurs when the material is perfused with normal medium, containing 5 m M KCI. The relative rate of this spontaneous efflux correlates well with the Vmax for high-affinity uptake into the preparations 13,3° and is consistent with a bidirectional carrier for GABAla,20,3L When short pulses of 15 m M KC1 are delivered to the cells, a stimulated release of [3H]GABA takes place which cannot be duplicated by substitution of sodium chloride or choline chloride. The relative K+-stimulation compared to spontaneous efflux is approximately similar for glial cells and synaptosomes and a little lower for neurons (Table I). Examination of the Ca2÷-dependence of the K÷-stimulated release, in our hands, reveals a considerable inhibition of the [3H]GABA release in the case of purified synaptosomes, an even more marked inhibition for neuronal perikarya, but no dependence on calcium in the case of bulk-isolated glial cells (Table II). Glutamate release ~from spinal roots, which may be a function of either the axonal or Schwann cell compartment, has also been shown to be independent of the Ca2+-concentrations 36. Minchin and Iversen 25, in contrast, found Ca2+-dependence for K+-stimulated

196 release of GABA from glial cells in dorsal root ganglia. These discrepancies are as yet unclear. Ca2+-dependence has been used as a criterion for establishing the validity of transmitter release from nerve terminals. Presumably, the lack of Ca"+-dependence for GABA release from glia argues in favor of a non-vesicle-mediated phenomenon, but the marked Ca2+-dependence of neuronal perikaryal release (Table ll) is puzzling if CaZ+-dependence implies release from a vesicle-board pool in the synaptosomal compartment. Of interest is that a strict parallelism has been observed with respect to the stimulating influence of Ca z+ withdrawal on high-affinity GABA uptake and potassium accumulation in the glial compartment; also bulk-isolated glial cells behave quite differently from synaptosomes with respect to the effect of Ca 2~ on GABAuptake10,29. Ouabain at 10-SM has been found to markedly stimulate spontaneous GABA efflux from synaptosomes, neuronal perikarya and glia in a previous study 30. However, although the fractions had been perfused in an ouabain-containing medium for 15 rain prior to the potassium stimulation, the K + pulse stimulated the GABA efflux 4-fold for glia and synaptosomes and approximately 3-fold for neurons. Since ouabain at 10-SM abolishes the existing K+/Na + gradient in the fractions, the relative stimulation caused by a 10 m M rise in K* will be far more pronounced than a similar shift in K + with the normal K+/Na +gradient present. Similar results for synaptosomes had been reported earlierL A dose response curve of GABA release as a function of increasing concentration of extracellular potassium has been determined for glial cells, neuronal perikarya and synaptosomes. In the lower range of" stimulation, i.e. with potassium concentrations lower than 15 mM, there is a linear increase in the GABA efflux as a function of the K+-concentration; however, above 15 mM, there seems to be a saturation of the increase in the GABA effiux. What might be observed is a saturation of the 'mechanism' responsible for the release. Maximally stimulated GABA release for glial cells of dorsal root ganglion 25 has been noted at very high K+-concentrations, approximately 100 raM. The discrepancy between data obtained with cell fractions and those obtained with the ganglia might be due either to a slower penetration rate of potassium into ganglia or to reduced membrane potentials in the isolated cell fractions 5,1°. The model of a bidirectional sodium-dependent GABA carrier to explain uptake and release of GABA ~9,2°,32 is consistent with what is known for GABA uptake into neurons, glial cells and synaptosomes 23,29. Optimal GABA uptake requires the intra- and extracellular concentrations of sodium and potassium which occur during a normal membrane potential. Increase of the extracellular potassium concentrations may stimulate the GABA carrier to work from inside out. The potassium-induced release of GABA is a rapid process working downwards along a concentration gradient. A dynamic transport system for GABA, the magnitude and direction of which depends upon the membrane potential, fulfills requirements both for a rapid mechanism of release and for re-uptake of a transmitter. In response to the action potential, enough GABA can be released to induce inhibition at the postsynaptic site, since GABAergic terminals contain 50-150 m M GABA 9. Glial cells probably have considerably lower GABA concentrations. However, GABA released from glial

197 cells m a y be superimposed o n presynaptically released G A B A a n d c o n t r i b u t e in this way by m o d u l a t i n g the n e u r o n a l excitability. ACKNOWLEDGEMENTS We are grateful to Mr. G u n n a r S u n d b e r g for his skillful technical assistance. Dr. Charles T. Weiler is sincerely acknowledged for discussing a n d c o m m e n t i n g on the manuscript. This work was supported by a g r a n t from the Swedish Medical Research C o u n c i l ( G r a n t B75-12X-164-11B).

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