Depolarization of cultured astrocytes by glutamate and aspartate

0306-4522/79/l

Neuro.wirncr Vol. 4, pp. 1593 to 1598 Pergamon Press Ltd 1979. Printed in Great Britain 0 IBRO

DEPOLARIZATION OF CULTURED ASTROCYTES GLUTAMATE AND ASPARTATE

101.1593SO2.00,Q

BY

L. H&I, P. F. ANLXI?Sand ELMBETH H&LI Department of Physiology, University of Base.1,Vesalgasse 1, CH-4051 Basel, Switzerland

Abstract-The excitatory transmitter substances glutamate and aspartate are known to have a depolarizing action on cultured CNS neurones, the &polarization being associated with an increase in membrane conductance. When the effects of these amino acids (at a concentration of lo-’ M) were studied on the membrane potential and resistance of cultured glial cells, they also caused a depolarization of many astrocytes but without producing significant changes in membrane resistance. The majority of glial cells depolarized by glutamate and aspartate were lying in the vicinity of neurones in the dense zone of the cultures, whereas isolated astrocytes in the outgrowth zone were usually not affected by the amino acids. CAminopyridine (5 m@, a substance known to block K+conductance in various excitable membranes, reversibly reduced or abolished the depolarization caused by glutamate and aspartate on glial cells, but had no or only a small effect on the depolarization of neurones caused by these amino acids. These results suggest that the depolarization of glial cells by glutamate and aspartate is caused by an increase in the concentration of extracellular K+ which is released from neighbouring neurones during their activation by the amino acids.

PROBABLY due to the difficulty of identifying glial cells on the basis of electrophysiological criteria, there are

only a few studies of the action of neurotransmitters on glial cells in vim (Kmumc! & Scxiwmx-5, 1967). The method of tissue culture, where cells can be identified by morphological criteria, is an excellent tool to study the effects of neurotransmitters on the membrane potential and resistance of astrocytes under direct visual control (H~SLI & H&I, 1978). Furthermore, tissue culture techniques have also proved to be a suitable model for investigating neuronc+glia interactions by means of electrophysiological methods (H~sLI, AND& & H&LX, 1978). In a recent publication we have reported that y-aminobutyrate (GABA) caused a depolarization of cultured glial cells which was not associated with a change in membrane resistance (HBSLIet al., 1977; 1978). Furthermore, Caminopyridine which blocks K+conductance in excitable tissue, reversibly abolished the depolarization of cultured glial cells by GABA, but did not affect, or only slightly affected, the action of this amino acid on neurones (H&I et al., 1979). From these results it was suggested that the depolarization of glial cells by GABA is an indirect effect due to an increase of the extracellular concentration of Kf which is released from neurones during their activation by the amino acid. We have now looked to see whether the excitatory amino acid transmitters glutamate and aspartate also induce a &polarization of cultured astrocytes and whether this depolarization might be caused by the elTlux of K+ from adjacent neurones.

Abbreviation:GABA, y-aminobutyrate.

EXPERIMENTAL

PROCEDURES

The method of preparing the cultures and the electrophysiological techniques used in this study have been described in detail in previous papers (H&I, H&I, AN&S & WOLFF,1975; H&I et nf., 1976a; 1978). Explants prepared from the lumbosacral part of the spinal cord, the medulla oblongata and the pons of fetal (18 days in oitro) and newborn rats were cultured in Maximov doublecoverslip assemblies for 10-35 days (Hi)sLl et al, 1975; 1976a). For the electrophysiological studies, the cultures were placed into a perfusion chamber mounted on an inverse microscope and constantly perfused with Gey’s solution (pH 7.3-7.4) at a temperature of 36°C f 1°C (Hall et al. 19760; 1978). Intracellular recordings were made with glass microelectrodes (tip diameter < 1 pm, resistance 40-100 Mn) filled with 4 M K acetate. Membrane potentials were displayed on an oscilloscope and on a rectilinear ink recorder. The membrane resistance was measured either by passing depolarizing or hyperpolarizing current pulses of a constant strength (0.5 or 1 nA; 5OOms duration) or by varying the intensity of the pulses over a range of +0.25-2 nA (30 ms duration, see Figs 2 and 3). Glutamate and aspartate were added to the bathing fluid in a concentration of lo-’ M. 4-Aminopyridine was tested in a concentration of 5 mM. RESULTS Electrophysiological properties of cultured astrocytes The morphological properties of cultured glial cells have been described in detail in previous publications (H&I et al., 1975; 19766; H&.x & H&I, 1978). Intrace&&u microele43ode recordings were made from giial cells lying in the dense zone of the culture, where many neurones are present as well as from isolated astrocytes which have migrated into the outgrowth zone where almost no neurones are found. The mem-

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brane potentials of cultured astrocytes ranging from - 40 to - 82 mV (mean f SD.: - 62.79 mV + 9.04 mV. n = 110) were higher than those recorded from neurones ( - 54.76 mV + 11.97 mV, n = 89) and from satellite glial cells in cultured dorsal root ganglia (-56.6mV & 10.2mV, n = 68, HSSLI et al., 1978). The membrane resistance of the astrocytes (14.06 MSZ Ifr 9.86 MQ, n = 24) was similar to that of satellite glial cells (15.6 MR t_ 9 MR, n = 28, Hbs~r et al., 1978) but higher than the resistance measured of neurones (7.61 MQ 4 5.18 MR, n = 14). The finding that cultured astrocytes have higher resting potentials and membrane resistances than neurones is consistent with studies on glial cells in uiuo in the cortex by KRNJEVI(:& SCHWARTZ(1967) and in the spinal cord by SOMJEN (1970), which also observed higher values for glial cells than for neurones. In contrast to neurones, cultured glial cells never showed spontaneous or evoked action potentials nor injury discharges (HBSLI er al., 1976b; 1978). Action of glutumute und aspartate on the membrane potential and resistance of astrocytes

Application of glutamate and aspartate (lO--4 M) to the bathing fluid caused a depolarization of 2-17 mV of a large number of astrocytes (mean + S.D. for glutamate 6.65 mV + 3.24 mV; n = 39; for aspartate 6.28 mV f 2.87 mV, n = 25). The action of the amino acids was clearly dependent on the location of the giial cells in the culture. All astrocytes lying in the vicinity of neurones in the dense zone of the culture were depolarized by glutamate (39 cells) and aspartate (25 cells) (Fig. 1A). In contrast, neither amino acids had any effect on the majority of isolated glial cells (16 out of 20 cells for glutamate, 7 out of 8 cells for aspartate) located in the outgrowth zone where no, or

and

ELBABETH

H~SLI

only few, neurones are found (Fig. 13). in a previous study from our laboratory, in which most of the recordings have been made from isolated astrocytes lying in the outgrowth zone, glutamate had no etlect on 33 out of 35 glial cells, whereas aspartate depolarized 5 out of 14 cells (HBSLIet al.. 1976h). A comparison of the action of the amino acids using equal concentrations ( 10U4 M) 011 the same glial cell revealed that the amplitude of the depolarization induced by glutamate was greater than that induced by aspartate on 10 astrocytes; on 5 cells aspartate was more effective and on 6 astrocytes both amino acids had an equal potency. There were no signs of desensitization on neurones and glial cells when the amino acids were tested for a long period of time OI after repeated applications at short intervals. When the actions of glutamate and aspartate were studied on the membrane conductance of cultured neurones, there was a reversible decrease m membrane resistance (by 20 to 40%) during the amino acid depolarization. In contrast, the depolarization of glial cells by glutamate (9 cells) and aspartate (10 cells) was not accompanied by a change in membrane conductance. This is illustrated in Fig. 2 which show> the voltage-current relationship before. during and after the application of glutamate and aspartate on two different astrocytes. The membrane resistance remained unchanged, although glutamate and aspartate caused a depolarization of 5 and 4 mV respectively. Depolarizations by glutamate and aspartate without concomitant changes in cell input resistance were also reported by CONSTANTIRr GALVAN(1978) for inexcitable cells in guinea-pig olfactory cortex slices. As was observed on cultured satellite glial cells (H&LI et al., 1978), there was sometimes a progressive

A Asp 10-4hl

Glut lO%l

rrN

B

Asp 10-4M

Glut 10*~tvl

FIG- 1. Comparison of the effectsof aspartate (Asp) and glutamate (Glut) on the membrane potential (A) of a glial cell lying in the dense zone of the culture and (B) of an isolated astroeyte iocated in the outgrowth zone (spinal cord cultures, 35 and 2d days in oitro respectively). Duration of p&fusion with the amino acids (concentrations W’M) is indicated by horizontal bars above tracings. Ordinate: membrane potential in mV.

Action of glutamate and aspartatc on cultured astrocytes

A

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FIG. 2. Action of glutamate (Glut) and aspartate (Asp) on the membrane resistance of cultured astrocytes. 0: before, Cl: during and A: after the application of the amino acids (lo-•M) (A: brain stem culture, 16 days in titro; B: spinal cord culture, 23 days in vitro). The voltagwcurrent relation was studied by injecting currents of various strength (&-0.25-l nA, 3Oms pulse duration) into the cells through the recording electrode. Glutamate and aspartate did not produce changes in membrane resistance although the amino acids caused a depolarization of 5 mV and 4 mV respectively. The membrane potentials were - 70 mV for (A) and -66 mV for (B).

increase of the membrane resistance throughout the whole recording period which did not depend on the application of the amino acids. Action of an increase of the extracellular K+ concentration on the membrane potential and resistance of astrocytes

Raising the K+ concentration in the bathing solution from 5 to 10 and 15m~ caused a depolarization ofallg+lccllstest&Tkmean.valuesoftheamplitudes of th8 depolarizations after perfusing with lOm~K+was8S9mVf3.48mV,s~.(t1=19)and with 15 mu K+ 15.8mV + 2.49mV (n = 10). The

depolarizations were usually not accompanied by a significti change in membrane resistance. In 10 mu K+ solution, the membrane resistance remained unchanged in 16 astrocytes (Fig. 3), whereas in 2 cells a small decxase (12 and 17%) was observed. in 15m~ K+ solution 7 out of 10 cells showed no change in membrane resistamx but in 3 glial cells there was a decrease of U-200/,. K+-induced

Action of kminopyridine

on the depolarization by glutamate and aspartate of newones and glial cells

It has been, observed that the quaternary ammonium compound 4-aminopyridine selectively

I596

L. HGSLI,P. F. ANURBS and ELISABETH HOSLI A

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Asp 10-4M

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FIG. 3. Action of an increase of the extracellular K’ concentration from 5 mM (normal) to 10 mM on the membrane resistance of a cultured astrocyte. 0: before, Cl: during and A: after perfusion with K+ (spinal cord culture, 23 days in vitro). The voltage-current relation was studied by injecting currents of various strength (f0.25-1 nA, 30 ms pulse duration) into the cell through the recording electrode. Although K+ 10mM caused a depolarization of 7 mV, there was no change in membrane resistance. The membrane potential of the cell was -70 mV. blocks branes

K+-conductance on various excitable mem(MWES & PICHON, 1977). Previous studies

from our laboratory have demonstrated that 4-aminopyridine (5 mM) reversibly abolished the depolarizing action of GABA on satellite glial cells of cultured dorsal root ganglia, but did not affect the GABAinduced depolarization of dorsal root ganghon neurones (Hbs~r et al., 1979).

FIG. 5. Effect of 4-aminopyridine (5 rnM) on the depolarization by aspartate (Asp, 10W4M) of an astrocyte (spinal cord culture, 34 days in vitro). The membrane potential of this cell was -60 mV. (A) Depolarization by aspartate in

normal bathing fluid. (B) After perfusion with 4-aminopyridine for 3.5 min the depolarization by aspartate was markedly reduced. (C) Almost full recovery of the depolarizing action of aspartate was observed 3 min after returning to normal bathing solution. The duration of perfusion with aspartate is indicated by horizontal bars.

Caminogyridine (5 mM) added to the bathing fluid had no or only a slight action on the amplitude of the depolarization by glutamate (3 cells) and aspartate (3 cells) on neurones, whereas the depolarization by both amino acids on glial cells (glutamate 10 cells, aspartate 4 cells) was blocked within I-4 min after perfusion with 4-aminopyridine (Figs 4 and 5). On 4 astrocytes the depolarization induced by aspartate ta3JFmE

GLIA

A

Glut 10-4M

Glut 10%

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C

f--c‘----

-J----ww shec

FIG. 4. Action of 4-aminopyridine (5 ma) on the depolarization by glutamate (Glut, W4 M) of a glial cell (left) and of a neurone (right) (spinal cord cultures, 34 and 24 days in vitro respectively). The resting potential of the astrocyte was -62 mV and that of the neurone -50 mV. (A) Dep&rization by glutamate in normal bathing fluid. (B) 4-At+oppidine (5 mru) added to the bath&g ‘?iuN ab&%Bed the glutamate depolariz&on ofthe glti ail &r’1.5 min witbout affecting the a&on of the adno acid on the neurone after 3.5 mtn. (C) Becovery of the glutamate depoi.@%ion of the @ii o&l was observed 2 min after perfusing with normal bathing .soIution. The duration of perfusion with &tamateis indicated by horizontal bars.

Action of glutamate and aspartatc on cultured astrocytes

1597

&odes have demonstrated an increase in [K’] in the vicinity of neurones during eleetrieal activity (Lux, 1976; GALVAN,TEN &WXENCATE & !%NE~WITSCH, 1979). An efeux of K+ has also been observed during the depolarization of dorsal root ganglion neurones by GABA (m & FEE=, 1976) and during the activation of motoneurones of the isolated frog spinal cord by glutamate, GABA and glycine (Kun~ & FIJKUDA, 1976; SONNHOF,GRAFE, RICH’IER,PAREKH, KRUMNXL& LINDER,1978). Finally, depolarizations of glial cells after neuronal stimulation (KUPFLER& NICHOLLS,1966; &xCEN, 1970; VYKLrCKk,SYKOV& KRfZ & UJEC, 1972) and during spreading depression (GROSSMAN & HAMPTON,1968; HIGASH~DA,MIYAEE, DISCUSSION TAEAO & WATANABE,1971; KEAIG & NICHOLSON, 1978) were also attributed to an eflIux of K+ from In the present study it has been demonstrated that neurones. the excitatory amino acid transmitters glutamate and Our experiments have shown that the mean ampliaspartate cause a depolarization of cultured glial cells. tudes of the amino acid-induced depolarizations of In slices of olfactory bulb, glutamate and aspartate were also found to produce depolarizations of inexci- glial cells are similar to or lower than those observed after perfusion with 10mru K+ solution suggesting tabk cells (OJNSLWTI & GUVAN, 1978). However, that the increase of the local extracellular K+ concenglutamate had no effect on cortical glial cells in situ tration produced by the amino acids is in the order of (KRNJEVzd & W~WARTZ, 1%7). We have no explanation for the difference of the action of glutamate on 3-5m~. The observation that the changes in glial glial cells in situ (KRNJEvIc’ & SCHWARTZ, 1967) and membrane potentials after raising the extracellular K+ concentration are smaller than predicted by the in vitro (CONSTANTI & GALVAN,1978; and the present study), although the discrepancy might be due to the Nemst equation may indicate that the membrane permeability to other ions cannot be neglected. Similar diffbrent methods of drug application used. observations were made by SCHLUE8z WALZ (1979) Possible mechanism of the depolarization on glial cells in the CNS of the leech. As was observed with GABA (H&I et al., 1978), In contrast to neurones, where these amino acids the action of glutamate and aspartate on cultured usually cause a deerease in membrane resistari~ (KRNJEVIC! & SCHWARTZ, 1967; BERNARDI, ZIBGL- astrocytes was dependent on the proportion of neurGtiseeRGEE, HEEZ & Purr, 1972; H&I & H&LI, ones located in the neighbourhood of the glial cells tested. Thus, astrocytes in the dense zone of the cul1978), there was no signitkant change in membrane ture, where a great number of neurones are present, conductance during the amino acid-induced depolarizations of glial cells in tissue culture (present study), were depolarized by the amino acids, whereas isolated in the cortex in situ (KRNJLWIC!& SalwA~lz, 1967) or glial cells in the outgrowth zone of the culture, where in slices of the olfactory bulb (CONSTANTI & GALVAN, no or only few neurones are found were usually unaffected. In earlier investigations from our laboratory, 1978). These observations suggest that the amino acid-induced depolarizations of glial cells are not pro- most of the recordings were made from isolated glial duced by the activation of receptors which alter the cells in the outgrowth zone and consequently almost ionic permeability of the glial membrane but might be no effects of amino acid transmitters could be observed (H&LI et al., 1976b). Similar results have caused indirectly by the e&x of K+ from adjacent neurones. This suggestion is supported by the obser- been reported by WARDELL(1966) who also found no vation that 4-aminopyridine-a substance known to effects of glutamate applied microelectrophoretically affect K+ conductance in excitable tissue (MEvE.s & to cultured astrocytes lying in the marginal zone of PKXON, 1977~reversibly blocked the action of the culture. Thus, the tinding that astrocytes can only GABA on satellite glial cells (H&I et al., 1979) and be depolarized when they are close to neurones is also of glutamate and aspartate on cultured astrocytes but consistent with our conclusion that depolarization of had no or only a slight action on the amino acid- astrocytes is caused by the release of Kt from activated neurones. induced depolarization of neurones. Furthermore, depolarizations of cultured glial cells caused by an increase of the extracellular K+ concentration up to PossibleJirnctional implications 1Omnr were not accompanied by significant changes Our studies on cultured glial cells together with in membrane resistance either, and were also reversmany other observations suggest that there is a funcibly blocked by 4-aminopyridine. tional coupling between neurones and glial cells, the An eRlux of K+ from neurones during electrical or neurones being able to inlluence the glial membrane amino acid-induced activation has been shown by potential by releasing K+ during their activation (for several authors. Studies using K+-sensitive microelecreferences see HEE~Z, 1965; KUFPLW & NICHOLLS,

was not completely abolished but was markedly reduced. Recovery of the amino acid-induced depolarization was usually observed 2-5 min after returning to normal bathing solution. A comparison of the etkcts of 4-aminopyridine on the glutama~induccd dtpol~ixation of a neurone and of a glial cell is illustrated in Fig. 4. 4-aminopyridine reversibly abolished the action of glutamate on the glial cell (Fig. 4A) without a&cting that of the neurone (Fig. 4B). The depolarization of glial cells (n = 3) caused by an increase in the K+ concentration of the bathing fluid (from 5 to 10 nip) was also reversibly blocked by 4-aminopyridine.

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L. H&L], P. F.

AND&S

1966; VARON, 1975, H&LI et ul., 1978). On the other hand, there is also evidence that glial cells can intluence neuronal activity. It has been shown that glial cells are able to accumulate and to release transmitter substances (for references see SCHRER & THOMPSON, 1974; SCHOLJSBOE, SVENNEBY& HERTZ, 1977; HBSLI & HBSLI, 1978) thereby controlling the levels of neuro-

and ELIZABETH HGSLI transmitters ones.

in the extracellular

environment

of neur-

Acknowledgements-This work was supported III part by the Sandoz-Stiftung zur FGrderung der medizinisch-biologischen Wissenschaften, Basel.

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