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Effects of 4-aminopyridine and tetraethylammonium on the depolarization by GABA of cultured satellite glial cells
Neuroscience ~etters, 11 (1979) 193--196 © Elsevier/North-Holland Scientific Publishers Ltd.
193
EFFECTS OF 4-AMINOPYRIDINE AND TETRAETHYLAMMONIUM ON THE DEPOLARIZATION BY GABA OF CULTURED SATELLITE GLIAL CELLS L. HOSLI, P.F. ANDRES and ELISABETH HC]SI,I Depa,tment of Physiology, University of Basp.l Ve.~algasse I, CH-4051 Basel (Switzerland) (Received October 19th, 1978) (Revised version received November 3rd, 1978) (Accepted November 6th, 1978)
SUMMARY
4-Aminopyridine (4-AP) which selectivelyblock: K+-conductance of exc~ ~ble membranes, reversibly abolished the depolarization by 7-aminobutyric acid (GABA) of cultured satelliteglial(SG) cells,but did not or only slightlyaffect the action of G A B A on dorsal root ganglion (DRG) neurons. It is therefore suggested that the depolarization of glialcellsby G A B A is an indirect effect due to the accumulation of K* which is released from adj-cent neurons during their depolarization by the amino acid. Tetraethylammonium (TEA) had no effect on the G A B A depolarization at a concentration of 15 m M but produced a slightreduction at higher covcentrations (60--70 mM).
The method of tissue culture provides a good model system to study neurone-glia interactions allowing intracellv~ar recordings from individual cells under visual control. Since in dorsal root ganglia (DRG) the small satellite glial (SG) cells are lying very close to the large neurones and completely surround their cell bodies, we have used this preparation in order to investigate the influence of neurones depolarized by 7-aminobutyric acid (GABA) on glial cells. There is strong evidence that the depolarization of D RG neurones by GABA is associated with an increase in CI-- and K÷~onductance causing an efflux of both ions from the cells [2,3,6,9 ]. Using K÷-sensitive electrodes, Deschenes and Feltz [3] have demonstrated that the GABA depolarization of DRG neurones is accompanied by an increase of the extracellular K÷-concentration. Recently we have shown that GABA caused a depolarization of SG celts of cultured DRG without producing changes in membrane conductance [ 7 ] suggesting that this depolarization isan indirect effect due to the release of K÷ from adjacent DRG neurones during their depolarization by the amino acid [5,7]. To further support this hypothesis~ we have studied the effects of
194
4-aminopyridine (4-AP) anc~ tetraethylammonium (TEA), two substances which selectively block K÷-conductance of various excitable membranes (see refs. 1,4,8) on the GABA depolarization of SG cells. The techniques for Culturing d0rsalroot ganglia~and for the electr0physiological " e x t e n t s have been described in detail ::Ln:pre~,ious :papers [5,7 ]. Intracellular recordings were made from cultured SG cells and neurones of rat DRG (8-24 days in vitro) Using glass microelectrodes filled with 1--4 M K-acetate. The resistance of the elechodes ranged from 4 0 - 1 5 0 Mrl. Membrane potentials were recorded on an oscilloscope and on a rectilinear pen writer. The perfusing solution (Gey's) consisted of: (raM) NaCI 137, KCI 5, CaCI= 2.4, }~gCl2, 2.2, Na2HPO4 1.0, KH=PO4 0.18, NaHCO~ 2.9, glucose 11.1. The pH of the bathing fluid ranged from 7.3--7.4 and the temperature was maintained at 36 ° ~ 1°C. GABA, 10-4 M (FLUKA), tetmethylammoniumchlorid, 15, 60 and 70 mM (FLUKA) and 4-AP, 5 mM (FLUKA) were added to the bathing fluid.
GLIA
A
NEURONE
GABA 10"4M
GABA 10"4M -
B
. -
4AP -
C
-
I IIIIII I
5mM
I
III
I III
m
,l mv
2u sec
Fig. I. Effect of 4-AP on the depoisrization by GABA (I0"4 M) of a SG cell (left) and a l~eurone (right) of cultured DRG (12 and 16 days in vitro respectively). Initial resting potentials were'--'] I m V (SG cell)and " 6 5 ~ V (neurone). A: depolarization (upward deflection) caused by-GABA. B: 4;BP(5 raM) add,~,to t~E; bathinlfluid, for 2 and 3 rain re6pactively, abolkhed the :GABA depolarization of the 8G cell without affecting that of ~ e neurone. C: GABA depolarization 5 rain after retum:ing to normal bathing solution. Duration of perfusio n with GABA is indicated by bars.
195
The identification of the various cell ';ypes (DXG neurones and the surrounding SG cells) in culture~ of rat D]:C~ was made on the basis of their morphological appearance a s we~ as on their electrophysiological properties [ 5 , 7 ] . A s w a s described previously, GABt~ ]L0-4 M caused a depolarization of all DRG neurones (n = 57) and SG cells (n = 68) tested [5,7]. 4-AP at a concentration of 5 mM reversibly abolished the GABA depolarization o'. SG cells, but did not or only slightly affect the depolarization of DRG neurones. As illustrated in Fig. 1A (left), GABA 1 0 -4 M c~tused a depolarization of SG cell by approx. 10 inV. After perfusing with 4.AP (5 mM) for 2 min, the GABA effect was completely abolished (Fig. 1B, left). Almost complete recovery was observed 5 min after returning to normal bathing solution (Fig. 1C, lett). Similar results of the action of 4-AP on the GABA depolarization werc~ obtained on all SG cells (n = 8) studied. In contrast, the depolarization by GABA o f a DRG nem'one was not affected by 4-AP (5 raM) after peffusilg for 3 min (Fig. 1, right). Similar observations were made on half of the DRG neurones tested whereas on the other half, there was a slight reduction of the GABA depolarization by 4-AP which was often reversible. TEA which also bk~cks K+.eonductance [1,4] usually had no effect on t!~e amplitude of the GABA depolarization of both SG cells (n = 6) and DRG neurones (n = 5) at a concentration o~ I 5 mM, although the time course and shape of the falling phase was often altered. At higher concentrations (60 A
GABA 10"4M
B
C 10'
III
TEA
15 mM
TEA 60 mM
10'
45'
~/~~'~
! 5mY 20 sec
Fig. 2. Effect of TEA on the depolarization by GABA (10 -4 M) of a cultured SG cell (12 days in vitro). A: depolarization (upward deflectiL,~) caused by GABA. B: depolarization by GABA of the sanie cell 10 and 45 rain after adaition of TEA (15 mM) to the perfusion fluid. C: action of GABA on this cell 10 and 20 rain after increasing the TEA concentration to 60 raM, Recovery could not be observed because the cell was lost. The resting potential of this cell was - 6 3 mV. Duration of perfusion wi~h GABA is indicated by bars above tracings.
196
and 70 mM), TEA caused a reduction of the amplitude of the GABA depolarization on all SG cells (n = 4) tested. Fig, 2 illustrates the action of TEA on the depolarization by GABA of a SG cell. After perfusing with TEA (15 raM)for 45 rain, the amplitude of the GABA depolarization was not affected, b u t increasing the concentration of TEA to 60 mM reduced the depolarizing effect of GABA after 20 rain. The difference in effectiveness and time course of action betwen 4-AP and TEA might be due to the fact that 4-AP acts from both the inside and outside of the cell membrane [ 8], whereas TEA is only effective from the inside. This is consisten~ with studies on the squid giant axon showing that TEA acts only from the inside of the cell membrane [ 1,4]. There is considerable evidence timt the GABA-induced depolarization of DRG neurones is ma:-~ly due to an increased Cl--conductance [2,3,9]. Our finding that 4-AP which sel~ctively blocks K÷-conductance [8] did not or only slightly affect the GABA depolarization of DRG neurones provides further evidence that mainly CI- and not K ÷ are involved in producing this depolarization. Furthermore, it has been shown by Deschenes and Feltz [3] that the depolarization of DRG neurones by GABA also causes an efflux of K ÷ thereby increasing the extracellular K÷-concentration in the vicinity of the activated neurones. Our results indicate that 4-AP abolished the GABAdepolarization on SG cells by blocking the efflux of K ÷ from depolarized neur,mes. It is suggested that the action of the amino acid on glial cells is an indirect effect due to the accumulation of K ÷ which is released from adjacent DRG neurones during their depul~rization by GABA. REFERENCES 1 Armstrong, C.M. and Binstock, L., Anomalous rectification in the squid giant w o n injected with tetraethylammonium chloride, J. gen. Physiol., 48 (1965) 859--872. 2 Deschenes, M., Feltz, P. and Lamour, Y., A model for an estimate in vivo of the ionic basi~, of presynaptic inhibition: an intra~ellular analysis of the GABA-indueed depolarization in rat dorsal root ganglia, Brain Res., 118 (1976) 486--493. 3 Deschenes, M. and Feltz, P., GABA-induced rise of extraeellular p o t ~ i u m in rat dorsal root ganglia: a n electrophysiological study in vivo, Brain Re~., 118 (1976) 494--499 4 Hille~B., Ionic channels in nerve membranes, Prog. Biophys. Mol. Biol., 21 (1970) 1--32. 5 H6sli, L., AndrOs, P.F. and H6sli, E., Action of GABA on neurones a~!d satellite glial cells of cultured rat dorsal root ganglia, Neurosci. Lett., 6 (1977) 79--83. 6 Hosli, L., Andrbs, P.F. and H6sli, E., Ionic mechanisms associated with the action of amino acid transmitters on spinal neurones in tissue culture. In Ryal! and Kelly (Eds.), Satellite Symposium "Iontophoresis and Transmitter Mechanisms i~ the Mammalian Central Nervous System", Elsevier/North Holland, Amsterdam, 1978, pp. 215--217. 7 ~[~sii, L., Andrbs, P.F. and Hissli, E., Neuron-glia interactions: indirect effect of GABA on cultured glial cells, Exp. Brain Res., 34 (1979) in press. 8 M~vez, H. and Pichon, Y., The effect of internal and external 4-aminopyridine on the potassium currents in intracellularly perfused squid giant axons, J. Physiol. (Lond.), 268 (1977) 511--532. 9 Ni~hi, S., Minota, S. and Karczmar, A.G., Primary afferent neurones: The ionic mechanism of GABA-mediated depolarization, Neuropharmaeology, 13 (1974) 215--219.
193
EFFECTS OF 4-AMINOPYRIDINE AND TETRAETHYLAMMONIUM ON THE DEPOLARIZATION BY GABA OF CULTURED SATELLITE GLIAL CELLS L. HOSLI, P.F. ANDRES and ELISABETH HC]SI,I Depa,tment of Physiology, University of Basp.l Ve.~algasse I, CH-4051 Basel (Switzerland) (Received October 19th, 1978) (Revised version received November 3rd, 1978) (Accepted November 6th, 1978)
SUMMARY
4-Aminopyridine (4-AP) which selectivelyblock: K+-conductance of exc~ ~ble membranes, reversibly abolished the depolarization by 7-aminobutyric acid (GABA) of cultured satelliteglial(SG) cells,but did not or only slightlyaffect the action of G A B A on dorsal root ganglion (DRG) neurons. It is therefore suggested that the depolarization of glialcellsby G A B A is an indirect effect due to the accumulation of K* which is released from adj-cent neurons during their depolarization by the amino acid. Tetraethylammonium (TEA) had no effect on the G A B A depolarization at a concentration of 15 m M but produced a slightreduction at higher covcentrations (60--70 mM).
The method of tissue culture provides a good model system to study neurone-glia interactions allowing intracellv~ar recordings from individual cells under visual control. Since in dorsal root ganglia (DRG) the small satellite glial (SG) cells are lying very close to the large neurones and completely surround their cell bodies, we have used this preparation in order to investigate the influence of neurones depolarized by 7-aminobutyric acid (GABA) on glial cells. There is strong evidence that the depolarization of D RG neurones by GABA is associated with an increase in CI-- and K÷~onductance causing an efflux of both ions from the cells [2,3,6,9 ]. Using K÷-sensitive electrodes, Deschenes and Feltz [3] have demonstrated that the GABA depolarization of DRG neurones is accompanied by an increase of the extracellular K÷-concentration. Recently we have shown that GABA caused a depolarization of SG celts of cultured DRG without producing changes in membrane conductance [ 7 ] suggesting that this depolarization isan indirect effect due to the release of K÷ from adjacent DRG neurones during their depolarization by the amino acid [5,7]. To further support this hypothesis~ we have studied the effects of
194
4-aminopyridine (4-AP) anc~ tetraethylammonium (TEA), two substances which selectively block K÷-conductance of various excitable membranes (see refs. 1,4,8) on the GABA depolarization of SG cells. The techniques for Culturing d0rsalroot ganglia~and for the electr0physiological " e x t e n t s have been described in detail ::Ln:pre~,ious :papers [5,7 ]. Intracellular recordings were made from cultured SG cells and neurones of rat DRG (8-24 days in vitro) Using glass microelectrodes filled with 1--4 M K-acetate. The resistance of the elechodes ranged from 4 0 - 1 5 0 Mrl. Membrane potentials were recorded on an oscilloscope and on a rectilinear pen writer. The perfusing solution (Gey's) consisted of: (raM) NaCI 137, KCI 5, CaCI= 2.4, }~gCl2, 2.2, Na2HPO4 1.0, KH=PO4 0.18, NaHCO~ 2.9, glucose 11.1. The pH of the bathing fluid ranged from 7.3--7.4 and the temperature was maintained at 36 ° ~ 1°C. GABA, 10-4 M (FLUKA), tetmethylammoniumchlorid, 15, 60 and 70 mM (FLUKA) and 4-AP, 5 mM (FLUKA) were added to the bathing fluid.
GLIA
A
NEURONE
GABA 10"4M
GABA 10"4M -
B
. -
4AP -
C
-
I IIIIII I
5mM
I
III
I III
m
,l mv
2u sec
Fig. I. Effect of 4-AP on the depoisrization by GABA (I0"4 M) of a SG cell (left) and a l~eurone (right) of cultured DRG (12 and 16 days in vitro respectively). Initial resting potentials were'--'] I m V (SG cell)and " 6 5 ~ V (neurone). A: depolarization (upward deflection) caused by-GABA. B: 4;BP(5 raM) add,~,to t~E; bathinlfluid, for 2 and 3 rain re6pactively, abolkhed the :GABA depolarization of the 8G cell without affecting that of ~ e neurone. C: GABA depolarization 5 rain after retum:ing to normal bathing solution. Duration of perfusio n with GABA is indicated by bars.
195
The identification of the various cell ';ypes (DXG neurones and the surrounding SG cells) in culture~ of rat D]:C~ was made on the basis of their morphological appearance a s we~ as on their electrophysiological properties [ 5 , 7 ] . A s w a s described previously, GABt~ ]L0-4 M caused a depolarization of all DRG neurones (n = 57) and SG cells (n = 68) tested [5,7]. 4-AP at a concentration of 5 mM reversibly abolished the GABA depolarization o'. SG cells, but did not or only slightly affect the depolarization of DRG neurones. As illustrated in Fig. 1A (left), GABA 1 0 -4 M c~tused a depolarization of SG cell by approx. 10 inV. After perfusing with 4.AP (5 mM) for 2 min, the GABA effect was completely abolished (Fig. 1B, left). Almost complete recovery was observed 5 min after returning to normal bathing solution (Fig. 1C, lett). Similar results of the action of 4-AP on the GABA depolarization werc~ obtained on all SG cells (n = 8) studied. In contrast, the depolarization by GABA o f a DRG nem'one was not affected by 4-AP (5 raM) after peffusilg for 3 min (Fig. 1, right). Similar observations were made on half of the DRG neurones tested whereas on the other half, there was a slight reduction of the GABA depolarization by 4-AP which was often reversible. TEA which also bk~cks K+.eonductance [1,4] usually had no effect on t!~e amplitude of the GABA depolarization of both SG cells (n = 6) and DRG neurones (n = 5) at a concentration o~ I 5 mM, although the time course and shape of the falling phase was often altered. At higher concentrations (60 A
GABA 10"4M
B
C 10'
III
TEA
15 mM
TEA 60 mM
10'
45'
~/~~'~
! 5mY 20 sec
Fig. 2. Effect of TEA on the depolarization by GABA (10 -4 M) of a cultured SG cell (12 days in vitro). A: depolarization (upward deflectiL,~) caused by GABA. B: depolarization by GABA of the sanie cell 10 and 45 rain after adaition of TEA (15 mM) to the perfusion fluid. C: action of GABA on this cell 10 and 20 rain after increasing the TEA concentration to 60 raM, Recovery could not be observed because the cell was lost. The resting potential of this cell was - 6 3 mV. Duration of perfusion wi~h GABA is indicated by bars above tracings.
196
and 70 mM), TEA caused a reduction of the amplitude of the GABA depolarization on all SG cells (n = 4) tested. Fig, 2 illustrates the action of TEA on the depolarization by GABA of a SG cell. After perfusing with TEA (15 raM)for 45 rain, the amplitude of the GABA depolarization was not affected, b u t increasing the concentration of TEA to 60 mM reduced the depolarizing effect of GABA after 20 rain. The difference in effectiveness and time course of action betwen 4-AP and TEA might be due to the fact that 4-AP acts from both the inside and outside of the cell membrane [ 8], whereas TEA is only effective from the inside. This is consisten~ with studies on the squid giant axon showing that TEA acts only from the inside of the cell membrane [ 1,4]. There is considerable evidence timt the GABA-induced depolarization of DRG neurones is ma:-~ly due to an increased Cl--conductance [2,3,9]. Our finding that 4-AP which sel~ctively blocks K÷-conductance [8] did not or only slightly affect the GABA depolarization of DRG neurones provides further evidence that mainly CI- and not K ÷ are involved in producing this depolarization. Furthermore, it has been shown by Deschenes and Feltz [3] that the depolarization of DRG neurones by GABA also causes an efflux of K ÷ thereby increasing the extracellular K÷-concentration in the vicinity of the activated neurones. Our results indicate that 4-AP abolished the GABAdepolarization on SG cells by blocking the efflux of K ÷ from depolarized neur,mes. It is suggested that the action of the amino acid on glial cells is an indirect effect due to the accumulation of K ÷ which is released from adjacent DRG neurones during their depul~rization by GABA. REFERENCES 1 Armstrong, C.M. and Binstock, L., Anomalous rectification in the squid giant w o n injected with tetraethylammonium chloride, J. gen. Physiol., 48 (1965) 859--872. 2 Deschenes, M., Feltz, P. and Lamour, Y., A model for an estimate in vivo of the ionic basi~, of presynaptic inhibition: an intra~ellular analysis of the GABA-indueed depolarization in rat dorsal root ganglia, Brain Res., 118 (1976) 486--493. 3 Deschenes, M. and Feltz, P., GABA-induced rise of extraeellular p o t ~ i u m in rat dorsal root ganglia: a n electrophysiological study in vivo, Brain Re~., 118 (1976) 494--499 4 Hille~B., Ionic channels in nerve membranes, Prog. Biophys. Mol. Biol., 21 (1970) 1--32. 5 H6sli, L., AndrOs, P.F. and H6sli, E., Action of GABA on neurones a~!d satellite glial cells of cultured rat dorsal root ganglia, Neurosci. Lett., 6 (1977) 79--83. 6 Hosli, L., Andrbs, P.F. and H6sli, E., Ionic mechanisms associated with the action of amino acid transmitters on spinal neurones in tissue culture. In Ryal! and Kelly (Eds.), Satellite Symposium "Iontophoresis and Transmitter Mechanisms i~ the Mammalian Central Nervous System", Elsevier/North Holland, Amsterdam, 1978, pp. 215--217. 7 ~[~sii, L., Andrbs, P.F. and Hissli, E., Neuron-glia interactions: indirect effect of GABA on cultured glial cells, Exp. Brain Res., 34 (1979) in press. 8 M~vez, H. and Pichon, Y., The effect of internal and external 4-aminopyridine on the potassium currents in intracellularly perfused squid giant axons, J. Physiol. (Lond.), 268 (1977) 511--532. 9 Ni~hi, S., Minota, S. and Karczmar, A.G., Primary afferent neurones: The ionic mechanism of GABA-mediated depolarization, Neuropharmaeology, 13 (1974) 215--219.