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Amino acid-evoked depolarization of electrically inexcitable (neuroglial?) cells in the guinea pig olfactory cortex slice
Brain Research, 153 (1978) 183-187 C() Elsevier/North-Holland Biomedical Press
183
Amino acid-evoked depolarization of electrically inexcitable (neuroglial?) cells in the guinea pig olfactory cortex slice
ANDREW CONSTANTI and MARTIN GALVAN* Department of Pharmacology, The School of Pharmacy, 29/39 Brunswick Sq., London WCIN l A X (Great Britain)
(Accepted April 6th, 1978)
The membrane potential of mammalian central neurones is known to be sensitive to microiontophoretically applied amino acids such as y-aminobutyric acid (GABA), L-glutamic acid or L-aspartic acid 1°. However, the membrane potential of neuroglial cells is generally thought to be unaffected directly by these agents 1,12,19. Neuroglial cells in the mammalian central nervous system are usually characterized by their high stable resting membrane potential ( > 50 mV), their failure to show spike discharges on electrode penetration and intracellular current injection and by their ability to depolarize slowly during repetitive neuronal activity 5-7. In some instances, these electrically inexcitable cells have been identified histologically as neuroglia 3,6,9. During the course of some recent experiments on neurones in the guinea pig olfactory cortex slice, a convenient in vitro brain preparation s, we frequently impaled elements whose electrical properties accorded with those of neuroglia. This enabled us to study the effects of some drugs on the membrane potential of these cells in a manner not possible in vivo. We found that bath-application of a number of amino acids produced a reversible membrane depolarization; in this preliminary report, we describe these responses and suggest some possible mechanisms by which they could arise. Surface slices of guinea pig olfactory cortex were cut to a thickness of about 600/zm using a recessed Perspex guide and bow cutter as previously described s. After several hours preincubation, the slice was placed (pial surface uppermost) between nylon meshes in a Perspex bath 15, and superfused continuously with Krebs solution at room temperature (20-24 °C) bubbled with 95 ~o 02/5 ~ Coz. The composition of the Krebs solution was (raM): NaC1, 118; KC1, 4.8; CaC12 2.52; NaHCO3, 25; KH2PO4, 1.18; MgSO4.7H20, 1.19; D-glucose, 11 (pH 7.4). Intracellular recordings were made with a single 100-300 Mf~ microelectrode, filled with potassium acetate (neutralized with glacial acetic acid) 15. Cells were discarded if their membrane potential was < 50 mV or was not stable for at least 3 rain. Orthodromic activation of cortical neurones in the slice was effected via supramaximal stimuli (20-30 V; 0.1 msec) de* Present address: PhysiologischesInstitut, 8000Mtinchen 2, Pettenkoferstrasse 12, Mfinchen,G.F.R.
184 livered through a pair of Pt stimulating electrodes placed across the cut end of the lateral olfactory tract (LOT) 16. All drugs were dissolved in Krebs solution in fixed concentrations and applied to the preparation via the superfusing solution. Electrical properties. The cells studied in these experiments had the following electrical characteristics" (1) in contrast to the pyriform neurones ~z, they showed no injury discharge on initial impalement; (2) their resting potential (measured from the point of electrode withdrawal) was relatively high (-- 66 ~::i-_-9 mV, mean ~ S.D. of 17 cells in 10 slices) and stable for up to 3 h or more. In 5 cells where [K' ]o of the superfusing solution was varied over the range 6-18 mM(KC1 added directly to the medium), the membrane potential was linearly related to loglo [K ~]0 with a slope of 40 ~ 14 mV (mean ~- S.D.) per decade change of [K~]0 (Figs. 2 and 3, see also refs. 1, 3, 5 and 14); (3) their apparent input resistance was rather low (~< 30 Mf~: pyriform neurones usually showed input resistances in the range 9-280 M ~ , see ref. 15); (4) injection of depolarizing current pulses (up to 0.5 hA) through the recording microelectrode always failed to generate a direct spike" moreover, single stimuli applied to the LOT (using current strengths sufficient to evoke excitatory postsynaptic potentials and action potentials in the pyriform neurones) did not produce any visible response in these cells" (5) repetitive stimulation (1-16 Hz) of the LOT for 15-30 sec consistently produced a slow depolarization (1.5-12 mV), which plateaued after about 10 sec, then declined with a half-time of 5-30 sec at the end of stimulation (Fig. 1, see also refs. 1, 7 and 13). No such response occurred when the recording electrode was extracellular. These characteristics are quite different from those of the pyriform neurones TM 16, but strongly resemble those previously described for identified neuroglia6,~2,t:L Histological identification of the inexcitable cells by intracellular injection of dye was impracticable with the very high resistance microelectrodes that we used: however, on the basis of their electrical properties, we feel justified in regarding them as neuroglia. Drug-evoked responses. Bath-application of 1 m M G A B A or 3 m M L-glutamate invariably produced a reversible low amplitude depolarization of 4.6 0.7 mV (n -- 10) and 8.5 % I mV (n :: 10) respectively (means :~ S.E.M.). In 2 cells. 3 m M stim/sec
4.I"'---I 5mY
1 ,,eerrrv,~,;;"7~-- 15 see Fig. I. Effect of repetitive stimulation of the lateral olfactory tract (LOT) on the membrane potential of an inexcitable cell in the olfactory cortex slice (resting potential = --73 mV; depolarization in this and all other figures is indicated by upward deflections). Three 15 sec trains of stimuli at 1,2 and 4 Hz were applied respectively (stimulus parameters were 20 V; 0.1 msec, and were supramaximal for orthodromic activation of underlying cortical neurones: see text). Note that the amplitude of the cell depolarization increased with increasing stimulus frequency. At 4 Hz, a plateau depolarization of about 9 mV was attained. (Downward deflections are shock artefacts.)
185
•
[7~ 6
• I
[77-7] 12
•
LOT
V-~
Kcl
m
GABA
1~
GLUTAMATE
[]
•
3
I
..... ~ 3
16 Hz
20mY 6
4rain
Fig. 2. Comparison of the effect of repetitive LOT stimulation with the effects of bath-applied GABA, glutamate or KCI (in order to increase [K+]0) on the membrane potential of an inexcitable cell (resting potential at point of electrode withdrawal --49 mV). Continuous intracellular recording shows initially a small depolarization produced by a 15 sec train of LOT stimuli (16 Hz) (filled circle) followed by responses to two different concentrations of KC1 (6 and 12 raM; hatched bars). In this cell, the relation between membrane potential and log10 [K~]0 had a slope of 31 mV. Bath-applications 6f GABA (1 mM, filled bars) or glutamate (3 mM, open bars) also produced reversible and reproducible depolarizations. Note the afterhyperpolarization following responses to glutamate, but not those of GABA or KCI (see ref. 18). Irregularities in the baseline were due to flow 'surges' in the bath. Numbers below bars indicate drug concentrations (raM) applied via the bathing fluid.
L-aspartate also p r o d u c e d a d e p o l a r i z a t i o n (Figs. 2 and 3) however, these depolarizations were not a c c o m p a n i e d by a clear reduction in the cell input resistance. This c o n t r a s t s with findings on pyriform neurones, which always showed a depolarization and fall in resistance in response to the applied a m i n o acids (ref. 17; also A.C. and M.G., unpublished observations). Large i o n t o p h o r e t i c doses o f G A B A (but not glutamate) have previously been r e p o r t e d to depolarize cat cortical ' u n r e s p o n s i v e ' cells in vivo without changing the m e m b r a n e resistance 12, and this has been a t t r i b u t e d to an active uptake o f G A B A coupled to an influx o f N a + (refs. I 1 and 12). On this basis, an a m i n o acid that is not a substrate for the glial G A B A carrier would not be expected to depolarize the cell: however we f o u n d t h a t 3 - a m i n o p r o p a n e s u l p h o n i c acid (3-APS, 300 # M ) , a potent G A B A - m i m e t i c agonist with very low affinity for glial G A B A carriers 2, was a much m o r e p o t e n t depolarizing agent t h a n G A B A (1 m M ; cf. Fig. 3), an o b s e r v a t i o n previously n o t e d on a variety o f m a m m a l i a n neurones, including those of the olfactory cortex ~v. M o r e o v e r , the involvement o f a non-specific n e u t r a l a m i n o acid carrier seems unlikely, since l m M L-leucine did not p r o d u c e a d e p o l a r i z a t i o n (2 cells).
186
20mV L O T 8 H z GLU 3
3APS 0"3
KC1 12
4min
KCI 6
ASP 3
Fig. 3. Comparison of depolarizations produced by repetitive LOT stimulation (8 Hz; 15 sec) and bath-applications of glutamate, aspartate, GABA, 3-APS or KCt on the membrane potential of a single inexcitable cell (resting potential = --75 mV) (continuous intracellular record - - see legend of Fig. 2). The slope of the membrane potential vs. loglo [K+]o relation was 42 mV. Note that 3-APS was more potent than GABA (on a molar basis). Glutamate and aspartate were approximately equipotent and had a comparable rate of onset and offset.
The possibility that the drug-evoked depolarizations are due to a direct receptormediated action on the cell m e m b r a n e cannot be excluded. However, the fact that m a m m a l i a n glial cells in culture do not respond to iontophoretically applied G A B A or glutamate 19 argues against such a hypothesis. In m a m m a l i a n central preparations, increases in [K÷]0 following repetitive nerve stimulation have been measured directlyS,14; furthermore, bath applications of G A B A to rat dorsal r o o t ganglia 4 or glutamate to frog spinal cord 18 have been reported to increase [K÷]0 by about 1 m M (although this m a y be an underestimate of the true rise in [K+]0 see ref. 4). It is therefore considered more likely that the depolarizations we observe following either repetitive L O T stimulation or application o f a m i n o acids are due to local accumulation o f K + released from adjacent depolarized neurons: indeed, a 1-5 m M increase in [K+]0 would be sufficient to account for these effects. In conclusion, our present in vitro data confirms an earlier report by Krnjevi6 and Schwartz 1~ that G A B A depolarizes cortical 'unresponsive' cells without a measurable change in input resistance. However in contrast to their results, we find that glutamate also has a similar depolarizing action. At present, we c a n n o t explain this discrepancy, although the different methods o f drug application m a y be relevant.
187 W e t h a n k D r . C. N . Scholfield for i n s t r u c t i o n in i n t r a c e l l u l a r r e c o r d i n g f r o m b r a i n slices. T h i s r e s e a r c h was s u p p o r t e d by M . R . C . g r a n t s t o P r o f e s s o r D. A. B r o w n a n d P r o f e s s o r D. W. S t r a u g h a n , a n d we are grateful to D . A . B . f o r his helpful c o m m e n t s o n the m a n u s c r i p t . M G is a n M . R . C . scholar.
1 Adams, P. R. and Brown, D. A., Some observations on electrically inexcitable cells (neuroglia) in rat sympathetic ganglia, Brit. J. Pharmacol., 51 (1974) 131-132P. 2 Bowery, N. G., Transport and Depolarizing Actions of Amino Acids in the Superior Cervical Sympathetic Ganglion of the Rat, Ph.D. Thesis, University of London, 1974. 3 Dennis, M. J. and Gerschenfeld, M. H., Some physiological properties of identified mammalian neuroglial cells, J. Physiol. (Lond.), 203 (1969) 211-222. 4 Deschenes, M. and Feltz, P., GABA-induced rise of extracellular potassium in rat dorsal root ganglia; an electrophysiological study in vivo, Brain Research, 118 (1976) 494-499. 5 Futamachi, K. J. and Pedley, T. A., Glial cells and extracellular potassium: their relationship in mammalian cortes, Brain Research, 109 (1976) 311-322. 6 Grossman, R. C. and Hampton, T., Depolarization of cortical glial cells during electrocortical activity, Brain Research, 11 (1968) 316-324. 7 Grossman, R. C., Whiteside, L. and Hampton, T., The time course of evoked depolarization of cortical glial cells, Brain Research, 14 (1969) 401-415. 8 Harvey, J. A., Scholfield, C. N. and Brown, D. A., Evoked surface-positive potentials in isolated mammalian olfactory cortex, Brain Research, 76 (1974) 235-245. 9 Kelly, J. S., Krnjevi6, K. and Yim, G. K. W., Unresponsive cells in cerebral cortex, Brain Research, 6 (1967) 767-769. 10 Krnjevi6, K., Chemical nature of synaptic transmission in vertebrates, Physiol. Rev., 54 (1974) 418-540. 11 Krnjevi6, K., Puil, E. and Werman, R., GABA and glycine actions on spinal motoneurones, Canad. J. Physiol. Pharmaeol., 55 (1977) 658-669. 12 Krnjevi6, K. and Schwartz, S., Some properties of unresponsive cells in the cerebral cortex, Exp. Brain. Res., 3 (1967) 306-319. 13 Kuffler, S. W., Neuroglial cells: physiological properties and a K*-mediated effect of neuronal activity on the glial membrane potential, Proc. roy. Soc. B, 168 (1967) 1-21. 14 Lothman, E. W. and Somjen, G. G., Extracellular K + activity, intracellular and extracellular potential responses in the spinal cord, J. Physiol. (Lond.), 252 (1975) 115-136. 15 Scholfield, C. N., Electrical properties of neurones in the olfactory cortex slice in vitro, J. Physiol. (Lond.), 275 (1978) 535 546. 16 Scholfield, C. N., A depolarizing inhibitory potential in neurones of the olfactory cortex in vitro, J. Physiol. (Lond.), 275 (1978) 547-557. 17 Scholfield, C. N., The action of depressant amino acids on neurones in the isolated olfactory cortex. In J. S. Kelly and R. W. Ryall (Eds.), lontophoresis and Transmitter Mechanisms in the Mammalian Central Nervous System, 1978. 18 Sonnhof, U., Grafe, P., Richter, D. W., Parekh, N., Krumnikl, G. and Linder, M., Investigations of the effects of glutamate on motoneurones of the isolated frog spinal cord. In J. S. Kelly and R. W. Ryall (Eds.), lontophoresis and Transmitter Mechanisms in the Mammalian Central Nervous System, 1978. 19 Wardell, W. M., Electrical and pharmacological properties of mammalian neuroglial cells in tissue culture, Proc. roy. Soc. B, 165 (1966) 326-361.
183
Amino acid-evoked depolarization of electrically inexcitable (neuroglial?) cells in the guinea pig olfactory cortex slice
ANDREW CONSTANTI and MARTIN GALVAN* Department of Pharmacology, The School of Pharmacy, 29/39 Brunswick Sq., London WCIN l A X (Great Britain)
(Accepted April 6th, 1978)
The membrane potential of mammalian central neurones is known to be sensitive to microiontophoretically applied amino acids such as y-aminobutyric acid (GABA), L-glutamic acid or L-aspartic acid 1°. However, the membrane potential of neuroglial cells is generally thought to be unaffected directly by these agents 1,12,19. Neuroglial cells in the mammalian central nervous system are usually characterized by their high stable resting membrane potential ( > 50 mV), their failure to show spike discharges on electrode penetration and intracellular current injection and by their ability to depolarize slowly during repetitive neuronal activity 5-7. In some instances, these electrically inexcitable cells have been identified histologically as neuroglia 3,6,9. During the course of some recent experiments on neurones in the guinea pig olfactory cortex slice, a convenient in vitro brain preparation s, we frequently impaled elements whose electrical properties accorded with those of neuroglia. This enabled us to study the effects of some drugs on the membrane potential of these cells in a manner not possible in vivo. We found that bath-application of a number of amino acids produced a reversible membrane depolarization; in this preliminary report, we describe these responses and suggest some possible mechanisms by which they could arise. Surface slices of guinea pig olfactory cortex were cut to a thickness of about 600/zm using a recessed Perspex guide and bow cutter as previously described s. After several hours preincubation, the slice was placed (pial surface uppermost) between nylon meshes in a Perspex bath 15, and superfused continuously with Krebs solution at room temperature (20-24 °C) bubbled with 95 ~o 02/5 ~ Coz. The composition of the Krebs solution was (raM): NaC1, 118; KC1, 4.8; CaC12 2.52; NaHCO3, 25; KH2PO4, 1.18; MgSO4.7H20, 1.19; D-glucose, 11 (pH 7.4). Intracellular recordings were made with a single 100-300 Mf~ microelectrode, filled with potassium acetate (neutralized with glacial acetic acid) 15. Cells were discarded if their membrane potential was < 50 mV or was not stable for at least 3 rain. Orthodromic activation of cortical neurones in the slice was effected via supramaximal stimuli (20-30 V; 0.1 msec) de* Present address: PhysiologischesInstitut, 8000Mtinchen 2, Pettenkoferstrasse 12, Mfinchen,G.F.R.
184 livered through a pair of Pt stimulating electrodes placed across the cut end of the lateral olfactory tract (LOT) 16. All drugs were dissolved in Krebs solution in fixed concentrations and applied to the preparation via the superfusing solution. Electrical properties. The cells studied in these experiments had the following electrical characteristics" (1) in contrast to the pyriform neurones ~z, they showed no injury discharge on initial impalement; (2) their resting potential (measured from the point of electrode withdrawal) was relatively high (-- 66 ~::i-_-9 mV, mean ~ S.D. of 17 cells in 10 slices) and stable for up to 3 h or more. In 5 cells where [K' ]o of the superfusing solution was varied over the range 6-18 mM(KC1 added directly to the medium), the membrane potential was linearly related to loglo [K ~]0 with a slope of 40 ~ 14 mV (mean ~- S.D.) per decade change of [K~]0 (Figs. 2 and 3, see also refs. 1, 3, 5 and 14); (3) their apparent input resistance was rather low (~< 30 Mf~: pyriform neurones usually showed input resistances in the range 9-280 M ~ , see ref. 15); (4) injection of depolarizing current pulses (up to 0.5 hA) through the recording microelectrode always failed to generate a direct spike" moreover, single stimuli applied to the LOT (using current strengths sufficient to evoke excitatory postsynaptic potentials and action potentials in the pyriform neurones) did not produce any visible response in these cells" (5) repetitive stimulation (1-16 Hz) of the LOT for 15-30 sec consistently produced a slow depolarization (1.5-12 mV), which plateaued after about 10 sec, then declined with a half-time of 5-30 sec at the end of stimulation (Fig. 1, see also refs. 1, 7 and 13). No such response occurred when the recording electrode was extracellular. These characteristics are quite different from those of the pyriform neurones TM 16, but strongly resemble those previously described for identified neuroglia6,~2,t:L Histological identification of the inexcitable cells by intracellular injection of dye was impracticable with the very high resistance microelectrodes that we used: however, on the basis of their electrical properties, we feel justified in regarding them as neuroglia. Drug-evoked responses. Bath-application of 1 m M G A B A or 3 m M L-glutamate invariably produced a reversible low amplitude depolarization of 4.6 0.7 mV (n -- 10) and 8.5 % I mV (n :: 10) respectively (means :~ S.E.M.). In 2 cells. 3 m M stim/sec
4.I"'---I 5mY
1 ,,eerrrv,~,;;"7~-- 15 see Fig. I. Effect of repetitive stimulation of the lateral olfactory tract (LOT) on the membrane potential of an inexcitable cell in the olfactory cortex slice (resting potential = --73 mV; depolarization in this and all other figures is indicated by upward deflections). Three 15 sec trains of stimuli at 1,2 and 4 Hz were applied respectively (stimulus parameters were 20 V; 0.1 msec, and were supramaximal for orthodromic activation of underlying cortical neurones: see text). Note that the amplitude of the cell depolarization increased with increasing stimulus frequency. At 4 Hz, a plateau depolarization of about 9 mV was attained. (Downward deflections are shock artefacts.)
185
•
[7~ 6
• I
[77-7] 12
•
LOT
V-~
Kcl
m
GABA
1~
GLUTAMATE
[]
•
3
I
..... ~ 3
16 Hz
20mY 6
4rain
Fig. 2. Comparison of the effect of repetitive LOT stimulation with the effects of bath-applied GABA, glutamate or KCI (in order to increase [K+]0) on the membrane potential of an inexcitable cell (resting potential at point of electrode withdrawal --49 mV). Continuous intracellular recording shows initially a small depolarization produced by a 15 sec train of LOT stimuli (16 Hz) (filled circle) followed by responses to two different concentrations of KC1 (6 and 12 raM; hatched bars). In this cell, the relation between membrane potential and log10 [K~]0 had a slope of 31 mV. Bath-applications 6f GABA (1 mM, filled bars) or glutamate (3 mM, open bars) also produced reversible and reproducible depolarizations. Note the afterhyperpolarization following responses to glutamate, but not those of GABA or KCI (see ref. 18). Irregularities in the baseline were due to flow 'surges' in the bath. Numbers below bars indicate drug concentrations (raM) applied via the bathing fluid.
L-aspartate also p r o d u c e d a d e p o l a r i z a t i o n (Figs. 2 and 3) however, these depolarizations were not a c c o m p a n i e d by a clear reduction in the cell input resistance. This c o n t r a s t s with findings on pyriform neurones, which always showed a depolarization and fall in resistance in response to the applied a m i n o acids (ref. 17; also A.C. and M.G., unpublished observations). Large i o n t o p h o r e t i c doses o f G A B A (but not glutamate) have previously been r e p o r t e d to depolarize cat cortical ' u n r e s p o n s i v e ' cells in vivo without changing the m e m b r a n e resistance 12, and this has been a t t r i b u t e d to an active uptake o f G A B A coupled to an influx o f N a + (refs. I 1 and 12). On this basis, an a m i n o acid that is not a substrate for the glial G A B A carrier would not be expected to depolarize the cell: however we f o u n d t h a t 3 - a m i n o p r o p a n e s u l p h o n i c acid (3-APS, 300 # M ) , a potent G A B A - m i m e t i c agonist with very low affinity for glial G A B A carriers 2, was a much m o r e p o t e n t depolarizing agent t h a n G A B A (1 m M ; cf. Fig. 3), an o b s e r v a t i o n previously n o t e d on a variety o f m a m m a l i a n neurones, including those of the olfactory cortex ~v. M o r e o v e r , the involvement o f a non-specific n e u t r a l a m i n o acid carrier seems unlikely, since l m M L-leucine did not p r o d u c e a d e p o l a r i z a t i o n (2 cells).
186
20mV L O T 8 H z GLU 3
3APS 0"3
KC1 12
4min
KCI 6
ASP 3
Fig. 3. Comparison of depolarizations produced by repetitive LOT stimulation (8 Hz; 15 sec) and bath-applications of glutamate, aspartate, GABA, 3-APS or KCt on the membrane potential of a single inexcitable cell (resting potential = --75 mV) (continuous intracellular record - - see legend of Fig. 2). The slope of the membrane potential vs. loglo [K+]o relation was 42 mV. Note that 3-APS was more potent than GABA (on a molar basis). Glutamate and aspartate were approximately equipotent and had a comparable rate of onset and offset.
The possibility that the drug-evoked depolarizations are due to a direct receptormediated action on the cell m e m b r a n e cannot be excluded. However, the fact that m a m m a l i a n glial cells in culture do not respond to iontophoretically applied G A B A or glutamate 19 argues against such a hypothesis. In m a m m a l i a n central preparations, increases in [K÷]0 following repetitive nerve stimulation have been measured directlyS,14; furthermore, bath applications of G A B A to rat dorsal r o o t ganglia 4 or glutamate to frog spinal cord 18 have been reported to increase [K÷]0 by about 1 m M (although this m a y be an underestimate of the true rise in [K+]0 see ref. 4). It is therefore considered more likely that the depolarizations we observe following either repetitive L O T stimulation or application o f a m i n o acids are due to local accumulation o f K + released from adjacent depolarized neurons: indeed, a 1-5 m M increase in [K+]0 would be sufficient to account for these effects. In conclusion, our present in vitro data confirms an earlier report by Krnjevi6 and Schwartz 1~ that G A B A depolarizes cortical 'unresponsive' cells without a measurable change in input resistance. However in contrast to their results, we find that glutamate also has a similar depolarizing action. At present, we c a n n o t explain this discrepancy, although the different methods o f drug application m a y be relevant.
187 W e t h a n k D r . C. N . Scholfield for i n s t r u c t i o n in i n t r a c e l l u l a r r e c o r d i n g f r o m b r a i n slices. T h i s r e s e a r c h was s u p p o r t e d by M . R . C . g r a n t s t o P r o f e s s o r D. A. B r o w n a n d P r o f e s s o r D. W. S t r a u g h a n , a n d we are grateful to D . A . B . f o r his helpful c o m m e n t s o n the m a n u s c r i p t . M G is a n M . R . C . scholar.
1 Adams, P. R. and Brown, D. A., Some observations on electrically inexcitable cells (neuroglia) in rat sympathetic ganglia, Brit. J. Pharmacol., 51 (1974) 131-132P. 2 Bowery, N. G., Transport and Depolarizing Actions of Amino Acids in the Superior Cervical Sympathetic Ganglion of the Rat, Ph.D. Thesis, University of London, 1974. 3 Dennis, M. J. and Gerschenfeld, M. H., Some physiological properties of identified mammalian neuroglial cells, J. Physiol. (Lond.), 203 (1969) 211-222. 4 Deschenes, M. and Feltz, P., GABA-induced rise of extracellular potassium in rat dorsal root ganglia; an electrophysiological study in vivo, Brain Research, 118 (1976) 494-499. 5 Futamachi, K. J. and Pedley, T. A., Glial cells and extracellular potassium: their relationship in mammalian cortes, Brain Research, 109 (1976) 311-322. 6 Grossman, R. C. and Hampton, T., Depolarization of cortical glial cells during electrocortical activity, Brain Research, 11 (1968) 316-324. 7 Grossman, R. C., Whiteside, L. and Hampton, T., The time course of evoked depolarization of cortical glial cells, Brain Research, 14 (1969) 401-415. 8 Harvey, J. A., Scholfield, C. N. and Brown, D. A., Evoked surface-positive potentials in isolated mammalian olfactory cortex, Brain Research, 76 (1974) 235-245. 9 Kelly, J. S., Krnjevi6, K. and Yim, G. K. W., Unresponsive cells in cerebral cortex, Brain Research, 6 (1967) 767-769. 10 Krnjevi6, K., Chemical nature of synaptic transmission in vertebrates, Physiol. Rev., 54 (1974) 418-540. 11 Krnjevi6, K., Puil, E. and Werman, R., GABA and glycine actions on spinal motoneurones, Canad. J. Physiol. Pharmaeol., 55 (1977) 658-669. 12 Krnjevi6, K. and Schwartz, S., Some properties of unresponsive cells in the cerebral cortex, Exp. Brain. Res., 3 (1967) 306-319. 13 Kuffler, S. W., Neuroglial cells: physiological properties and a K*-mediated effect of neuronal activity on the glial membrane potential, Proc. roy. Soc. B, 168 (1967) 1-21. 14 Lothman, E. W. and Somjen, G. G., Extracellular K + activity, intracellular and extracellular potential responses in the spinal cord, J. Physiol. (Lond.), 252 (1975) 115-136. 15 Scholfield, C. N., Electrical properties of neurones in the olfactory cortex slice in vitro, J. Physiol. (Lond.), 275 (1978) 535 546. 16 Scholfield, C. N., A depolarizing inhibitory potential in neurones of the olfactory cortex in vitro, J. Physiol. (Lond.), 275 (1978) 547-557. 17 Scholfield, C. N., The action of depressant amino acids on neurones in the isolated olfactory cortex. In J. S. Kelly and R. W. Ryall (Eds.), lontophoresis and Transmitter Mechanisms in the Mammalian Central Nervous System, 1978. 18 Sonnhof, U., Grafe, P., Richter, D. W., Parekh, N., Krumnikl, G. and Linder, M., Investigations of the effects of glutamate on motoneurones of the isolated frog spinal cord. In J. S. Kelly and R. W. Ryall (Eds.), lontophoresis and Transmitter Mechanisms in the Mammalian Central Nervous System, 1978. 19 Wardell, W. M., Electrical and pharmacological properties of mammalian neuroglial cells in tissue culture, Proc. roy. Soc. B, 165 (1966) 326-361.