Physiological identification of GABA as the inhibitory transmitter for mammalian cortical neurons in cell culture

Brain Research, 190 (1980) 111-121 © Elsevier/North-Holland Biomedical Press

111

P H Y S I O L O G I C A L I D E N T I F I C A T I O N OF GABA AS T H E I N H I B I T O R Y T R A N S M I T T E R F O R M A M M A L I A N C O R T I C A L N E U R O N S I N CELL CULTURE

MARC A. DICHTER Department of Neurology, Beth Israel Hospital, Children's Hospital Medical Center and Harvard Medical School, CHMC, 300 Longwood Avenue, Boston, Mass. 02115 (U.S.A.)

(Accepted October 25th, 1979) Key words: Gaba - - inhibitory transmitter - - neuron cell culture - - cortical neurons

SUMMARY (1) Rat cortical neurons grown in dissociated cell culture exhibit IPSPs which appear to be generated by an increase in membrane conductance to chloride. (2) The neurons are all sensitive to GABA in micromolar concentrations and GABA mimics the inhibitory transmitter. (3) The neurons are much less sensitive to glycine and insensitive to taurine. (4) Bicuculline and strychnine both block essentially all IPSPs and at the same concentrations block GABA effects. (5) It is concluded that GABA is the main, or only, inhibitory transmitter utilized by the cortical neurons in vitro. The relevance of this conclusion to in situ transmitter identification is discussed.

INTRODUCTION The identification of chemical excitatory and inhibitory transmitter substances in the mammalian central nervous system and the mechanisms by which these substances produce their effects are among the most challenging problems currently being pursued by neurobiologists. Several transmitters have been unequivocally identified at specific invertebrate synapses, while in the mammalian CNS only acetylcholine (ACh) in motor neurons and gamma-aminobutyric acid (GABA) in Purkinje cells have been so established. It is likely that GABA also serves as the inhibitory transmitter at cortical and cerebellar basket cell synapses and in the spinal cord. In addition, although ACh, norepinephrine, dopamine, serotonin, glutamate, and a variety of peptides are found in various regions of the mammalian CNS and have been

112 shown to excite and/or inhibit neuronal firing when applied in the vicinity of neurons, the specific demonstration of their transmitter function has not been fully established. The complexity of the mammalian CNS makes such proof very difficult because of the inability to apply agents at known concentrations to isolated cells and the great difficulty in studying direct membrane effects with intracellular recordings. The limited specificity of the many pharmacologic agents used to antagonize various transmitters, either by synthesis blockade or receptor blockade, is an additional problem. The use of mammalian cortical neurons grown in cell culture allows us to overcome many of the problems inherent in the intact brain. The neurons are relatively isolated from one another, are not covered by glial cells and are thus exposed to the bathing medium, and can be impaled by microelectrodes while other micropipettes are positioned just outside the cell for the application of putative transmitters or blocking agents (or various combinations thereof) in known concentrations to the cell. The major limitation of the system is the question of whether neurons in culture use the same transmitters and have the same sensitivities as their in situ counterparts. This question can best be answered by a detailed analysis of the culture system followed by a comparison, as far as it is possible with more limited techniques, with comparable cells in the cortex. Analysis of the basic electrophysiological properties of the cortical neurons in culture has demonstrated that they have properties which are quite similar to their in vivo counterparts 1I. GABA is the best documented cortical inhibitory transmitter20, 22,25,26. GABA promptly inhibits most cortical neurons to which it is applied. GABA, and its specific synthesizing enzyme, glutamic acid decarboylase, are found in the cortex. GABA can be shown to be released from cortex by neuronal activity and from cortical tissue in vitro in response to depolarization, by a Ca-dependent mechanism. Drugs such as bicucultine and picrotoxin, which are known to block GABA inhibition, can be shown to block cortical inhibition. Inhibitory as well as excitatory synaptic connections develop extensively between rat cortical neurons maintained in cell culture H. In this series of papers we will attempt to demonstrate that GABA is the inhibitory transmitter used in almost all of the inhibitory synapses and establish a quantitative estimate of GABA sensitivity on cortical neurons. Both chemical and physiological measures are required to demonstrate a transmitter role for GABA. This paper presents physiological data. The second paper presents chemical data and a general discussion of the criteria we believe to be the most valid zg, and the third paper in this series identifies GABAergic neurons by GABA uptake autoradiography 3~. METHODS

Tissue culture

Rat cortical cell cultures were established from 15-day embryos as previously described la, except that some cultures were grown on a polylysine-collagen surface instead of on feeder layers (see ref. 29). The neurons were studied between 3 and 7 weeks in vitro.

113

Electrophysiology Intracellular recordings were made with 3.5 M potassium acetate (and occasionally, potassium citrate or 3 M KC1) microelectrodes (resistance = 30-90 M~)) 11. Cells were accepted for analysis if they maintained resting potentials larger than --40 mV and action potentials > 50 mV during the recording. The agents tested in these studies were applied in known concentrations by a 'puffer' microperfusion technique 5. Micropipette tips were broken to 5-20 ffm i.d. and were filled with the drug dissolved in physiological saline at the concentration to be delivered to the cell. The micropipette was connected to air pressure at between 1 and 10 psi (exact pressure needed for a good flow was established for each pipette before use). There was little leakage from the pipettes since when the puffer was placed within 30-40 #m of a cell no detectable effect from the drug was observed, even when substantially suprathreshold concentrations were used. When the pressure was applied, a gentle steady stream of medium containing the drug at a known concentration bathed the cell. When the pressure was removed, the small quantity of drug (or GABA) applied diffused away into the 3-5 ml of bathing media. To monitor input resistance during the perfusion, constant (50-100 msec) pulses of hyperpolarizing current were applied to the cell and the resulting change in membrane potential measured. Input resistance was calculated directly from the AV/A I, leaving out any correction for current leakage in the dendrites. These cells have generally linear I-V curves for hyperpolarizations up to 30 mV 11. During the experiments described below we routinely positioned up to three puffer pipettes near neurons to be studied. This allowed us to perform dose-response curves with three different concentrations of GABA or glycine or any other putative agonists on single cells. In addition, we were able to test the effect of a known concentration of antagonist on a known concentration of agonist by perfusing, for example, first with GABA, then with bicuculline and finally with GABA and bicuculline. We were also concerned about the time course of the effects of transmitter antagonists. We compared bath application of antagonists (minutes to hours of equilibration) with microperfusions lasting between several seconds and several minutes. In addition, we tested the effects of antagonist pretreatment on the effectiveness of blockade and found results which indicated different time courses of action for different agents. These will be discussed in the Results section below. We emphasize these technical points because the results obtained in the cell culture system may appear at odds with some of the results from in situ multibarrel iontophoresis experiments and the kinds of drug interactions we can specify in this simple system, and which might not be discernible in the in situ system, might explain these discrepancies. For certain specific experiments, GABA was also applied by iontophoresis from a fine-tipped ( < 1 #m) micropipette filled with 1 M GABA (pH 7) positioned just outside a cell. Currents were kept between 1 and 8 nA.

114 RESULTS

(A) GABA mimics the inhibitory transmitter The majority of neurons from cultures used in these experiments show spontaneously occurring IPSPs. They range in size between 1 and 25 mV and are usually simple and discrete but may be complex. Spontaneous EPSPs are often seen in the same cells. If two cells are impaled simultaneously, stimulation of one can sometimes evoke an IPSP in the other 11. All the cortical neurons in the culture are sensitive to exogenously applied GABA (Fig. 1A). This can be demonstrated qualitatively by iontophoresis of GABA from a 1 M pipette or quantitatively from the puffer (see section below). Both GABA and the inhibitory transmitter produce an increased membrane conductance which usually produces a hyperpolarization of the membrane potential. (In some cells, GABA produces a depolarization which probably results from leakage of CI from the medium into a partially injured cell (see below).) By artificially depolarizing and hyperpolarizing the cell while measuring the evoked (or spontaneous) IPSP and the GABA response, the equilibrium potentials for these responses can be obtained (Fig. 1B, C). In fact, the ease with which these potentials are changed in size, and even inverted, suggests that they are generated on, or close to, the neuron soma. In different cells the equilibrium potential varies somewhat between --50 and --90 mV (presumably because of differences in internal CI) but in each case, it is the same for the GABA response and the IPSP (Fig. lC). In order to determine the ionic dependence of the IPSP and the GABA response, various ion substitutions were performed. Replacing the external C1 by sulfate caused

A

~ -54 [ ~ l ~ w ~ q m m q l l q ~

C

-100 8O

EIpsP

.i

-60

B

-62 v

-68, . . . . . -90

,

~

~

~

' .....

-4C

-20

i

i -20

' 40

' -~o

'

-10

' x~o

F GAIaA

Fig. 1. G A B A responses and IPSPs at different resting membrane potentials. In this and subsequent figures, all electrophysiological illustrations are penwriter records of intracellular recordings. A: response to miniperfusion with 10 # M G A B A (bar over trace). Small downward deflections are spontaneous IPSPs. R M P -- --54 mV. B: same cell during background hyperpolarization, indicating diminution of G A B A response and IPSPs and inversion of both at --90 inV. Calibrations 20 mV and 5 sec. C: graph of IPSP equilibrium potential and G A B A equilibrium potential for 13 different neurons.

115

-. . . . . . . . . .

1--

l

B-

~Jtlt,I

,

I -

LOW CL

~

l ....

I " ....

"-':':"-'-'-'-'=

~- -

l

---1

see

L

GABA

1111~! rll rll lli~l ' ..... ~v,, ~ll fill II1111 I1 I1! 1! II111111111 I

lii I Illll'l III I-/[1II IU Illll GABA,

LOW

CL

5

see

Fig. 2. Chloride dependence of IPSPs and GABA response. A: regularly recurring IPSPs invert when cell is perfused with low chloride media. B: initial GABA (10 #M) response is hyperpolarizing and is associated with an increased conductance. (Each large downward deflection is the voltage change induced by a 50 msec constant current pulse.) When GABA (10/~M) is subsequently applied in low CI media, the response becomes depolarizing and there is no increased conductance. (In fact, the decrease in conductance is due to the CI replacement.) The thick baseline in both traces is due to the frequent spontaneous synaptic potentials. Calibration 50 inV.

both the IPSP and GABA response to become depolarizing (Fig. 2) and also reduced the GABA-induced increase in membrane conductance. Similarly, raising the internal Cl by leakage from a KC1 electrode caused both responses to become depolarizing. Raising external K does not decrease either response. Lowering external Na does not effect either response. Thus, both the IPSP and the GABA response appear to be due to an increased conductance of C1.

( B) GA BA dose-response relationship In order to determine the specific dose-response relationship for the cortical neurons, GABA was applied in known concentrations from our puffer micropipettes. Responses were measured as changes in conductance. Fig. 3 illustrates the dose-response curve for GABA. Threshold was approximately 1 # M GABA. Below that level it was difficult to appreciate a change in conductance by our techniques. The response increased between 1 and 50 # M and appeared to flatten out above that. It is impossible for us to determine whether concentrations of GABA above 50/~M continued to increase conductance because of the limitations of our measuring techniques. Although the average responses at each concentration were generally consistent, a small proportion of cells could show responses which varied by as much as an order of magnitude from the mean. The sensitivity of these cortical neurons to GABA is approximately equivalent to that seen in chick spinal cord neurons 4 and is somewhat higher than that reported for crayfish stretch receptor 16.

(C) Other possible amino acid transmitters Glycine is another amino acid mentioned prominently as a possible inhibitory transmitter, especially in spinal cord, possibly also in cortex or forebrain. Cortical

116 neurons in culture are much less responsive to glycine than G A B A . Moreover, the variability o f the responsiveness is much greater. Fig. 3 also illustrates the dose-response curve for glycine. A few cells are responsive to 50 # M , and the response becomes m a x i m u m at between 250 and 1000 tiM (1 mM). At 1 m M approximately 10"i, of neurons remain insensitive, and for most cells the maximum conductance change induced by glycine is less than that obtainable with G A B A . Neurons with very low sensitivities to glycine m a y still exhibit large IPSPs. It is therefore unlikely that at least these IPSPs are generated by glycinergic synapses. Taurine, a sulfur-containing amino acid, is also mentioned as a possible inhibitory transmitter since it is present in cortex and has been shown to reduce firing when applied by iontophoresis to cortical neurons in situ. Taurine at 100-1000 /~M produced no detectable effects on the cortical neurons.

(D) Response to inhibitory blocking agents Bicuculline and picrotoxin are t h o u g h t to act by specifically blocking G A B A

1000

LLI CO ~C W rr" C) Z

100

LU C)

Z .,~ ~-~O "7 0 Z

10

o c.)

i

i

....

i

10

. . . . . . . .

I

100

CONCENTRATION (IJM)

1ooo

eGABA &GLYCINE

Fig. 3. Graph of conductance change versus applied GABA (circles) or glycine (triangles) concentration. GABA response is barely detectable at 1/~M and cannot be measured above 50 FM. Insert illustrates recording from one neuron during successive miniperfusions with 9, 25 and 50/zM GABA. Each large division is 10 inV. Constant hyperpolarizing potentials were passed into cell to monitor input resistance during GABA application. Each point is mean and standard error. Conductance responses to 25 and 50 #M GABA were often large enough to be difficult to measure and the values on the graph for these points should be considered more approximate that the others. Note marked shift to the right of the glycine curve.

117

GABA ( l O u M )

GABA (lOuM)

GABA (lOuM) AND BICUCULLINE (IOuM)

~

iljilrliilliilfilnliJIilliiiW GABA ( l O u M ) AND STRYCHNINE (50uM)

Fig. 4. Examples of the effects of bicuculline (at 10 #M) and strychnine (at 50 #M) on GABA effects. Each pair is from one cell showing the membrane potential and conductance response to GABA (10 #M) and then GABA (10 #M) plus blocking agent. responses in several systems 22. We tested these drugs at low concentrations on the spontaneous IPSPs in the cortical neurons in two different ways. By recording the spontaneous synaptic activity from a relatively large number of neurons (n ~ 20) while bathing the neurons in media containing increasing concentrations (0.5, 1,3,5, 10,50,100/aM) of blocking agents, it was determined that more than 95 ~ of the IPSPs were blocked by 3/~M bicuculline or 10 # M picrotoxin. These drugs did not block electrical excitability and the neurons showed no gross alterations in electrophysiological behavior. There was an increased tendency to fire in bursts which appeared related to increased reverberatory excitatory synaptic activity. In both cases, as soon as the drugs were washed out, the spontaneous IPSPs returned. A second technique involved perfusion of bicuculline onto individual cells which exhibited [PSPs. In these experiments, brief exposure to 1/~M bicuculline attenuated IPSPs by greater than 50 ~o in 3 of 8 cells, and by less than 50 ~ in 4 of the remaining 5 cells, while l0 # M attenuated IPSPs by more than 5 0 ~ in 19 of 20 cells. Strychnine is a drug which is thought to specifically block glycinergic synapses while having little or no effect on GABAergic synapses. Bathing the cells in 10 # M strychnine greatly attenuated or blocked spontaneous IPSPs in approximately 80 ~ of the neurons in the cortical cultures without affecting other electrophysiological properties. This would indicate that either these IPSPs were being generated by a nonGABA mechanism (i.e. glycine, taurine, etc.) or that strychnine, in fact, blocked GABA in the cortical neurons. In order to test the pharmacological specificity of bicuculline and strychnine, we applied known concentrations of GABA and glycine to the neurons, first the agonist alone and then combined with the antagonist. (In some experiments the cells were pretreated for 15-60 sec with antagonist before application ofagonist plus antagonist.) Fig. 4 illustrates an experiment in which we had two 'puffers' just outside the neuron impaled. The first contained GABA at l0/~M and produced a large, typical response. The second contained GABA (10/~M) and bicuculline (10 #M). It can be seen that the GABA response was greatly attenuated by the bicuculline. Experiments performed in

118 this manner allowed each neuron to be its own control and responses to the GABAbicuculline were often compared with and without 30-60 sec bicuculline pretreatment. Average membrane conductance went from 25 nS in control to 106 nS in response to 10 #M GABA but to only 31 nS in the presence of 10 #M bicuculline. Inhibition by bicuculline was somewhat less pronounced but still quite effective when the GABA and bicuculline were presented without pretreatment with bicuculline. Similar experiments were performed with strychnine and GABA as illustrated in Fig. 4. Strychnine at 10 #M markedly attenuates the GABA response, just as bicuculline does, and at 50 #M the response to GABA is not apparent (Fig. 5). Interestingly, the strychnine effect appears somewhat less dependent on brief pretreatment. Although the GABA blocking effect of strychnine is quite strong, strychnine is even more effective as a glycine blocking agent. At 0.5 #M strychnine produces a 70-90°0 reduction in the glycine (200/zM)-induced membrane conductance increase (Fig. 5). Thus, when measured against the selected doses of GABA and glycine, strychnine appears to be approximately 10-20 fold more potent against glycine. However, when unknown, and possibly quite large, amounts of strychnine are being applied to cortex either directly or by iontophoresis, it is doubtful that this difference in selectivity of strychnine would be useful in separating glycinergic from GABAergic synapses. DISCUSSION

The evidence presented in this paper strongly suggests that the rat cortical neurons in vitro utilize GABA as their main (and possibly exclusive) inhibitory transmitter. This is based on the universal postsynaptic sensitivity to GABA, the fact

100

GABA (10 uM)

80

z
I

GLYCINE (200~uM)

60

i

,3 c~ z O o

40

O o

f J 1

10

BICUC.

10

50

STRYCH.

uM

10

50

BICUC.

0.5

pM

STRYCH.

Fig. 5. Graph of per cent reduction in G A B A (10/~M) or gtycine (200/zM) induced conductance increase by bicueulline or strychnine. Neurons were first treated with agonist and then miniperfused with antagonist for 30-60 sec before being treated with agonist plus antagonist.

119 that GABA at low concentrations mimics the action of the inhibitory transmitter in producing an increased C1 conductance and the demonstration of the identical pharmacological effects of inhibitory blocking agents on both the IPSP and GABA response. Further biochemical evidence for the role of GABA as the inhibitory transmitter are provided 29 by demonstrating: (1) the presence of a GABA synthesizing system in the culture (GAD), (2) the ability of the neurons to synthesize GABA from glucose, (3) the ability of the neurons to release GABA in response to a depolarization by a Ca-dependent mechanism, (4) the presence of a high affinity uptake system for GABA, and (5) the development of GABA receptors. Is there evidence for any other inhibitory transmitter in addition to GABA? Since all the cortical neurons are sensitive to GABA, there is no necessity to postulate another agent. In addition, the neurons are markedly less sensitive to glycine and almost insensitive to taurine. In the presence of 10 #M bicuculline in the bath there are occasional neurons which continue to exhibit IPSPs, but these were too infrequent to be adequately studied and could still represent GABAergic IPSPs which would be sensitive to slightly higher bicuculline concentrations. It could be argued that since we are only studying spontaneous IPSPs, there could exist a population of glycinergic neurons which are not spontaneously active. We feel this is unlikely, because in the presence of bicuculline, the spontaneous activity in the culture often increased substantially, but significant numbers of bicuculline resistant IPSPs were not seen. If it is true that GABA represents almost the exclusive inhibitory transmitter in cortex in culture, does this accurately reflect the situation for intracortical inhibition in situ? Careful analysis of existing data suggests that it is not necessary to postulate additional inhibitory transmitters. As in vitro, all cortical neurons in situ appear to be inhibited by GABAT,I~,z2. Other putative neurotransmitters have also been shown to inhibit the firing of cortical neurons, although usually less effectively than GABA: glycine 2A4,21,2a, taurine s, dopamine (DA) and norepinephrine (NE) la,17,1s. However, DA and NE producing neurons are not found in cortex, so these agents could not be intracortical inhibitory transmitters (except by secondary release). Furthermore, neither the concentrations of glycine and taurine delivered from concentrated solutions by iontophoresis nor their transmembrane effects on cortical neurons are known. Another approach utilized in vivo to identify transmitters employs specific blocking agents. Much intracortical inhibition is bicuculline-sensitive 9,1°, although the extent by which GABA blocking agents are able to block intrinsic inhibition in cortex has not been adequately studied. Some investigators have reported reduction in cortical inhibition by strychnine~0, while others have reported no effects of strychnine on cortical inhibition1,2,6, 24. However, the specificity of either bicuculline as a GABA antagonist or strychnine as a glycine antagonist in situ has been extensively questioned3,15,19. The data presented here demonstrate that concentrations of bicuculline which block IPSPs are also able to block GABA effects while having no direct effect on the membrane and little effect on glycine responses. We have also demonstrated that low concentrations of strychnine block GABA effects on the cortical neurons, as well as the IPSPs, although glycine responses are even more sensitive. Thus, in this system, the

120 specificity of each blocking agent against both IPSPs and putative transmitter agents can be established. Based on the apparent limited concentration range over which strychnine can specifically block glycine responses but not GABA responses, it would seem impossible to deliver such precise amounts of strychnine by iontophoresis in situ that glycinergic inhibition would be blocked while GABAergic inhibition would be spared. Thus, it is possible to propose the hypothesis that all or almost all intracortical inhibitory connections utilize GABA as the transmitter. The relatively exclusive utilization of GA BA for inhibitory transmission between the cortical ceils in vitro is unlikely to be an artifact of the culture conditions since both chick 5 and mouse "~7 spinal cord cells in similar cultures probably utilize both glycine and GABA as their inhibitory transmitters. In fact, glycine is more commonly used as the cultures mature. It has also been suggested in chick spinal cord cells ~ that strychnine is more selective as a glycine blocking agent than we have seen in the mammalian cortical neurons. Both the predominance of glycinergic inhibition and the selectivity of strychnine are also characteristics of in situ spinal cord, as opposed to cortex. Given the prominence of GABAergic inhibition in the cortical cultures, it could be asked which cells are GABAergic. In situ GAD immunohistochemistry indicates that aspinous and sparsely spinous stellate cells utilize GABA 2s. Autoradiographic studies utilizing high affinity uptake of tritiated GABA to identify GABAergic neurons in the cultures will be presented in the third paper of this series 3°. The identification of GABA as the primary, if not exclusive, inhibitory transmitter utilized by these cortical neurons in culture provides us with a powerful model with which to study the ontogenetic development of GABAergic neurons and GABA receptors and the molecular mechanisms involved in cell to cell recognition in this system. Furthermore, we hope to utilize this model to study the effects of drugs which are thought to act directly on cortical GABA inhibition, such as certain anticonvulsants, and the effects of chronic drug exposure (especially those neuroactive drugs commonly prescribed for young children) on the developing GABAergic systems. ACKNOWLEDGEMENTS I would like to thank Mr. Bernard Biales and Ms. Sara Vasquez for technical assistance with the tissue cultures and the electrophysiological experiments and Ms. Diane Kilday for typing this manuscript. Dr. Dichter is supported by RCDA NS00130, NS15362, a grant from the Esther A. and Joseph Klingenstein Fund and the Children's Hospital Mental Retardation Center Core Grant HD06276.

REFERENCES 1 Anderson, P., Eccles, J., Loyning, Y. and Voorhoeve, P., Strychnine-resistant inhibition in the brain, Nature (Lond.), 200 (1963) 843-845. 2 Biscoe, T. and Curtis, D., Strychnine and cortical inhibition, Nature (Lond.), 214 (1967) 914-915. 3 Briscoe, J., Duggan, A. and Lodge, D., Antagonism between bicuculline, strychnine, and

121 picrotoxin and depressant amino acids in the rat nervous system, Comp. gen. PharmacoL, 3 (1972) 423433. 4 Choi, D., Pharmacological Evidence for GABA as a Neurotransmitter in Dissociated Spinal Cord Cell Cultures, P h . D . Thesis, Harvard University, 1978. 5 Choi, D. W., Farb, D. H. and Fischbach, G. D., Chlordiazepoxide selectively augments GABA action in spinal cord cell cultures, Nature (Lend.), 269 (1977) 342-344. 6 Crawford, J., Curtis, D., Voorhoeve, P. and Wilson, V., Strychnine and cortical inhibition, Nature (Lend.), 200 (1963) 845-846. 7 Curtis, D. and Crawford, J. M., Central synaptic transmission-microelectrophoretic studies, Ann. Rev. Pharmacol., 9 (1969) 209-240. 8 Curtis, D., Duggan, A., Felix, D., Johnston, G. and McLennan, H., Antagonism between bicuculline and GABA in the cat brain, Brain Research, 33 (1971) 57-73. 9 Curtis, D. and Felix, D., The effect of bicuculline upon synaptic inhibition in the cerebral and cerebellar cortices of the cat, Brain Research, 34 (1971) 301-321. 10 Curtis, D., Felix, D. and McLennan, H., GABA and hippocampal inhibition, Brit. J. Pharmacol., 40 (1970) 881-883. 11 Dichter, M., Rat cortical neurons in cell culture: culture methods, cell morphology, electrophysiology and synapse formation, Brain Research, 149 (1978) 279-293. 12 Dreifuss, J., Kelly, J. and Krnjevic, K., Cortical inhibition and v-aminobutyric acid, Exp. Brain Res., 9 (1969) 137-154. 13 Frederickson, R., Jordan, L. and Phillis, J., The action of noradrenaline on central neurons, Brain Research, 35 (1971) 556-560. 14 Hill, R., Simmonds, M. and Straughan, D., A comparative study of some convulsant substances as y-aminobutyric acid antagonists in the feline cerebral cortex, Brit. J. PharmacoL, 49 (1973) 37-51. 15 Hill, R., Simmonds, M. and Straughan, D., Antagonism of 7-aminobutyric acid and glycine by convulsants in the cuneate nucleus of cat, Brit. J. Pharmacol., 56 (1976) 9-19. 16 Hori, N., Ikeda, K. and Roberts, E., Muscimol, GABA and picrotoxin: effects of membrane conductance of a crustacean neuron, Brain Research, 141 0978) 364-370. 17 Johnson, E., Roberts, M. and Straughan, D., The responses of cortical neurons to monoamines under differing anesthetic conditions, J. Physiol. (Lend.), 203 (1969) 261-280. 18 Johnson, E., Roberts, M. and Sobrezak, A., Noradrenaline cells in the cat cerebral cortex, Brit. J. NeuropharmacoL, 8 (1969) 549-566. 19 Johnson, E., Roberts, M. and Straughan, D., Amino acid-induced depression of cortical neurones, Brit. J. Pharmacol., 38 (1970) 659-666. 20 Johnston, G., Amino acid inhibitory transmitters in the central nervous system. In C. Hockman and D. Bieger, (Eds.), Chemical Transmission in the Mammalian Central Nervous System, University Park Press, Baltimore, 1976. 21 Kelly, J. and Krjnevic, K., The action of gtycine on cortical neurones, EAT. Brain Res., 9 (1969) 155-163. 22 Krnjevic, K., Chemical nature of synaptic transmission in vertebrates, PhysioL Rev., 54 (1974) 418-540. 23 Krnjevic, K. and Phillis, J., Iontophoretic studies of neurones in the mammalian cerebral cortex, J. Physiol. (Lend.), 165 (1963) 274-304. 24 Krnjevic, K., Randic, M. and Straughan, D., Pharmacology of cortical inhibition, J. PhysioL (Lend.), 184 (1966) 78-105. 25 Krnjevic, K. and Schwartz, S., ~,-Aminobutyric acid, an inhibitory transmitter? Nature (Lend.), 211 (1966) 1372-1374. 26 Krnjevic, K. and Schwartz, S., The action of~'-aminobutyric acid on cortical neurones, Exp. Brain Res., 3 (1967) 320-326. 27 Nelson, P., Ransom, B., Henkart, M. and Bullock, P., Mouse spinal cord in cell culture: IV. Modulation of inhibitory synaptic function, J. Neurophysiol., 40 (1977) 1178-1186. 28 Riback, C., Aspinous and sparsely-spinous stellate neurons in the visual cortex of rats contain glutamic acid decarboxylase, J. Neurocytol., 7 (1978) 461-478. 29 Snodgrass, S. R., White, W. F., Biales, B. and Dichter, M., Biochemical correlates of GABA function in rat cortical neurons in culture, Brain Research, 190 (1980) 123-137. 30 Suzuki, H. and Tsukahara, Y., Recurrent inhibition of the Betz cell, Jap. J. Physiol., 13 (1963) 386-398. 31 White, W. F., Snodgrass, S. R. and Dichter, M., Transmitter autoradiography in a rat cortical culture system, Brain Research, 190 (1980) 139-152.