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1 J. Phy8iol. (1975), 245, pp With 8 text-figures Printed in Great Britain STDES ON CONVLSANTS N THE SOLATED FROG SPNAL CORD.. ANTAGONSM OF AMNO ACD RESPONSES By J. L. BARKER,* R. A. NCOLL,t AND A. PADJENJ From the Laboratory of Neuropharmacology, National nstitute of Mental Health, St Elizabeths Hospital, Washington, D.C. 232,.S.A. (Received 5 April 1974) SMMARY 1. The isolated frog spinal cord was used to study the effects of picrotoxin, bicuculline, and strychnine on the responses of primary afferents to amino acids. Recording was by sucrose gap technique. 2. A series of neutral amino acids was found to depolarize primary afferents. Optimal activity was obtained by an amino acid whose carboxyl and amino groups were separated by a three-carbon chain length (i.e. GABA). Amino acids with shorter (i.e. f-alanine, glycine) or longer (i.e. a-aminovaleric acid, e-aminocaproic acid) distances between the charged groups were less potent. midazoleacetic acid was the most potent depolarizing agent tested. 3. Picrotoxin and bicuculline antagonized the primary afferent depolarizations of a number of amino acids tested with equal specificity. Depolarizing responses to standard (1-3 M) concentrations of fl-alanine and taurine were completely blocked by these convulsants, while depolarizations to 13 M y-aminobutyric acid (GABA) were only partially antagonized. Glycine responses were unaffected by these agents. 4. Strychnine completely blocked,8-alanine and taurine depolarizations and incompletely antagonized several other neutral amino acids. GABA, glutamate, and glycine depolarizations were not affected. 5. These results suggest that there are at least three distinct populations of neutral amino acid receptors on primary afferent terminals: a GABAlike receptor, a taurine/f,-alanine receptor, and a glycine-like receptor. The * National nstitute of Child Health and Human Development, Bethesda, Maryland 214,.S.A. t Present address: Laboratory of Neurobiology, SNY at Buffalo, Amherst, New York 14226,.S.A. t Visiting Scientist, on leave from Ruder Boskovic nstitute, Zagreb, Yugoslavia.

2 522 J. L. BARKER, R. A. NCOLL AND A. PADJEN strychnine resistance of the glycine responses indicates that the primary afferent receptors for glycine differ from those on the somata of spinal neurones. NTRODCTON The use of specific antagonists as a tool in identification of transmitters has received wide support in characterizing amino acid and synaptic inhibitory responses in the vertebrate central nervous system (C.N.S.). The 'inhibitory' neutral amino acids have been classified into two broad categories depending on whether they can be antagonized by strychnine on the one hand, or by picrotoxin or bicuculline on the other: glycine and glycine-like amino acids are antagonized by strychnine, while GABA and GABA-like amino acids are antagonized by bicuculline and picrotoxin (cf. Curtis, Duggan, Felix & Johnston, 1971). Research on amino acid agonistantagonist interactions throughout the vertebrate C.N.S. (e.g. Curtis, Hosli & Johnston, 1968; Galindo, 1969; Obata & Highstein, 197; Curtis et al. 1971; Curtis & Felix, 1971; Nicoll, 1971) has provided evidence to support the hypothesis that glycine is a principal inhibitory transmitter in the spinal cord (Werman, Davidoff & Aprison, 1968), while GABA is a major inhibitory transmitter at supraspinal sites (Krnjevic & Schwartz, 1967; Obata, to, Ochi & Sato, 1967). More recent work has demonstrated that (presynaptic) primary afferent fibres in the amphibian spinal cord are depolarized by the neutral amino acids (Tebecis & Phillis, 1969; Davidoff, 1972a, b; Barker & Nicoll, 1972, 1973). These findings have led us to investigate amino acid agonistantagonist interactions on the primary afferent fibres of the frog spinal cord. n the present study the amino acid responses on primary afferent fibres of the isolated frog spinal cord have been recorded with sucrose gap technique (cf. Koketsu, Karczmar & Kitamura, 1969). This experimental approach should allow one to make quantitative studies since the isolated preparation permits the use of known concentrations of drugs and the sucrose gap technique provides a stable and high resolution recording of electrical events. A preliminary account of some of the results has appeared (Nicoll & Barker, 1973). METHODS Preparation. Frogs (Rana pipiens) were chilled on ice to an anaesthetic state, decapitated and the spinal cord and attached roots carefully removed to a dissecting dish where the cord was hemisected sagittally. The hemisected cord with attached 8th and 9th dorsal roots was then placed in a sucrose gap apparatus, schematically presented on Fig. 1. The spinal cord was placed in the central compartment (R1) and both roots (DR8 and DRY) were led out through the sucrose compartment (S) into separate pools of Ringer solution (Rz and R8). Ringer-agar bridges connected R2 and

3 CONVLSANTS, AMNO ACDS AND SPNAL CORD 523 R3 with corresponding pools of KCl solution (K1 and K2). To ensure a leak-proof separation of compartments all slits through which roots were led, as well as a closely fitted cover, were coated with Vaseline. The sucrose and Ringer solution (R1) compartments were continuously perfused (S = 1 ml./min; R1 = 3 ml./min). The experiments were carried out with the room temperature between 17 and 2 C. Higher temperature tended to shorten the viability of the preparation and soften the Vaseline coating, thusproducing unstable recording. A properly maintained preparation easily survived more than 1 hr. The Ringer solution (115 mm-nacl, 2 mn-cacl2, 2 mm-kcl, and 1 m Tris (hydroxymethyl) DR& R2 DR, R S Fig. 1. Diagram of sucrose gap recording of amino acid responses on the dorsal roots. See text for further details. aminomethane buffer adjusted to ph 7.3) was continuously bubbled with 1% oxygen. This solution will be referred to as a 'normal Ringer'. When drug responses were examined, 2 mx-mgso4 was added and this solution will be referred to as 'Mg Ringer'. The drugs were applied to the spinal cord by turning a stopcock which replaced the flow of Ringer solution with the drug solution. The small volume of the Ringer compartment R1 ( 1 ml. with spinal cord present) allowed rapid exchange of fluid. Recording. The recording of electrical activity was accomplished by monitoring the potential difference between two calomel electrodes: one was in contact with the bath (R1) through a Ringer bridge and the other was placed in the KC pool (K1 or K2). For drug responses a high frequency filter which did not distort the shape of the amino acid response was usually employed. Approximately 45-6 min were required for the sucrose gap to completely form. After this period the drug potentials remained constant. Structure-activity 8tudiea. sing GABA in concentrations between 5 x 14 and 2-5 x 1- M, a dose-response curve was constructed for each preparation. The responses of other amino acids were compared to the GABA dose-response curve to determine the concentration of GABA producing the response of the same size. The equipotent concentration was defined as the dose of GABA which produced the same depolarization as the test amino acid and the relative activity was expressed as the ratio of the equipotent concentrations. Antagoni8m of amino acid response. When quantitative studies were done on the interaction of convulsants with GABA and fl-alanine, the amino acids were prepared in the convulsant solution, in order to ensure that the convulsant was not washed

4 524 J. L. BARKER, R. A. NCOLL AND A. PADJEN from the preparation during the drug application. The results of a group of observations is presented as the mean s.e. of the mean (n = number of samples). Drug abbreviation. AHB: f-amino-y-hydroxybutyric acid; BALA: f8-alanine; BGP: fl-guanidinopropionic acid: DAV = 8-aminovaleric acid; EAC = c-aminocaproic acid: GABA = y-aminobutyric acid; GL = glutamic acid; GLY = glycine; MA = imidoazolacetic acid; PRO = proline; TAR = taurine. RESLTS Amino acid depolarization of primary afferent terminals Exposure of the entire hemisected cord to a drug solution could affect the primary afferents indirectly by activating pathways which synapse on to the primary afferent fibres. To test for indirect synaptic effects, the responses in normal Ringer solution were compared with responses obtained in a Ringer solution containing 2 mm-mgso4, which entirely blocked synaptic transmission. The addition of Mg ions slightly but A Control Control BALA GL GABA BALA GABA GL 2 mm-mg"" Picrotoxin 5 min 2 mv Fig. 2. The effect of Mg and picrotoxin on amino acid responses in normal Ringer solution. A: addition of 2 mx-mgso4 to the normal Ringer solution reduces the size of the glutamate depolarization. 2-5 x 1-3 M concentration of amino acids used. n B, 5 x 14 M picrotoxin reduces all of the amino acid responses in normal Ringer including glutamate to some extent. Concentration of amino acids 2-5 x 1-3 M; calibration is the same in A and B. significantly increased the size of the GABA (1 1 8 / 3-1, n = 6) and /J-alanine (118 % ± 4-3, n = 5) responses, but reduced the glutamate response to 39-3 % of control (± 2 4, n = 6) (Fig. 2A). These results suggest that a significant proportion of the glutamate response occurred indirectly. This might occur by glutamate depolarizing the neurones involved in generating the dorsal root potential. This notion is supported

5 CONVLSANTS, AMNO ACDS AND SPNAL CORD 525 by the observation that the glutamate response in normal Ringer solution was often reduced by picrotoxin (Fig. 2B), as previously reported by Tebecis & Phillis (1969), whereas the glutamate response obtained in magnesium Ringer solution was never reduced by picrotoxin (see below). To avoid these indirect effects, Mg ions were always present when the actions of amino acids on primary afferent terminals were investigated. A 2 mv 5 min GLY BALA GABA DAV EAC 1 mml mm 1 mm 1 mm 1 mm 1 B cis -5/ No. of carbon atoms between amino and carboxyl groups Fig. 3. Relationship between the depolarizing potency and the structure of a series of neutral amino acids. A, samples of records. Note higher concentration of glycine and e-aminocaproic acid. B, graphic presentation of the effect of charge separation, expressed as number of carbon atoms between amino and carboxyl groups, on the depolarizing potency of neutral amino acids relative to GABA. Amino acids arranged in the same order as in A. Comparison of structurally related amino acids Most of the amino acids tested depolarized the fibres with varying degrees of potency (Table 1, Figs. 3, 4 and 5). No hyperpolarizing response was seen in Mg Ringer with any of the amino acids tested except for glycine which on occasion showed a small hyperpolarizing component (Tebecis & Phillis, 1969; Davidoff, 1972a; Barker & Nicoll, 1973). There was a clear correlation between the distance of charged moieties of amino acid and depolarizing potency as presented on Fig. 3. The optimal distance was a chain of three carbon atoms, i.e. GABA molecule. Both the amino and carboxyl group were necessary for significant activity and removal of either of them (e.g. amino group: butyric acid;

6 526 J. L. BARKER, R. A. NCOLL AND A. PADJEN bo, m o h z C) C) C) P.,^ C) N Ml _e > o b Ca... o t- co o * - - o o o * 6 v V -) 42 C3 C; C) 1 bq.h o1 Eq 2 C) 4C') cc z 1 n u i Z Z ' L u z u 1 1Z z 1] 14 z x 1 F O u o 1 O -Z 14 1 c X ' V C 6 -Z o~~ "-1 O - 4 S- -C)a-23 d q~~~. &q ;>~~~~~~

7 ~~~~~~~ CONVLSANTS, AMNO ACDS AND SPNAL CORD 527 z E 4 o z x x r - o o o -V -l cm 74 v x : -Z 2: x 2 x x ) cl 2: 2 2: -Z :r o o 2 O o i : 2 2: 2 u -Z 2 #4 z x -z - = -Z -Z -O - -Z z X / \ Z /z ~z 2: u -' i -o z. z LL-Lt Z = 2 2: C)~~~~~~~~~~~~~C o * n.~~~~~~~~~~~~ce C o *', <,> E & g S~ $-,-kk D>- E C~~~~.2 ~~~~~~ Q~G

8 528 J. L. BARKER, R. A. NCOLL AND A. PADJEN carboxyl group: propylamine) drastically decreased potency of the compound. Since the sulphonate group could replace the carboxyl group (e.g. taurine) and the guanidino group could replace the amino group (e.g. fl-guanidinopropionic acid) it seems that the charges carried by these groups determine the activity of the substance, as was already noted in earlier studies (cf. Curtis & Watkins, 196). However, replacing the carboxyl group with a sulphonate moiety increased the time required to reach a maximum response (cf. fl-alanine and taurine in Figs. 4 and 5). This type of slow response was also seen with serine and alanine (not illustrated) and, therefore, was not dependent only on the presence of a sulphonate moiety. A GABA TAR BALA MA B ~ ~ ~ H Picrotoxin (1-4 M) 3 mv 6 mv 5 min Fig. 4. Picrotoxin antagonism of depolarizing responses to amino acids. A: control responses: The amino acids (1-3M) were applied until they produced a maximal response. Duration of application is indicated by bar. B: record B was 2 min after 14M picrotoxin. t is evident that the responses to all but glutamate (GL) and glycine (GLY) are either reduced or abolished in the presence of picrotoxin. Note different gain of MA record (for abbreviations see Methods). Of all ac-amino acids tested only alanine and glycine had any substantial depolarizing effect (Table 1). The addition of an imidazole ring increased the activity relative to GABA (i.e. imidazole acetic acid). Cystathionine had essentially no effect when applied in a saturated solution. Frequently, the glutamate response exhibited a prolonged hyperpolarization following the depolarization (e.g. Fig. 4). GABA and closely related amino acids (e.g. fl-guanidinopropionic acid and fi-amino-yhydroxybutyric acid) often produced responses which faded during the drug application (e.g. Fig. 5). None of the amino acids (whether in the absence or presence of magnesium) caused the primary afferents to fire action potentials, as is the case with glutamate action on motoneurones (Curtis, Phillis & Watkins, 1961).

9 j ~~~~~~~1 CONVLSANTS, AMNO ACDS AND SPNAL CORD 529 The action of convulsants on amino acid responses Picrotoxin (and bicuculline, not illustrated) reversibly antagonized the depolarizations of most of the neutral amino acids by varying degrees (Table 1, Fig. 4). There was, however, a specificity in their actions because the depolarizations produced by glycine and glutamate were not reduced by either antagonist, and because neither antagonist reduced the depolarization produced by a fivefold increase in extracellular potassium (not illustrated). These results suggested that all the compounds which were antagonized by these agents are acting at the same receptor complex and that the differences in relative potency between amino acids might be due to differences in the ability of a particular agonist to combine with the receptor and/or elicit an effect. A 6< < E D X~~~~~ < <_ < - _< Strychnine -(1-4 M) 22 mv 14 mv Fig. 5. Strychnine antagonism of depolarizing responses to amino acids. A, controls: all amino acid concentrations 1-3M except as noted for glycine. B: responses obtained 3 min after start of strychnine (1-4 M). The GABA response is increased while,8-alanine and taurine responses are abolished. The remainder are reduced slightly except for glycine and glutamate. Note different gain for imidazoleacetic acid (MA) record (for abbreviations, see Methods). However, the selective action of strychnine on the amino acid responses (Fig. 5) suggest that all of the amino acids do not share the same receptors. Strychnine antagonism of the amino acid responses depended on the concentration of strychnine used. At a concentration of 14 M, strychnine abolished the 8-alanine and taurine responses and also reduced 6-aminovaleric acid, imidazoleacetic acid, /?-guanidinopropionic acid, and /8- amino-y-hydroxybutyric acid depolarizations, but did not reduce glycine, GABA and glutamate responses. n fact, the GABA response and to a lesser extent glutamate and glycine responses were increased (Fig. 5). 23 PH Y 245

10 @ -;5s *~~~~~ GABA 53 J. L. BARKER, R. A. NCWOLL AND A. PADJEN Lower concentrations of strychnine (e.g. 1-5 M) blocked /-alanine and taurine depolarization with little effect on the other amino acid responses. The amino acids L-alanine and L-serine were similar to glycine in not being antagonized significantly by either picrotoxin or strychnine. The action of strychnine was reversible. The action of the antagonists was examined more quantitatively by studying the antagonism over a wide range of antagonist concentrations. For this purpose GABA was selected from the second group and,-alanine was selected from the third group over taurine because of its more rapid responses, thus making it more amenable to study i Ad i ~~obala ~ ii 8 AGL 6. 2 g Picrotoxin (14 M) Fig. 6. Effect of increasing concentrations of picrotoxin on GABA, f-alanine and glutamate depolarizations. The sizes of the amino acid (1-3 M) depolarizations, expressed as a percent of control, are plotted against the log of the picrotoxin concentration. Each point represents the average of twelve to twenty values ( ± S.E.). fl-alanine is more sensitive than is GABA, which is only partially reduced, while glutamate is actually increased slightly. The interaction between increasing concentrations of picrotoxin and 1-3 -GABA,,-alanine, and glutamate depolarizations is shown in Fig. 6.,8-alanine was reduced at a lower antagonist concentration (5 x 1- M) than GABA and was completely blocked at 14 M. As the concentration of picrotoxin was increased the antagonism of the GABA depolarization became saturated at about 3 % of control. The action of glutamate increased slightly with increasing concentrations of picrotoxin. Similar results were obtained with bicuculline (Fig. 7), though this antagonism was 5-1 times more potent. Fig. 8 shows the effect of increasing concentrations of strychnine on fi-alanine and GABA depolarizations. The,8-alanine response decreased as the strychnine dose was increased and was abolished at 5 x 1-5 M, while the GABA response increased at high concentrations of strychnine. The

11 CONVLSANTS, AMNO ACDS AND SPNAL CORD 531 BALA GABA V a D N.P 1w BALA GABA Bicuculline (1-' M) Fig. 7. Effect of increasing concentrations of bicuculline on GABA and f-alanine depolarizations. The size of the GABA (1-3 M) and fl-alanine (1-3 m) depolarizations, expressed as the percent of control, are plotted against the log of the concentration of bicuculline. Records are shown above the graph. fl-alanine is more sensitive than GABA, which is only partially reduced. Calibration: 4 min, 6 mv. C o 6-6 C oj %a Strychnine (1-' M) Fig. 8. Effect of increasing strychnine concentrations on GABA and fialanine depolarizations. The size ofthe GABA (1-3 M) andf-alanine (1-3M) depolarizations, expressed as the percent of control, are plotted against the log of the concentration of strychnine. Each point represents the average of eight to twelve values (± s.e.). The depolarization by fi-alanine is reduced and that by GABA increases at high strychnine concentration. 23-2

12 532 J. L. BARKER, R. A. NCOLL AND A. PADJEN GABA response may have been decreased slightly at concentrations of 1-5 to 5 x 1-5, but this was not significant. Glutamate followed a curve similar to GABA. DSCSSON The blockade of synaptic transmission with Mg ions showed that the depolarization of the primary afferents to the neutral amino acids did not involve an indirect synaptic component, while approximately 5 % of the glutamate response was indirect, presumably arising from the depolarization of neurones involved in generating the dorsal root potential since picrotoxin blocked this indirect component. Although the presence of Mg ions eliminates indirect synaptic effects, the possibility that the responses might result from increased extracellular K secondary to drug effects on membranes other than the primary afferent membranes should be considered. n the case of the neutral amino acids it is clear that the primary afferents definitely possess receptors for these amino acids, since the isolated dorsal root and dorsal root ganglion are depolarized by GABA (Obata, 1972; DeGroat, Lalley & Saum, 1972; Nishi, Minota & Karczmar, 1973; R. A. Nicoll & A. Padjen, unpublished observations). n the case of the acidic amino acids (e.g. glutamate) this argument cannot be used, since the primary afferent sensitivity is limited to the intraspinal portion of these fibres (Nishi, Soeda & Koketsu, 1965; R. A. Nicoll & A. Padjen, unpublished observations). However, the presence of glutamate receptors on excitatory terminals of invertebrates (sherwood & Machili, 1966; Kerkut & Walker, 1967; Florey & Woodcock, 1968; Dowson & sherwood, 1973), which probably release glutamate as their transmitter, suggests that a similar phenomenon may exist on the primary afferent fibres. n general, the results of the structure activity study were similar to the post-synaptic responses in the mammalian C.N.S. (Curtis & Watkins, 196), the crayfish stretch receptor (Edwards & Kuffler, 1959) and earlier work on frog spinal cord (Curtis et al. 1961), and indicate that a compound with positively charged and negatively charged groups separated by three carbon atoms is necessary for optimal activity. However, the depolarizing potency of glycine relative to GABA on primary afferents was considerably less than its hyperpolarizing potency on mammalian neurones (Curtis & Watkins, 196; Werman et al. 1968). The extremely potent depolarizing action of imidazoleacetic on primary afferent fibres agrees with the strong post-synaptic inhibitory action of this substance (McGeer, McGeer & McLennan, 1961; Phillis, Tebecis & York, 1968; Haas, Anderson & Hosli, 1972; Godfraind, Krnjevic, Maretic & Pumain, 1973; Swagel, keda & Roberts, 1973). The recent detailed structure-activity study of depolarizing amino acid responses on superior cervical ganglion are in remarkable

13 CONVLSANTS, AMNO ACDS AND SPNAL CORD 533 agreement with the present results (Bowery & Brown, 1974). They differ in that glutamate has no action on the ganglion and the action of glycine on the ganglion was considerably weaker. The effects of picrotoxin and bicuculline proved to be relatively nonspecific, antagonizing to varying degrees most of the neutral amino acids except glycine. These results are similar to those obtained with picrotoxin at the neuromuscular junction (Dudel, 1965) and at the superior cervical ganglion (Bowery & Brown, 1974); and to those obtained with bicuculline on neurones in the superior cervical ganglion (Bowery & Brown, 1974) and in the cerebral cortex (Curtis, Duggan, Felix, Johnston & McLennan, 1971). The action of bicuculline on amino acid responses of spinal interneurones (Curtis, Duggan, Felix & Johnston, 1971) is, however, more selective than that obtained on the primary afferents, since bicuculline has no action on the inhibition of interneurones by fl-alanine and taurine. The fact that high concentrations of strychnine completely blocked fl-alanine responses with little antagonism of GABA responses strongly suggests that the action of these two amino acids occurs through distinct receptor mechanisms. Thus a difference of only one carbon atom in distance between the amino and carboxyl groups of these two amino acids prevent any crossover and interaction between their respective receptors. The slight strychnine antagonism of f-guanidinopropionic acid, imidazoleacetic acid, f-amino-y-hydroxybutyric acid and d-amino valeric acid responses suggest that these molecules may cross over, to some extent, to the fl-alanine receptor. The relative non-specificity of the action of strychnine in the superior cervical ganglion (Bowery & Brown, 1974) is in contrast to the present results which clearly separate the actions of GABA and fl-alanine. The ineffectiveness of strychnine in blocking glycine depolarizing responses may be contrasted with its well described antagonism of glycine hyperpolarizations on post-synaptic membranes (i.e. somata of spinal neurones) (e.g. Curtis, Duggan & Johnston, 1971). This suggests that the post-synaptic hyperpolarizing response and primary afferent depolarizing response are mediated by different receptors. Furthermore, the ability of strychnine to antagonize /?-alanine (and taurine) depolarizations but not glycine depolarizations on the primary afferents indicates that either glycine utilizes a different receptor than fl-alanine (or taurine) or that the action of strychnine on the fl-alanine receptor is such that it is unable to prevent the access of glycine to /J-alanine receptors. The present results with antagonists suggest that the primary afferent depolarizing and motoneurone hyperpolarizing responses to glycine are mediated by different receptors. Whether the depolarizing and hyperpolarizing responses of GABA and f-alanine are mediated by separate can

14 534 J. L. BARKER, B. A. NCOLL AND A. PADJEN receptors is not entirely clear. Since picrotoxin and bicuculline antagonize the GABA depolarization, this depolarization cannot be the result of an amino acid uptake process, which is known to be relatively unaffected by these convulsants (versen & Johnston, 1971). n conclusion, on the basis of the present results the neutral amino acids exerting a depolarizing action on the primary afferent fibres fall into three broad categories: (1) those not affected by any of the antagonists (e.g. glycine); (2) those affected by only bicuculline and picrotoxin (e.g. GABA) and (3) those affected by all three antagonists (e.g. /J-alanine). n addition, glutamic acid, an acidic amino acid, was not blocked by any of the antagonists and thus falls into a separate category. The significance of these results in relation to the participation of amino acids in the generation of dorsal root potentials in frog spinal cord is considered in the following paper (Barker, Nicoll & Padjen, 1975). We wish to thank Dr F. E. Bloom and Sir John C. Eccles for the helpful criticism of the manuscript, and Mr H. Poole for prints. REFERENCES BARKER, J. L. & NcOLL, R. A. (1972). GABA; role in primary afferent depolarization. Science N.Y. 176, BARKER, J. L. & NicouL, R. A. (1973). The pharmacology and ionic dependency of amino acid responses in the frog spinal cord. J. Physiol. 228, BARKER, J. L., NicouL, R. A. & PADJEN, A. (1975). Studies on convulsants in the isolated frog spinal cord.. Effects on root potentials. J. Physiol. 245, BOWERY, N. G. & BROWN, D. A. (1974). Depolarizing actions of y-aminobutyric acid and related compounds on rat superior cervical ganglia in vitro. Br. J. Pharmac. 5, CRTS, D. R., DGGAN, A. W. & JOHNSTON, G. A. R. (1971). The specificity of strychnine as a glycine antagonist in the mammalian spinal cord. Expl Brain Res. 12, CRTS, D. R., DGGAN, A. W., FELX, D. & JOHNSTON, G. A. R. (1971). Bicuculline, an antagonist of GABA and synaptic inhibition in the spinal cord of the cat. Brain Res. 32, CRTS, D. R., DGGAN, A. W., FELX, D., JOHNSTON, G. A. R. & McLENNAN, H. (1971). Antagonism between bicuculline and GABA in the cat brain. Brain Res. 33, CRTS, D. R. & FELX, D. (1971). The effect of bicuculline upon synaptic inhibition in the cerebral and cerebellar cortices of the cat. Brain Res. 34, CRTS, D. R., HoSL, L. & JOHNSTON, G. A. R. (1968). A pharmacological study of the depression of spinal neurones by glycine and related amino acids. Expl Brain Res. 6, CRTS, D. R., PHLLS, J. W. & WATKNS, J. C. (1961). Actions of amino acids on the isolated hemisected spinal cord of the toad. Br. J. Pharmac. Chemother. 16, CuRTis, D. R. & WATKNS, J. C. (196). The excitation and depression of spinal neurones by structurally related amino acids. J. Neurochem. 6,

15 CONVLSANTS, AMNO ACDS AND SPNAL CORD 535 DAVDOFF, R. A. (1972a). Gamma-aminobutyric acid antagonism and presynaptic inhibition in the frog spinal cord. Science, N.Y. 175, DAVDOFF, R. A. (1972b). The effect of bicuculline on the isolated spinal cord of the frog. Expl Neurol. 35, DEGROAT, W. C., LA.LEY, P. M. & SAM, W. R. (1972). Depolarization of dorsal root ganglia in the cat by GABA and related amino acids antagonism by picrotoxin and bicuculline. Brain Res. 44, DowsoN, R. J. & SHERWOOD, P. N. R. (1973). The effect of low concentrations of L-glutamate and L-aspartate on transmitter release at the lowest excitatory nervemuscle synapse. J. Physiol. 229, 13-14P. DDEL, J. (1965). Presynaptic and postsynaptic effect of inhibitory drugs on the crayfish neuromuscular junction. Pfiitgers Arch. ge8. Physiol. 283, EDWARDS, C. & KFFLER, S. W. (1959). The blocking effect of y-aminobutyric acid (GABA) and the action of related compounds on single nerve cells. J. Neurochem. 4, FLOREY, E. & WOODcocK, B. (1968). Presynaptic excitatory action of glutamate applied to crab nerve-muscle preparations. Comp. Biochem. Physiol. 26, GAMNDO, A. (1969). GABA-picrotoxin interaction in the mammalian central nervous system. Brain Res. 14, GODFRAND, J. M., KRNJEV6, K., MARETC, H. & PMAN, R. (1973). nhibition of cortical neurones by imidazole and some derivatives. Can. J. Physiol. Pharmacol. 51, HAAS, H. L., ANDERSON, E. G. & HOSL, L. (1972). Histamine and metabolites: Their effects and interactions with convulsants on brain stem neurones. Brain Res. 51, VERSEN, L. L. & JOHNSTON, G. A. R. (1971). GABA uptake in rat central nervous system: Comparison of uptake in slices and homogenates and the effects of some inhibitors. J. Neurochem. 18, KERKT, G. A. & WALKER, R. J. (1967). The effect of iontophoretic injection of glutamic acid and y-amino-n-butyric acid on the miniature end-plate potentials and contractures of the coaxial muscles of the cockroach Periplaneta americana L. Comp. Biochem. Physiol. 2, KoKETS, K., KARCZMAR, A. G. & KTAMRA, R. (1969). Acetylcholine depolarization of the dorsal root nerve terminals in the amphibian spinal cord. nt. J. Neuropharmac. 8, KRNJEV6, K. & SCHWARTZ, S. (1967). The action of y-aminobutyric acid on cortical neurones. Expl Brain Res. 3, McGEER, E. G., McGEER, P. L. & McLENNAN, H. (1961). The inhibitory action of 3-hydroxytyramine, gamma-aminobutyric acid (GABA) and some other compounds toward the crayfish stretch receptor neuron. J. Neurochem. 8, NcoLL, R. A. (1971). Pharmacological evidence for GABA as the transmitter in granule cell inhibition in the olfactory bulb. Brain Res. 35, NcOLL, R. A. & BARKER, J. L. (1973). Strychnine: effect on dorsal root potentials and amino acid responses in the frog spinal cord. Nature, New Biol. 246, NSH, S., MNOTA, S. & KARCZMAR, A. G. (1973). The GABA-mediated depolarization of primary afferent neurons. Physiologist 16, 41. NSH, S., SOEDA, H. & KOKETS, K. (1965). Effect of alkali-earth cations on frog spinal ganglion cell. J. Neurophysiol. 28, OBATA, K. (1972). Acetylcholine and gamma-aminobutyric acid action on tissue cultured cells from sympathetic ganglion, dorsal root ganglion and diaphragm muscle. Fedn Proc

16 536 J. L. BARKER, R. A. NCOLL AND A. PADJEN OBATA, K. & HGHSTEN, S. M. (197). Blocking by picrotoxin of both vestibular inhibition and GABA action on rabbit oculomotor neurones. Brain R"s. 18, OBATA, K., TO, M., OCH, R. & SATO, N. (1967). Pharmacological properties of postsynaptic inhibition by Purkinje cell axons and the action of y-aminobutyric acid on Deiters neurons. Expl Brain Res. 4, PHLLS, J. W., TEBECS, A. V. & YORE, D. H. (1968). Histamine and some antihistamines: Their action on cerebral cortical neurones. Br. J. Pharmac. 33, SWAGEL, M. W., KEDA, K. & ROBERTS, E. (1973). Effects of GABA imidazoleacetic acid, and related substances on conductance of crayfish abdominal stretch receptor. Nature, New Biol. 246, TEBECS, A. K. & PHASus, J. W. (1969). The use of convulsants in studying possible functions of amino acids in the toad spinal cord. (Jomp. Biochem. Physiol. 28, SHERWOOD, P. N. R. & MACHLl, P. (1966). Chemical transmission at the insect excitatory neuromuscular synapse. Nature, Lond. 21, WERMAN, R., DAVDOFF, R. A. & APRsoN, M. H. (1968). nhibitory action of glycine on spinal neurons in the cat. J. Neurophysiol. 31,