J. Phy8iol. (1975), 245, pp. 521-536 With 8 text-figures Printed in Great Britain
521
STUDIES ON CONVULSANTS IN THE ISOLATED FROG SPINAL CORD. I. ANTAGONISM OF AMINO ACID RESPONSES
By J. L. BARKER,* R. A. NICOLL,t AND A. PADJENJ From the Laboratory of Neuropharmacology, National Institute of Mental Health, St Elizabeths Hospital, Washington, D.C. 20032, U.S.A.
(Received 5 April 1974) SUMMARY
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. Imidazoleacetic 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 (10-3 M) concentrations of fl-alanine and taurine were completely blocked by these convulsants, while depolarizations to 103 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 Institute of Child Health and Human Development, Bethesda,
Maryland 20014, U.S.A.
t Present address: Laboratory of Neurobiology, SUNY at Buffalo, Amherst, New York 14226, U.S.A. t Visiting Scientist, on leave from Ruder Boskovic Institute, Zagreb, Yugoslavia.
522 J. L. BARKER, R. A. NICOLL 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. INTRODUCTION
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, 1970; 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, Ito, 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. In 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
CONVULSANTS, AMINO ACIDS AND SPINAL 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 200 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 10 hr. The Ringer solution (115 mM-NaCl, 2 mn-CaCl2, 2 mM-KCl, and 10 m Tris (hydroxymethyl)
DR&
DR,
R2
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 100% oxygen. This solution will be referred to as a 'normal Ringer'. When drug responses were examined, 20 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 (0 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 KCI 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 45-60 min were required for the sucrose gap to completely form. After this period the drug potentials remained constant.
Structure-activity 8tudiea. Using GABA in concentrations between 5 x 104 and 2-5 x 10- 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
524
J. L. BARKER, R. A. NICOLL 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; GLU = glutamic acid; GLY = glycine; IMA = imidoazolacetic acid; PRO = proline; TAUR = taurine.
RESULTS
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 20 mM-MgSO4, which entirely blocked synaptic transmission. The addition of Mg ions slightly but A BALA
Control GLU GABA
20 mM-Mg""
Control BALA GABA GLU
Picrotoxin
I2 mV 5 min
Fig. 2. The effect of Mg and picrotoxin on amino acid responses in normal Ringer solution. A: addition of 20 mx-MgSO4 to the normal Ringer solution reduces the size of the glutamate depolarization. 2-5 x 10-3 M concentration of amino acids used. In B, 5 x 104 M picrotoxin reduces all of the amino acid responses in normal Ringer including glutamate to some extent. Concentration of amino acids 2-5 x 10-3 M; calibration is the same in A and B.
significantly increased the size of the GABA (1 10 8 0/ + 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
CONVULSANTS, AMINO ACIDS AND SPINAL 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 I2 mV 5 min
GLY BALA GABA DAV EAC
10 mMl mm 1 mm 1 mm 10 mm
100
cis
B
0-50/
0
4 1 2 3 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;
J. L. BARKER, R. A. NICOLL AND A. PADJEN
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528 J. L. BARKER, R. A. NICOLL 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, 1960). 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
B
TAUR BALA
~
~
~
IMA
H
Picrotoxin (10-4 M)
3 mV
6 mV
5 min
Fig. 4. Picrotoxin antagonism of depolarizing responses to amino acids. A: control responses: The amino acids (10-3M) were applied until they produced a maximal response. Duration of application is indicated by bar. B: record B was 20 min after 104M picrotoxin. It is evident that the responses to all but glutamate (GLU) and glycine (GLY) are either reduced or abolished in the presence of picrotoxin. Note different gain of IMA 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).
j~ ~01
CONVULSANTS, AMINO ACIDS AND SPINAL 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
521
STUDIES ON CONVULSANTS IN THE ISOLATED FROG SPINAL CORD. I. ANTAGONISM OF AMINO ACID RESPONSES
By J. L. BARKER,* R. A. NICOLL,t AND A. PADJENJ From the Laboratory of Neuropharmacology, National Institute of Mental Health, St Elizabeths Hospital, Washington, D.C. 20032, U.S.A.
(Received 5 April 1974) SUMMARY
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. Imidazoleacetic 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 (10-3 M) concentrations of fl-alanine and taurine were completely blocked by these convulsants, while depolarizations to 103 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 Institute of Child Health and Human Development, Bethesda,
Maryland 20014, U.S.A.
t Present address: Laboratory of Neurobiology, SUNY at Buffalo, Amherst, New York 14226, U.S.A. t Visiting Scientist, on leave from Ruder Boskovic Institute, Zagreb, Yugoslavia.
522 J. L. BARKER, R. A. NICOLL 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. INTRODUCTION
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, 1970; 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, Ito, 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. In 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
CONVULSANTS, AMINO ACIDS AND SPINAL 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 200 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 10 hr. The Ringer solution (115 mM-NaCl, 2 mn-CaCl2, 2 mM-KCl, and 10 m Tris (hydroxymethyl)
DR&
DR,
R2
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 100% oxygen. This solution will be referred to as a 'normal Ringer'. When drug responses were examined, 20 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 (0 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 KCI 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 45-60 min were required for the sucrose gap to completely form. After this period the drug potentials remained constant.
Structure-activity 8tudiea. Using GABA in concentrations between 5 x 104 and 2-5 x 10- 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
524
J. L. BARKER, R. A. NICOLL 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; GLU = glutamic acid; GLY = glycine; IMA = imidoazolacetic acid; PRO = proline; TAUR = taurine.
RESULTS
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 20 mM-MgSO4, which entirely blocked synaptic transmission. The addition of Mg ions slightly but A BALA
Control GLU GABA
20 mM-Mg""
Control BALA GABA GLU
Picrotoxin
I2 mV 5 min
Fig. 2. The effect of Mg and picrotoxin on amino acid responses in normal Ringer solution. A: addition of 20 mx-MgSO4 to the normal Ringer solution reduces the size of the glutamate depolarization. 2-5 x 10-3 M concentration of amino acids used. In B, 5 x 104 M picrotoxin reduces all of the amino acid responses in normal Ringer including glutamate to some extent. Concentration of amino acids 2-5 x 10-3 M; calibration is the same in A and B.
significantly increased the size of the GABA (1 10 8 0/ + 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
CONVULSANTS, AMINO ACIDS AND SPINAL 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 I2 mV 5 min
GLY BALA GABA DAV EAC
10 mMl mm 1 mm 1 mm 10 mm
100
cis
B
0-50/
0
4 1 2 3 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;
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528 J. L. BARKER, R. A. NICOLL 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, 1960). 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
B
TAUR BALA
~
~
~
IMA
H
Picrotoxin (10-4 M)
3 mV
6 mV
5 min
Fig. 4. Picrotoxin antagonism of depolarizing responses to amino acids. A: control responses: The amino acids (10-3M) were applied until they produced a maximal response. Duration of application is indicated by bar. B: record B was 20 min after 104M picrotoxin. It is evident that the responses to all but glutamate (GLU) and glycine (GLY) are either reduced or abolished in the presence of picrotoxin. Note different gain of IMA 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).
j~ ~01
CONVULSANTS, AMINO ACIDS AND SPINAL 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
