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The effects of strychnine on the inhibition of interneurons by glycine and γ-aminobutyric acid
Inr. J. Neuropharmac.,
1969, 8, 191-194
Pergamon
Press.
Printed
in Ct. Britain.
PRELIMINARY
NOTE
THE EFFECTS OF STRYCHNINE ON THE INHIBITION OF INTERNEURONS BY GLYCINE AND r-AMINOBUTYRIC ACID* R. A. DAVIDOFF,M. H. APRISONand R. WERMAN The Institute of Psychiatric Research, The Department of Psychiatry, Neurology and Biochemistry The Veterans Administration Hospital, Indiana University Medical Center, Indianapolis, Indiana
and
(Accepted 12 November 1968) Summary-The effects of strychnine on the inhibition of spinal interneurons produced by glycine and GABA was studied by means of iontophoretic application from multibarrel electrodes. The actions of glycine were more effectively blocked by strychnine than were those of GABA. The data suggest that a non-competitive type of antagonism exists between strychnine and these inhibitory amino acids.
A FUNDAMENTAL condition proposed for the identification of a putative inhibitory transmitter in the vertebrate spinal cord has been antagonism by strychnine (CURTIS,1963, 1968; ECCLES, 1964). It has been commonly accepted, but never proven, that strychnine blocks inhibition by competing for the same receptor on the inhibitory postsynaptic membrane of the neuron as does the inhibitory transmitter, i.e. that a competitive antagonism corresponding to the effect of curare at the vertebrate neuromuscular junction occurs at the inhibitory synapse (BRADLEYet al., 1953; ECCLES,1957, 1964). CURTISand his associates (1968) have reported that the inhibition of firing of interneurons by iontophoretically administered glycine could be reversibly blocked by strychnine, while the depression produced by GABA (gamma-aminobutyric acid) was unaffected. The differential effect of this alkaloid on the two amino acids was used to satisfy the criterion of pharmacological identity for the inhibitory transmitter. Previous work from our laboratories had led to the proposal that glycine could be an inhibitory transmitter on the basis of its distribution in spinal cord (APRISONand WERMAN,1965), its association with interneurons (DAVIDOFFet al., 1967) and its action on the inhibitory postsynaptic membrane (WERMANet al., 1968). We have recently re-investigated the effect of strychnine on interneurons and its interactions with glycine and GABA and have found that the qualitative dichotomy between GABA and glycine reported by CURTIS(1968) usually does not hold, but instead there is only a quantitative difference in the interaction of strychnine and each of these amino acids. All experiments were performed on cats decerebrated by electrolytic transection of the midbrain during ether anesthesia. Amino acids and other substances were administered iontophoretically from five-barrel micropipettes as previously described (WERMANet al., *Supported in part by U.S. Public Health Service Grants NP 07301-02 and MH 03225-09 and Career Development Award NB 14,8 15-04. 191 H
192
R. A. DAVIDOFF, M. H. APRISON and R. WERMAN
1968). Extracellular spike potentials of L7 interneurons, either spontaneous or evoked by iontophoreti~ release of glutamate, were recorded from the central barrel of the electrode assembly and the rate of firing counted by means of an electronic counter coupled to a Grass ink writing oscillograph. Changes in cell firing were assessed by comparing the average firing frequency for ten consecutive I set periods before and during the application of inhibitory amino acids. Measurements of the latter frequency were generally made at sufficient time after the onset of the iontophoretic current so that the effect of the administered amino acid was constant, It is assumed that the amount of compound discharged from a given micropipette is proportional to the charge transferred (KRNJEVIC et al., 1963). The amounts of amino acid and strychnine administered are given in nanoamps (nA) of current passed through an electrode and therefore only relative amounts of a given substance are known. Furthermore, no comparison was made between the data collected from different neurons because of differences in transport number and electrode-cell geometric relationships when recording from different cells with different electrodes. Log dose-response curves for each amino acid before and during the administration of strychnine were plotted from the data obtained from a given neuron. Almost without exception, spinal interneurons were inhibited by both glycine and GABA. were more sensitive to As previously reported (WERMAN et al., 1968) most interneurons glycine than to GABA-smaller amounts of iontophoretic current had to be passed through the glycine microelectrode than through the GABA electrode to produce maximal inhibition of cell firing. In many cases, however, both compounds appeared to be equally potent as can be seen from the control log dose-response curves (filled circles) in Figure 1A and B where the inhibition of neuron firing produced by increasing amounts of amino acid were plotted against the logarithm of the nanoamps passed through the glycine or GABA electrode. There is a graded response to both amino acids which gradually increases with the amount of iontophoretic current and approaches some plateau level asymptotically; in this case approximately 80 y0 inhibition of firing. In confirmation of the work of CURTIS and his colleagues (1968) we found that electrophoretically applied strychnine (20 mlM strychnine HCI in 165 mM NaCl, pH 3.5) blocked the glycine-produced inhibition of interneuron firing. However, we found that the action of GABA was also usually decreased by the concomitant administration of strychnine. But a quantitative difference was almost always seen-strychnine blocked the effects of glycine more completely than those produced by GABA. For example, in Figure I, 30 nA of strychnine (closed triangles) reduced the maximal action of glycine to 5 % of control levels while the action of GABA was reduced to only 30%. Similar results were found in spontaneously firing neurons. It was clear (cf. Fig. 1) that the effects produced by strychnine were monotonically related to the amount of alkaloid released by iontophoresis. An increase in the amount of amino acid released could not overcome the decreased maximal inhibition produced by strychnine. This is shown in Figure 1. The decreases in the plateau levels and in the slopes of the straight-line portions of the log dose-response curves were directly related to the dose of strychnine. This type of interaction is the type that one would expect in a non-competitive antagonism (ARI~~NSand SIMONIS, 1964). If a competitive antagonism existed between strychnine and the inhibitory amino acids then increasing doses of glycine and GABA would result in a displacement of strychnine from receptors and the log dose-response curves would be shifted but parallel. The plateau values in a competitive antagonism would be the same in both the presence and absence of
The effects of strychnine on the inhibition of interneurons
i.0
1.5
by glycine and y-aminobutyric
acid
193
Z;O log nA
FIG. 1. Antagonistic action of strychnine on the inhibition of firing of a single interneuron (mean rate 46 spikes/xc, glutamate iontophoretic current 62 nA) produced by iontophoresis
of glycine (A) and GABA (B). The abscissa is scaled in log nA of amino acid iontophoretic current. The ordinate is scaled in percent inhibition (decrease in number of spikes/set produced by amino acid divided by control firing rate). 0 Control log dose-response curve; 0 during iontophoresis of strychnine 10 nA; A strychnine 20 nA; A strychnine 30 nA.
antagonist; this was not seen in our data (Fig. 1). Our results would therefore seem to be incompatible with the competitive theory of the action of strychnine (BRADLEY et al., 1953; ECCLES, 1957, 1964). It would be of interest to know whether the difference in results between the present is due to differences in the type of cat preparation data and those of Curms and collaborators used. We avoided the use of barbiturates and noted that in earlier preliminary experiments that more variability was obtained when these agents were employed. The interactions of barbiturates and amino acids are still to be studied. Acknowledgements-The acknowledged.
skilful technical assistance of Mr. L. THORNTONand Mr. C. HANCOCKis gratefully
REFERENCES APRISON,M. H. and WERMAN,R. (1965). The distribution of glycine in cat spinal cord and roots. Life &i. 4: 2075-2083. ARI~NS,E. J. and SIMONIS,A. M. (1964). A molecular basis for drug action. J. Pharm. Pharmac. 16: 137-157, 289-312.
194
R. A. DAVIDOFF,M. H. APRISONand R. WERMAN
BRADLEY,K., EASTON,D. M. and ECCLPS,J. C. (1953). An inv~tigation of primary or direct inhibition. J. Physio/., Land. 122: 474-488. CURTIS,D. R. (1963). The pharm~olo~ of central and peripheral inhibition. ~~~~/?~~c.Rev. 15: 333-364. CURTIS, D. R. (1968). Pharmacology and neur~hemistry of mammalian central inhibitory processes. In Srructure and Function of Inhibitory Neuronal Mechanisms (VON EULER, C., SKOGLUND,S. and SODERBERG,U., Eds.), p. 429-456. Pergamon, Oxford. CURTIS,D. R., H~SLI, L. and JOHNSTON,G. A. R. (1968). A pharmacological study of the depression of spinal neurons by glycine and related amino acids. Exp. Brain Res. 6: l-18. DAVIDOFF,R. A., GRAHAM,L. T., JR., SHANK,R. P., WERMAN,R. and APRISON,M. H. (1967). Changes in amino acid concentrations associated with loss of spinal interneurons. J. Neurochem. 14: 102.5-1031. ECCLES,J. C. (1957). The Physiolog.v of Nerve Cells. Johns Hopkins Press, Baltimore. 270 pp. ECCLES,J. C. (1964). The .PI+o~og~ ofsynapses. Springer, Berlin. 316 pp. KRNJEVIC,K., LAVERX, R. and SHARMAN,D. F. (1963). Iontophoretic release of adrenaline, noradrenaline and 5-hydroxytryptarnine from micropipettes. Br. J. Pharmac. 20: 491496. WERMAN,R., DAMDOFF,R. A. and APRISON,M. H. (1968). The inhibitory action of glycine on spinal neurons in the cat. J. Neuraphysiol. 31: 81-95.
1969, 8, 191-194
Pergamon
Press.
Printed
in Ct. Britain.
PRELIMINARY
NOTE
THE EFFECTS OF STRYCHNINE ON THE INHIBITION OF INTERNEURONS BY GLYCINE AND r-AMINOBUTYRIC ACID* R. A. DAVIDOFF,M. H. APRISONand R. WERMAN The Institute of Psychiatric Research, The Department of Psychiatry, Neurology and Biochemistry The Veterans Administration Hospital, Indiana University Medical Center, Indianapolis, Indiana
and
(Accepted 12 November 1968) Summary-The effects of strychnine on the inhibition of spinal interneurons produced by glycine and GABA was studied by means of iontophoretic application from multibarrel electrodes. The actions of glycine were more effectively blocked by strychnine than were those of GABA. The data suggest that a non-competitive type of antagonism exists between strychnine and these inhibitory amino acids.
A FUNDAMENTAL condition proposed for the identification of a putative inhibitory transmitter in the vertebrate spinal cord has been antagonism by strychnine (CURTIS,1963, 1968; ECCLES, 1964). It has been commonly accepted, but never proven, that strychnine blocks inhibition by competing for the same receptor on the inhibitory postsynaptic membrane of the neuron as does the inhibitory transmitter, i.e. that a competitive antagonism corresponding to the effect of curare at the vertebrate neuromuscular junction occurs at the inhibitory synapse (BRADLEYet al., 1953; ECCLES,1957, 1964). CURTISand his associates (1968) have reported that the inhibition of firing of interneurons by iontophoretically administered glycine could be reversibly blocked by strychnine, while the depression produced by GABA (gamma-aminobutyric acid) was unaffected. The differential effect of this alkaloid on the two amino acids was used to satisfy the criterion of pharmacological identity for the inhibitory transmitter. Previous work from our laboratories had led to the proposal that glycine could be an inhibitory transmitter on the basis of its distribution in spinal cord (APRISONand WERMAN,1965), its association with interneurons (DAVIDOFFet al., 1967) and its action on the inhibitory postsynaptic membrane (WERMANet al., 1968). We have recently re-investigated the effect of strychnine on interneurons and its interactions with glycine and GABA and have found that the qualitative dichotomy between GABA and glycine reported by CURTIS(1968) usually does not hold, but instead there is only a quantitative difference in the interaction of strychnine and each of these amino acids. All experiments were performed on cats decerebrated by electrolytic transection of the midbrain during ether anesthesia. Amino acids and other substances were administered iontophoretically from five-barrel micropipettes as previously described (WERMANet al., *Supported in part by U.S. Public Health Service Grants NP 07301-02 and MH 03225-09 and Career Development Award NB 14,8 15-04. 191 H
192
R. A. DAVIDOFF, M. H. APRISON and R. WERMAN
1968). Extracellular spike potentials of L7 interneurons, either spontaneous or evoked by iontophoreti~ release of glutamate, were recorded from the central barrel of the electrode assembly and the rate of firing counted by means of an electronic counter coupled to a Grass ink writing oscillograph. Changes in cell firing were assessed by comparing the average firing frequency for ten consecutive I set periods before and during the application of inhibitory amino acids. Measurements of the latter frequency were generally made at sufficient time after the onset of the iontophoretic current so that the effect of the administered amino acid was constant, It is assumed that the amount of compound discharged from a given micropipette is proportional to the charge transferred (KRNJEVIC et al., 1963). The amounts of amino acid and strychnine administered are given in nanoamps (nA) of current passed through an electrode and therefore only relative amounts of a given substance are known. Furthermore, no comparison was made between the data collected from different neurons because of differences in transport number and electrode-cell geometric relationships when recording from different cells with different electrodes. Log dose-response curves for each amino acid before and during the administration of strychnine were plotted from the data obtained from a given neuron. Almost without exception, spinal interneurons were inhibited by both glycine and GABA. were more sensitive to As previously reported (WERMAN et al., 1968) most interneurons glycine than to GABA-smaller amounts of iontophoretic current had to be passed through the glycine microelectrode than through the GABA electrode to produce maximal inhibition of cell firing. In many cases, however, both compounds appeared to be equally potent as can be seen from the control log dose-response curves (filled circles) in Figure 1A and B where the inhibition of neuron firing produced by increasing amounts of amino acid were plotted against the logarithm of the nanoamps passed through the glycine or GABA electrode. There is a graded response to both amino acids which gradually increases with the amount of iontophoretic current and approaches some plateau level asymptotically; in this case approximately 80 y0 inhibition of firing. In confirmation of the work of CURTIS and his colleagues (1968) we found that electrophoretically applied strychnine (20 mlM strychnine HCI in 165 mM NaCl, pH 3.5) blocked the glycine-produced inhibition of interneuron firing. However, we found that the action of GABA was also usually decreased by the concomitant administration of strychnine. But a quantitative difference was almost always seen-strychnine blocked the effects of glycine more completely than those produced by GABA. For example, in Figure I, 30 nA of strychnine (closed triangles) reduced the maximal action of glycine to 5 % of control levels while the action of GABA was reduced to only 30%. Similar results were found in spontaneously firing neurons. It was clear (cf. Fig. 1) that the effects produced by strychnine were monotonically related to the amount of alkaloid released by iontophoresis. An increase in the amount of amino acid released could not overcome the decreased maximal inhibition produced by strychnine. This is shown in Figure 1. The decreases in the plateau levels and in the slopes of the straight-line portions of the log dose-response curves were directly related to the dose of strychnine. This type of interaction is the type that one would expect in a non-competitive antagonism (ARI~~NSand SIMONIS, 1964). If a competitive antagonism existed between strychnine and the inhibitory amino acids then increasing doses of glycine and GABA would result in a displacement of strychnine from receptors and the log dose-response curves would be shifted but parallel. The plateau values in a competitive antagonism would be the same in both the presence and absence of
The effects of strychnine on the inhibition of interneurons
i.0
1.5
by glycine and y-aminobutyric
acid
193
Z;O log nA
FIG. 1. Antagonistic action of strychnine on the inhibition of firing of a single interneuron (mean rate 46 spikes/xc, glutamate iontophoretic current 62 nA) produced by iontophoresis
of glycine (A) and GABA (B). The abscissa is scaled in log nA of amino acid iontophoretic current. The ordinate is scaled in percent inhibition (decrease in number of spikes/set produced by amino acid divided by control firing rate). 0 Control log dose-response curve; 0 during iontophoresis of strychnine 10 nA; A strychnine 20 nA; A strychnine 30 nA.
antagonist; this was not seen in our data (Fig. 1). Our results would therefore seem to be incompatible with the competitive theory of the action of strychnine (BRADLEY et al., 1953; ECCLES, 1957, 1964). It would be of interest to know whether the difference in results between the present is due to differences in the type of cat preparation data and those of Curms and collaborators used. We avoided the use of barbiturates and noted that in earlier preliminary experiments that more variability was obtained when these agents were employed. The interactions of barbiturates and amino acids are still to be studied. Acknowledgements-The acknowledged.
skilful technical assistance of Mr. L. THORNTONand Mr. C. HANCOCKis gratefully
REFERENCES APRISON,M. H. and WERMAN,R. (1965). The distribution of glycine in cat spinal cord and roots. Life &i. 4: 2075-2083. ARI~NS,E. J. and SIMONIS,A. M. (1964). A molecular basis for drug action. J. Pharm. Pharmac. 16: 137-157, 289-312.
194
R. A. DAVIDOFF,M. H. APRISONand R. WERMAN
BRADLEY,K., EASTON,D. M. and ECCLPS,J. C. (1953). An inv~tigation of primary or direct inhibition. J. Physio/., Land. 122: 474-488. CURTIS,D. R. (1963). The pharm~olo~ of central and peripheral inhibition. ~~~~/?~~c.Rev. 15: 333-364. CURTIS, D. R. (1968). Pharmacology and neur~hemistry of mammalian central inhibitory processes. In Srructure and Function of Inhibitory Neuronal Mechanisms (VON EULER, C., SKOGLUND,S. and SODERBERG,U., Eds.), p. 429-456. Pergamon, Oxford. CURTIS,D. R., H~SLI, L. and JOHNSTON,G. A. R. (1968). A pharmacological study of the depression of spinal neurons by glycine and related amino acids. Exp. Brain Res. 6: l-18. DAVIDOFF,R. A., GRAHAM,L. T., JR., SHANK,R. P., WERMAN,R. and APRISON,M. H. (1967). Changes in amino acid concentrations associated with loss of spinal interneurons. J. Neurochem. 14: 102.5-1031. ECCLES,J. C. (1957). The Physiolog.v of Nerve Cells. Johns Hopkins Press, Baltimore. 270 pp. ECCLES,J. C. (1964). The .PI+o~og~ ofsynapses. Springer, Berlin. 316 pp. KRNJEVIC,K., LAVERX, R. and SHARMAN,D. F. (1963). Iontophoretic release of adrenaline, noradrenaline and 5-hydroxytryptarnine from micropipettes. Br. J. Pharmac. 20: 491496. WERMAN,R., DAMDOFF,R. A. and APRISON,M. H. (1968). The inhibitory action of glycine on spinal neurons in the cat. J. Neuraphysiol. 31: 81-95.