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Actions of microiontophoretically applied ibotenate on cat spinal interneuaones D e p a r t ~ ~ l e noft R e ~ e a r c hin An~lesthrsia,McGill Llnivsrsfty, Montreal, P.Q., Canada H3G H Y6 Received June 2. 1977
MACDONALD, J. F., and NISTRI,A. 1977. Actions of microiontophoretically applied ibatenate on cat spinal interneurones. Can. J. Physiol. Pharmacol. 55,965-967. Micrsiontophoretic applications of ibstenate, an amino acid analogue of glutamate, evoked biphasic responses from single dorsal horn interneurones of the cat spinal w r d , i.e., an initial increase in the firing rate followed by a sustained depression of spontaneous firing. A reduced cell sensitivity to excitatory amino acids or peripheral field stimulation was also found during the ibotenate-induced depression. These effects were not praduced by glutamate, quisqualate. o r kainate, although occasional transient reductions of spontaneous eel1 firing were observed after application af glutamate. It is suggested that ibotenate might act on inhibitory as well as excitatory receptors of cat spinal interne~iranes.
Iwtraducltiora Ibotenate, a rigidly extended analogue of Lglutamate, is supposed to activate a receptor which binds glutamate in an extended conforrnation (Buu el ak. 1976). In invertebrate preparations ibotenate activates a population of glutarnatc receptors mediating hyperpolarization of locust muscle fibres (cull-candy 1976) and of some molluscan central neurones (Walker et ak. 197%). In the cat spinal cord, however, the iontophoretic application of ibotenate has been reported to cause, exclusively, excitation of interneurones and Renshaw cells (Johnston et al. 1968). In the course of our experiments, carried out to determine the relative potencies of various glutamate analogues, we observed that, in addition to its excitatory actions on spinal interneurones, the iontophoretic application sf ibotenate evoked a long-lasting depression of spinal interneurones. This finding clearly differentiated ibotenate from other glutamate analogues. We present a preliminary report of this action of ibotenate. Methods Six cats were anaesthetized with intraperitoneal injections of Dial (Ciba) sected at the level of the lumbar cord was exposed activity of 27 single dorsal
and their cords were tranfirst lumbar vertebra. The and the extracellular spike horn interneurones (identi-
'Please address correspondence to: Dr. J. F. MacDonald, Department of Research in Anaesthesia, McGill University, McIntyre Medical Building, 3655 Drumrnond Street, Montreal, P.Q., Canada H3G IY6.
fied by depth and receptive field) was studied by conventional techniques (KrnjeviC and Phillis 1963) using multi-barrelled electrodes that permitted the iontophoretic application s f L-glutamate (Sigma), quisqualate (gift of Professor T. Takernoto), kainate (Sigma), and ibotenate (gift of Professor C Eugster) (barrel concentrations in the range from 0.1 to o,5 and adjusted to pH 8 ) .
Results The iontophoretic application of ibotenate evoked an excitatory response (increased firing rate) of greater latency and duration than that evoked by glutamate (Fig. 1B). Repetitive applications of constant ibotenate pulses were associated with a progressive reduction in the excitatory responses (but not in spike height) of spinal interneurones, as shown in Fig. 1A in the case of a nonspontaneously active unit. It is important to note that repeated iontophoretic applications of glutamate, quisqualate, or kainate produced neuronal responses which did not decline. Furthermore, during the loss of ibotenate responses the interneurones had a higher threshold for excitatory effects produced by either glutamate and related agents or peripheral field stimulation. When spontaneously active neurones were examined a clear biphasic response to ibotenate was apparent: excitation followed by depression (Fig. IB). The excitatory response to a prolonged (510 min) application of ibotenate was not maintained but gradually faded until spike activity disappeared (Fig. 1 B 9. This depres-
Can. J. Physiol. Pharmacol. Downloaded from www.nrcresearchpress.com by SAVANNAHRIVNATLABBF on 11/17/14 For personal use only.
966
CAN. J.
PHYSIQL.PHARMACOL. VOL. 55,
1977
120 s
FIG. 1. Effect of microiontophoretically applied ibotenate on a dorsal horn interneurone of the cat spinal cord. A. Repeated ejections of constant ibotenate currents (indicated by downward deflections on the top tracing) produced progressively smaller responses (measured as changes in the neurone firing rate, bottom tracing); recovery of the response to ibotenate is shown (obtained 5 min later). R. Three constant glutamate ejections (sce top tracing for currents) were applied before ( u ) and at different times ( c - f ) after a prolonged application of ibotenate ( b ) which evoked an excitatory response (peak similar to that of glutamate response) that gradually faded followed by a sustained depression of spontaneous firing and of sensitivity to glutamate; (d) 8 min, ( e ) 18 min, and ( f ) 30 min after ibotenate application, respectively. Note that the response to the first glutamate ejection of each pulse sequence was smaller than the subsequent two, probably because the glr~tamatebarrel needed to be 'warmed up.' The current used to release ibotenate was smaller than that used for glutamate: however, the ibotenate current had to be applied for a longer time to reach peak of excitation. When this factor is taken into account and 'dose' effect relations are plotted for the two amino acids, ibotenate is found t~ be equipotent t~ glutamate (MacBonald, J. F., and Nistri, A.: unpublished results). Same cell in A and B. Backing currents were 8-10 nA.
sion of activity was accompanied by a reduction in responsiveness to glutamate, quisqualate, or kainatc, and to stimuli applied to the receptive field of the neurone. Neurones with little or no spontaneous activity demonstrated a similar decrease in their sensitivity to amino acids and peripheral stimulation after a prolonged (2-5 inin) ejection of ibotenate. The duration of this depression was related to the amount of ibotenate passed. It is worth mentioning that during prolonged ibotenate
ejections the amplitude of recorded action potentials did not decline but rather showed an increase of 25-30% over the initial amplitude. Applications of quisqualate or kainate for similar periods in the same neurone did not produce such a sustained depression of spsntaneous or glutamate-evoked spike activity; however, occasionally a depression of such activity (four neurones) was noted during the intervals between pulses of glutamate which had clear excitatory actions. These depressions
Can. J. Physiol. Pharmacol. Downloaded from www.nrcresearchpress.com by SAVANNAHRIVNATLABBF on 11/17/14 For personal use only.
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were small and transient compared with those induced by ibotenate, which could reduce unit acitivity for periods of up to 1 h.
Discussion Ibotenate produced excitation of spinal interneurones as did glutamate and other related analogues. but it also had a marked depressant action on the firing rate and excitability, as judged by the sensitivity of the neurone to glutamate and its analogues or to peripheral stimulation. Such a depressant action of ibotenate has not been reported previously, possibly because of its long duration and latency in comparison with that produced by typical inhibitory substances such as GABA and glycine. Therefore, it is possible to mistake the ibotenate depression for loss of the cell (perhaps as a consequence of electrode movement). Furthermore, the biphasic action of ibotenate is best seen in interneurones with a steady rate of spontaneous discharge; such neurones are not easily found and held for long periods. Local anaesthetic actions or excessive depolarization could not account for ibotenateinduced depression as it was accompanied by an increase in spike height ( a finding which might suggest hyperpolarization rather than depolarization) . Furthermore, the reversibility of this depression, its dependence upon the intensity of the current releasing ibotenate, and the observation that the depressed neurones were still capable of responding to excitatory amino acids and peripheral stimulation makes it unlikely that a 'toxic' action was exerted by this amino acid. Biphasic (depolarizing-hyperpolarizing) responses to iontophoretically applied glutamate have been recorded intracellularly from cat spinal motoneurones by Bernardi et ui. ( 1972). From their results and those obtained with invertebrate preparations it is possible to hypothesize that the extracellularly recorded depression produced by ibotenate in cat interneurones was at least partly mediated by activation of inhibitory receptors. Of course other mechanisms (e.g., alterations in the activity of membrane ion pumps) might be responsible for such a prolonged inhibitory ef-
967
fect. Ibotenate might conceivably have reduced spontaneous firing by ( I ) reducing presynaptic transmitter release and (or) by (2) exciting neighbouring inhibitory interneurones. The first hypothesis is unlikely because of the profound increase in peripheral stimulation threshold coupled with an almost total disappearance of glutamate responses; this indirectly suggests a large decrease in neuronal excitability. The second hypothesis is made unlikely by the fact that other chemically related excitatory amino acids (e.g., quisqualate and kainate) did not induce a similar depression of unit firing rate. Unlike locust muscle fibres, where ibotenate activates selectively extrajunctional receptors mediating only hyperpolarizations, spinal interneurones probably possess excitatory glutamate receptors that are also activated by ibotenate. The existence of a glutamate receptor mediating hyperpolarization of vertebrate neurones (Buu et al. 1976) might be revealed by a more extensive examination of the actions of ibotenate.
Acknowledgments We thank Dr. K. KrnjeviC for helpful discussion, Professor C. H. Eugster, Professor T. Takemoto, and Dr. A. Padjen for drug samples, and the Medical Research Council of Canada for financial support. BERNARDI, G., ZIEGLGANSBERGER. W.. HERZ, A., and PUIL,E. 1972. Intracellular studies on the action of L-glutamic acid on spinal neurones in the cat. Brain Res. 39, 523-525. Buu, N. T., PUIL,E., and VANGELDER,N. M. 1976. Receptors for amino acids in excitable tissues. Gen. Pharmacol. 7, 5-14. CULL-CANDY, S. G. 1976. Two types of extrajunctional L-glutamate receptors in lscust muscle fibres. J. Physiol. (London), 255,449-464. JOHNSTON, G. A. R., CWRTIS,D. R., DEGROAT,W. C., and DUC~GAN, A. W. 1968. Central actions of ibotenic acid and muscimol. Biochem. Phamacal. 17, 24882489. KRNJEVIC,K., and PHILLIS,J. W. 1963. Iontophoretic studies of neurones in the mammalian cerebral cortex. J. Physiol. (London), 165,274-304. WALKER, R. J., WOODRUFF, G. N., and KERKUT,6. A. 1971. The effect of ibotenic acid and muscimol on single neurons of the snail, Helix aspersa. Comp. Gen. Pharmacol. 2, 168-174.
Actions of microiontophoretically applied ibotenate on cat spinal interneuaones D e p a r t ~ ~ l e noft R e ~ e a r c hin An~lesthrsia,McGill Llnivsrsfty, Montreal, P.Q., Canada H3G H Y6 Received June 2. 1977
MACDONALD, J. F., and NISTRI,A. 1977. Actions of microiontophoretically applied ibatenate on cat spinal interneurones. Can. J. Physiol. Pharmacol. 55,965-967. Micrsiontophoretic applications of ibstenate, an amino acid analogue of glutamate, evoked biphasic responses from single dorsal horn interneurones of the cat spinal w r d , i.e., an initial increase in the firing rate followed by a sustained depression of spontaneous firing. A reduced cell sensitivity to excitatory amino acids or peripheral field stimulation was also found during the ibotenate-induced depression. These effects were not praduced by glutamate, quisqualate. o r kainate, although occasional transient reductions of spontaneous eel1 firing were observed after application af glutamate. It is suggested that ibotenate might act on inhibitory as well as excitatory receptors of cat spinal interne~iranes.
Iwtraducltiora Ibotenate, a rigidly extended analogue of Lglutamate, is supposed to activate a receptor which binds glutamate in an extended conforrnation (Buu el ak. 1976). In invertebrate preparations ibotenate activates a population of glutarnatc receptors mediating hyperpolarization of locust muscle fibres (cull-candy 1976) and of some molluscan central neurones (Walker et ak. 197%). In the cat spinal cord, however, the iontophoretic application of ibotenate has been reported to cause, exclusively, excitation of interneurones and Renshaw cells (Johnston et al. 1968). In the course of our experiments, carried out to determine the relative potencies of various glutamate analogues, we observed that, in addition to its excitatory actions on spinal interneurones, the iontophoretic application sf ibotenate evoked a long-lasting depression of spinal interneurones. This finding clearly differentiated ibotenate from other glutamate analogues. We present a preliminary report of this action of ibotenate. Methods Six cats were anaesthetized with intraperitoneal injections of Dial (Ciba) sected at the level of the lumbar cord was exposed activity of 27 single dorsal
and their cords were tranfirst lumbar vertebra. The and the extracellular spike horn interneurones (identi-
'Please address correspondence to: Dr. J. F. MacDonald, Department of Research in Anaesthesia, McGill University, McIntyre Medical Building, 3655 Drumrnond Street, Montreal, P.Q., Canada H3G IY6.
fied by depth and receptive field) was studied by conventional techniques (KrnjeviC and Phillis 1963) using multi-barrelled electrodes that permitted the iontophoretic application s f L-glutamate (Sigma), quisqualate (gift of Professor T. Takernoto), kainate (Sigma), and ibotenate (gift of Professor C Eugster) (barrel concentrations in the range from 0.1 to o,5 and adjusted to pH 8 ) .
Results The iontophoretic application of ibotenate evoked an excitatory response (increased firing rate) of greater latency and duration than that evoked by glutamate (Fig. 1B). Repetitive applications of constant ibotenate pulses were associated with a progressive reduction in the excitatory responses (but not in spike height) of spinal interneurones, as shown in Fig. 1A in the case of a nonspontaneously active unit. It is important to note that repeated iontophoretic applications of glutamate, quisqualate, or kainate produced neuronal responses which did not decline. Furthermore, during the loss of ibotenate responses the interneurones had a higher threshold for excitatory effects produced by either glutamate and related agents or peripheral field stimulation. When spontaneously active neurones were examined a clear biphasic response to ibotenate was apparent: excitation followed by depression (Fig. IB). The excitatory response to a prolonged (510 min) application of ibotenate was not maintained but gradually faded until spike activity disappeared (Fig. 1 B 9. This depres-
Can. J. Physiol. Pharmacol. Downloaded from www.nrcresearchpress.com by SAVANNAHRIVNATLABBF on 11/17/14 For personal use only.
966
CAN. J.
PHYSIQL.PHARMACOL. VOL. 55,
1977
120 s
FIG. 1. Effect of microiontophoretically applied ibotenate on a dorsal horn interneurone of the cat spinal cord. A. Repeated ejections of constant ibotenate currents (indicated by downward deflections on the top tracing) produced progressively smaller responses (measured as changes in the neurone firing rate, bottom tracing); recovery of the response to ibotenate is shown (obtained 5 min later). R. Three constant glutamate ejections (sce top tracing for currents) were applied before ( u ) and at different times ( c - f ) after a prolonged application of ibotenate ( b ) which evoked an excitatory response (peak similar to that of glutamate response) that gradually faded followed by a sustained depression of spontaneous firing and of sensitivity to glutamate; (d) 8 min, ( e ) 18 min, and ( f ) 30 min after ibotenate application, respectively. Note that the response to the first glutamate ejection of each pulse sequence was smaller than the subsequent two, probably because the glr~tamatebarrel needed to be 'warmed up.' The current used to release ibotenate was smaller than that used for glutamate: however, the ibotenate current had to be applied for a longer time to reach peak of excitation. When this factor is taken into account and 'dose' effect relations are plotted for the two amino acids, ibotenate is found t~ be equipotent t~ glutamate (MacBonald, J. F., and Nistri, A.: unpublished results). Same cell in A and B. Backing currents were 8-10 nA.
sion of activity was accompanied by a reduction in responsiveness to glutamate, quisqualate, or kainatc, and to stimuli applied to the receptive field of the neurone. Neurones with little or no spontaneous activity demonstrated a similar decrease in their sensitivity to amino acids and peripheral stimulation after a prolonged (2-5 inin) ejection of ibotenate. The duration of this depression was related to the amount of ibotenate passed. It is worth mentioning that during prolonged ibotenate
ejections the amplitude of recorded action potentials did not decline but rather showed an increase of 25-30% over the initial amplitude. Applications of quisqualate or kainate for similar periods in the same neurone did not produce such a sustained depression of spsntaneous or glutamate-evoked spike activity; however, occasionally a depression of such activity (four neurones) was noted during the intervals between pulses of glutamate which had clear excitatory actions. These depressions
Can. J. Physiol. Pharmacol. Downloaded from www.nrcresearchpress.com by SAVANNAHRIVNATLABBF on 11/17/14 For personal use only.
COMMUNICATIONS
were small and transient compared with those induced by ibotenate, which could reduce unit acitivity for periods of up to 1 h.
Discussion Ibotenate produced excitation of spinal interneurones as did glutamate and other related analogues. but it also had a marked depressant action on the firing rate and excitability, as judged by the sensitivity of the neurone to glutamate and its analogues or to peripheral stimulation. Such a depressant action of ibotenate has not been reported previously, possibly because of its long duration and latency in comparison with that produced by typical inhibitory substances such as GABA and glycine. Therefore, it is possible to mistake the ibotenate depression for loss of the cell (perhaps as a consequence of electrode movement). Furthermore, the biphasic action of ibotenate is best seen in interneurones with a steady rate of spontaneous discharge; such neurones are not easily found and held for long periods. Local anaesthetic actions or excessive depolarization could not account for ibotenateinduced depression as it was accompanied by an increase in spike height ( a finding which might suggest hyperpolarization rather than depolarization) . Furthermore, the reversibility of this depression, its dependence upon the intensity of the current releasing ibotenate, and the observation that the depressed neurones were still capable of responding to excitatory amino acids and peripheral stimulation makes it unlikely that a 'toxic' action was exerted by this amino acid. Biphasic (depolarizing-hyperpolarizing) responses to iontophoretically applied glutamate have been recorded intracellularly from cat spinal motoneurones by Bernardi et ui. ( 1972). From their results and those obtained with invertebrate preparations it is possible to hypothesize that the extracellularly recorded depression produced by ibotenate in cat interneurones was at least partly mediated by activation of inhibitory receptors. Of course other mechanisms (e.g., alterations in the activity of membrane ion pumps) might be responsible for such a prolonged inhibitory ef-
967
fect. Ibotenate might conceivably have reduced spontaneous firing by ( I ) reducing presynaptic transmitter release and (or) by (2) exciting neighbouring inhibitory interneurones. The first hypothesis is unlikely because of the profound increase in peripheral stimulation threshold coupled with an almost total disappearance of glutamate responses; this indirectly suggests a large decrease in neuronal excitability. The second hypothesis is made unlikely by the fact that other chemically related excitatory amino acids (e.g., quisqualate and kainate) did not induce a similar depression of unit firing rate. Unlike locust muscle fibres, where ibotenate activates selectively extrajunctional receptors mediating only hyperpolarizations, spinal interneurones probably possess excitatory glutamate receptors that are also activated by ibotenate. The existence of a glutamate receptor mediating hyperpolarization of vertebrate neurones (Buu et al. 1976) might be revealed by a more extensive examination of the actions of ibotenate.
Acknowledgments We thank Dr. K. KrnjeviC for helpful discussion, Professor C. H. Eugster, Professor T. Takemoto, and Dr. A. Padjen for drug samples, and the Medical Research Council of Canada for financial support. BERNARDI, G., ZIEGLGANSBERGER. W.. HERZ, A., and PUIL,E. 1972. Intracellular studies on the action of L-glutamic acid on spinal neurones in the cat. Brain Res. 39, 523-525. Buu, N. T., PUIL,E., and VANGELDER,N. M. 1976. Receptors for amino acids in excitable tissues. Gen. Pharmacol. 7, 5-14. CULL-CANDY, S. G. 1976. Two types of extrajunctional L-glutamate receptors in lscust muscle fibres. J. Physiol. (London), 255,449-464. JOHNSTON, G. A. R., CWRTIS,D. R., DEGROAT,W. C., and DUC~GAN, A. W. 1968. Central actions of ibotenic acid and muscimol. Biochem. Phamacal. 17, 24882489. KRNJEVIC,K., and PHILLIS,J. W. 1963. Iontophoretic studies of neurones in the mammalian cerebral cortex. J. Physiol. (London), 165,274-304. WALKER, R. J., WOODRUFF, G. N., and KERKUT,6. A. 1971. The effect of ibotenic acid and muscimol on single neurons of the snail, Helix aspersa. Comp. Gen. Pharmacol. 2, 168-174.
