- Email: [email protected]
The excitatory effect of diphenylthiohydantoin on spinal reflex activity
THE EXCITATORY EFFECT OF DIPHENYLT~~OHYDANTOIN ON SPINAL REFLEX ACTIVITY* W. GROSSMANN, 1. JURNA and C. THERES lnstitut fur Pharmakologie und Toxikologie. Universittit des Saarlandes, D-665 Homburg (Saar), W. Germany
(Accepr&13February19?4) Summary-The effect of diphenylthiohydant~in (DPTH) 20 mgjkg on spinal reflexes was studied in cats and rats which were spinalized at the lower thoracic level and also in unspinalized rats. DPTH increased the amplitude of mono- and polysynaptic reflex responses in spinal cats and rats. The amplitude of the early component of the afferent volley to peripheral nerve stimulation was enhanced. In unspinalized rats DPTH increased as well as decreased mono- and poiysynaptic reflex responses. In spinal cats DPTH increased the number of alpha reflex discharges, but left the number of gamma reflex discharges unchanged. The results are discussed with respect to the conflicting findings obtained by other authors when studying the effect of diphenylhydantoin and DPTH on spinal reflex activity.
Diphenylthiohydantoin (DPTH), an anticonvulsant agent acting similarly to diphenylhydantoin (DPH) (SOHN, LEVITT and RAINES,1970). enters and leaves the central nervous system more rapidly than DPH (VEREBELY,KUTT, SOHN, LEVITT and RAINES,1970). Conflicting results have been obtained when the effect of DPH, as well as of DPTH, on spinal reflexes were investigated in different preparations (STILLE, 1955; ESPLIN, 1957; RAINES,SOHNand LEVITT,1971). It was the aim of the present investigation to determine whether the effect of DPTH on mass reflexes is different in spinal and unspinalized preparations. A study of the reflex discharges from c1and y motoneurones in spinal cats was included to assess the effect of DPTH on functionally different motoneurones. METHODS
The experiments were performed on 17 cats (2.3-3-7 kg body wt) and on 10 rats (Wistar strain, 250g body wt) operated under halothane anaesthesia. The spinal cord was transected in all cats and in some of the rats at the level of T12. The ventral roots and (apart from the experiments in which stimulation of peripheral nerves was performed), the dorsal roots LS-S2 were cut and prepared for recording or stimulation. For stimulation of peripheral nerves, the posterior biceps-semitendinosus (PBST), the gastrocnemius-soleus (GS) and the sural nerves were dissected free in five cats. The cats were intercollicularly decerebrated. The rats were kept at a level of anaesthesia which did not interfere with spinal reflex activity. This level was discovered in experiments in which all surgical wounds were instilled with procaine and the animals allowed to recover for a short period from anaesthesia. All preparations were immobilized with gallamine and maintained on artificial respiration The body temperature was maintained between 36.5 and 38°C. * Supported by the Sonderfors~hungsbereic~t 38 “Memhranen” of the Deutsche Forschungsgemeins~haft. 813
814
W.
GROSSMAN. I. JURKA and
C. THERM
Reflexes were elicited by stimulation of the dorsal roots L7 and Sl (cat) and L6 and Sl (rat) or of the peripheral nerves (cat) and recorded from the corresponding ventral roots. When peripheral nerves were stimulated, the afferent impulse volley was recorded with a chlorided silver ball electrode at the dorsal root entry zone. The stimulation strength was supramaximal for the monosynaptic reflex response in the case of dorsal root stimulation, and for the mono- or polysynaptic reflex respectively in the case of peripheral nerve stimulation (pulse duration 0.05 msec; frequency 0.4 pulsesjsec). Alpha and y reflex discharges were determined in six cats according to the method described by SIXG (1964) and JURMA. Ruz~rti. NELL and GROSSMANN (1972). Diphenylthiohydantoin (Chemical Procurement Laboratories, College Point, New York) was triturated and suspended in polyvinylpyrrolidone (Periston@, Bayer, Leverkusen) in a concentration of 1.5 g/20 ml. The suspension was twice homogenized at 2000 rev/min and 3 strokes/min for 10 min and centrifuged at 3000 rev/min for 10 min. The concentration of DPTH in the supernatant was determined spectrophotometrically (DILL, KAZENKO, WOLF and GLAZKO, 1956; VEREBELY rt al., 1970) and ranged between 14.6 and 19.3 mg/ml. The supernatant was injected intravenously at a rate of 1 ml/min until a total dose of DPTH 20 mg/kg was administered. Similar volumes of the suspension medium only were administered in control experiments. RESULTS In five spinal cats, 20 mg/kg DPTH increased the amplitude of the mono- and polysynaptic reflexes elicited by stimulation of dorsal roots. At 15 min after injection the inmonorynoptic
polysynoptic
% 100
80
spinolized
unspinalized
20
0 1
Fig. 1. Change in the amplitude of the mono- and polysynaptic reflex responses produced by 20 mgjkg DPTH in five spinal and in five unspinalized rats. The scale on the left indicates the change of the amplitude in “A of controls determined 10 min after drug injection. Each column represents the value obtained in one experiment (= one rat). Except in two cases ( x ), the changes were statistically significant (P < 0.05).
DPTH
and spinal reflex activity
815
crease in the amplitude of the monosynaptic reflex amounted to 17.6 + 6.4%, and that of the polysynaptic reflex to 25.7 + 9.2% of the controls. The effect of DPTH on the reflex amplitudes began 2-5 min after the administration, had fully developed 10 min after the injection and lasted for about 1 hr. Control experiments, in which a quantity of the suspension medium (equal to the volume of the suspension of DPTH administered) was injected, showed no changes in the mass reflex activity. In five spinal rats, 20 mg/kg DPTH increased the amplitude of mono- and polysynaptic reflexes evoked by dorsal root stimulation. However, when DPTH was injected to unspinalized preparations (five rats), it produced either an increase, a decrease, or no change of the reflex amplitudes. The result of these experiments is summarized by Figure 1. In five spinal cats, in which the reflexes were elicited by stimulation of the GS, PBST and sural nerves, the amplitudes of the monosynaptic reflexes to GS and to PBST stimulation increased by 16.2 f 7.1 and 9.9 + 3.6% of the controls respectively, and those of the polysynaptic reflexes to PBST and sural nerve stimulation by 34.4 + 8.5 and CONTROL
DPTH
20mg/kg
5 nisec Fig. 2. Effect of 20 mg/kg DPTH on the reflex response and the afferent volley to stimulation of the PBST nerve. Upper tracings: atlerent volley recorded from the dorsal root entry zone L7. Lower tracings: reflex responses recorded from the ventral root L7. The stimulation strength was 4.8 times threshold for the monosynaptic reflex response.
26.2 f 7.1% of the controls respectively. The effect of DPTH had fully developed 10 min after the injection. The latency of the monosynaptic responses remained unchanged after DPTH. Simultaneously with the increase in the reflex amplitudes, the amplitude of the early component of the afferent volley was enhanced by DPTH (Fig. 2). The number of reflex discharges of alpha and gamma motoneurones to dorsal root stimulation was determined in six spinal cats. Before the application of DPTH the number of a discharges amounted to 9.5 i 1.3 per sweep (sweep duration 10msec; the total number of sweeps counted was 68). Ten min after the injection of 20 mg/kg DPTH, the number of a reflex discharges had increased to 14.0 + 0.5 per sweep (the total number of sweeps counted was 63) while the number of y discharges remained unchanged. The
816
W. GROSSMAN,1. JURNAand C. THERES
latency between the stimulus artifact and the first x reflex discharge was not changed by DPTH. DISCUSSION
The results presented show that DPTH exerts an excitatory effect on reflex activity in spinal cats and rats. They are, however, not entirely in accord with the findings obtained with DPH and DPTH by other workers (STILLI:, 1955; ESPLIN. 19.57; RAINESet al., 1971) who, apart from activation, also observed inhibition of spinal reflex activity. This divergence may in part be due to the different type of spinalization employed, because activation as well as inhibition of reflex activity has been observed in this study after the administration of DPTH in unspinalized rats, whereas in spinal rats the drug always increased the reflex activity. Another point deserving consideration is the way of preparing DPTH for an intravenous administration. To dissolve the drug in water a rather high alkalinity (pH 11.5) must be established. Saline of high alkalinity influences spinal reflex activity and thus can mask the effect of the drug dissolved (JURNA and LANZER, 1969). Moreover, it was found in experiments performed with microelectrodes on single spinal motoneurones (GROSSMANN,JLJRNAand RICHTER, 1974) that an injection of saline with a high alkalinity produced depolarization of the ceh membranes which, by a reduction of the action potentials of the motoneurones, must decrease the amplitude of mass reflexes. In the present experiments such interference can be excluded because the medium in which DPTH was suspended did not influence the reflex activity in spinal animals. The increase in reflex activity may be ascribed to an excitatory action of the drug on motoneurones (GROSSMANN, JURNAand RICHTER,1971; GROSSMANN et al., 1974), but also to the excitatory effect on the primary afferent fibres observed in this study. In microelectrode studies, it has been found that not all motoneurones are activated by DPTH (GROSSMANNet al., 1974). In all probability, the motoneurones which are not affected by DPTH are small cells. This would be in accord with the finding that only the reflex discharge from SI but not from y motoneurones was increased by DPTH. A similar result has been obtained in an investigation of the effect of DPH on x and 7 reflex discharges in spinal rats (JURNAand THERES,1969). From the results presented it is concluded that DPTH enhances spinal reflex activity by an effect on CImotoneurones and on afferent fibres. REFERENCES DILL, W. A., KAZENKO, A.. WOLF, L. M. and GLAZKO. A. J. (1956). Studies
on 5.5-diphenylhydantoin (dilantin) in animals and man. J. Phc~,muc. cup. Thw. 118: 27s 279. ESPLIN. D. W. (1957). Effects of diphenylhydantoin on synaptic transmission in cat spinal cord and stellate ganglion. .I. Pharmac. exp. Thu. 120: 301-323. GROSSMANN,W., JURNA,1. and RICHTER, D. (1971). The action of diphenylthiohydantoin on the electrical activity of single spinal motoneurones. NauM~,1-S~h,niedehPrgs Arch. Pharmac. 270: Suppl. R 47. GROSSMANN. W., JURKA. I. and RICHTER. D. (1974). Activating effect of diphenylthiohydantoin on spinal motoneurones. ~Vcu~.o~~/~~~~~f~u~oIo~~~ 13: 803 -8 I I, JLJRNA, 1. and LANZER, G. (1969). Inhibition of the effect of rcserpine on motor control bv. drugs- which influence reserpine rigidity. Naunyn-Sckmirdehc~gs Arch. Pharmac. 262.309-324. JUKNA, I., RuZDI~, N.. NELL. T. and GROSSMANXW. (1972). The effect of x-mcthvl-n-tvrosine and substantia nigra lesions on spinal motor activity in the rat. Eur. J. ‘Pharkac. 20: 341-350. . ’ . JURNA, I. and THERFS, C. (1969). The effect of phenytoin and metamphetamine on spinal motor activity. NuunynSchmivdehergs Arch. Pharmac. 265: 244-259. RAINES, A., SOHN. Y. J. and LEVITT. B. (1971). Spinal excitatory and depressant effects of sodium diphenylthiohydantoin. J. Pharmac. cup. T/w. 177: 35@259.
DPTH and spinal reflex activity
817
SOHN,Y. J., LEVITT,B. and RAINES,A. (1970). Anticon~~ulsant properties of diphenylthiohydantoin. Pha~~~acod~~. T&r. 188: 284-289. STEG,G. (1964). Etferent muscle innervation and rigidity. Acru physiol. stand. 661: Suppl. 225. STILLE,G. (1955). Die Wirkung von Antikonvulsiva auf das Riickenmark. Nuunyn-SchmiedehePgs 226: 172-279.
Archs int.
Arch. Pharmac.
VEREBELY, K., KUTT,H., SOHN,Y. J.. LEVITT.B. and RAINES.A. (1970). Uptakeand distribution of diphenylthiohydantoin. Eur. J. Pharmuc. 10: 106-l 10.
(Accepr&13February19?4) Summary-The effect of diphenylthiohydant~in (DPTH) 20 mgjkg on spinal reflexes was studied in cats and rats which were spinalized at the lower thoracic level and also in unspinalized rats. DPTH increased the amplitude of mono- and polysynaptic reflex responses in spinal cats and rats. The amplitude of the early component of the afferent volley to peripheral nerve stimulation was enhanced. In unspinalized rats DPTH increased as well as decreased mono- and poiysynaptic reflex responses. In spinal cats DPTH increased the number of alpha reflex discharges, but left the number of gamma reflex discharges unchanged. The results are discussed with respect to the conflicting findings obtained by other authors when studying the effect of diphenylhydantoin and DPTH on spinal reflex activity.
Diphenylthiohydantoin (DPTH), an anticonvulsant agent acting similarly to diphenylhydantoin (DPH) (SOHN, LEVITT and RAINES,1970). enters and leaves the central nervous system more rapidly than DPH (VEREBELY,KUTT, SOHN, LEVITT and RAINES,1970). Conflicting results have been obtained when the effect of DPH, as well as of DPTH, on spinal reflexes were investigated in different preparations (STILLE, 1955; ESPLIN, 1957; RAINES,SOHNand LEVITT,1971). It was the aim of the present investigation to determine whether the effect of DPTH on mass reflexes is different in spinal and unspinalized preparations. A study of the reflex discharges from c1and y motoneurones in spinal cats was included to assess the effect of DPTH on functionally different motoneurones. METHODS
The experiments were performed on 17 cats (2.3-3-7 kg body wt) and on 10 rats (Wistar strain, 250g body wt) operated under halothane anaesthesia. The spinal cord was transected in all cats and in some of the rats at the level of T12. The ventral roots and (apart from the experiments in which stimulation of peripheral nerves was performed), the dorsal roots LS-S2 were cut and prepared for recording or stimulation. For stimulation of peripheral nerves, the posterior biceps-semitendinosus (PBST), the gastrocnemius-soleus (GS) and the sural nerves were dissected free in five cats. The cats were intercollicularly decerebrated. The rats were kept at a level of anaesthesia which did not interfere with spinal reflex activity. This level was discovered in experiments in which all surgical wounds were instilled with procaine and the animals allowed to recover for a short period from anaesthesia. All preparations were immobilized with gallamine and maintained on artificial respiration The body temperature was maintained between 36.5 and 38°C. * Supported by the Sonderfors~hungsbereic~t 38 “Memhranen” of the Deutsche Forschungsgemeins~haft. 813
814
W.
GROSSMAN. I. JURKA and
C. THERM
Reflexes were elicited by stimulation of the dorsal roots L7 and Sl (cat) and L6 and Sl (rat) or of the peripheral nerves (cat) and recorded from the corresponding ventral roots. When peripheral nerves were stimulated, the afferent impulse volley was recorded with a chlorided silver ball electrode at the dorsal root entry zone. The stimulation strength was supramaximal for the monosynaptic reflex response in the case of dorsal root stimulation, and for the mono- or polysynaptic reflex respectively in the case of peripheral nerve stimulation (pulse duration 0.05 msec; frequency 0.4 pulsesjsec). Alpha and y reflex discharges were determined in six cats according to the method described by SIXG (1964) and JURMA. Ruz~rti. NELL and GROSSMANN (1972). Diphenylthiohydantoin (Chemical Procurement Laboratories, College Point, New York) was triturated and suspended in polyvinylpyrrolidone (Periston@, Bayer, Leverkusen) in a concentration of 1.5 g/20 ml. The suspension was twice homogenized at 2000 rev/min and 3 strokes/min for 10 min and centrifuged at 3000 rev/min for 10 min. The concentration of DPTH in the supernatant was determined spectrophotometrically (DILL, KAZENKO, WOLF and GLAZKO, 1956; VEREBELY rt al., 1970) and ranged between 14.6 and 19.3 mg/ml. The supernatant was injected intravenously at a rate of 1 ml/min until a total dose of DPTH 20 mg/kg was administered. Similar volumes of the suspension medium only were administered in control experiments. RESULTS In five spinal cats, 20 mg/kg DPTH increased the amplitude of the mono- and polysynaptic reflexes elicited by stimulation of dorsal roots. At 15 min after injection the inmonorynoptic
polysynoptic
% 100
80
spinolized
unspinalized
20
0 1
Fig. 1. Change in the amplitude of the mono- and polysynaptic reflex responses produced by 20 mgjkg DPTH in five spinal and in five unspinalized rats. The scale on the left indicates the change of the amplitude in “A of controls determined 10 min after drug injection. Each column represents the value obtained in one experiment (= one rat). Except in two cases ( x ), the changes were statistically significant (P < 0.05).
DPTH
and spinal reflex activity
815
crease in the amplitude of the monosynaptic reflex amounted to 17.6 + 6.4%, and that of the polysynaptic reflex to 25.7 + 9.2% of the controls. The effect of DPTH on the reflex amplitudes began 2-5 min after the administration, had fully developed 10 min after the injection and lasted for about 1 hr. Control experiments, in which a quantity of the suspension medium (equal to the volume of the suspension of DPTH administered) was injected, showed no changes in the mass reflex activity. In five spinal rats, 20 mg/kg DPTH increased the amplitude of mono- and polysynaptic reflexes evoked by dorsal root stimulation. However, when DPTH was injected to unspinalized preparations (five rats), it produced either an increase, a decrease, or no change of the reflex amplitudes. The result of these experiments is summarized by Figure 1. In five spinal cats, in which the reflexes were elicited by stimulation of the GS, PBST and sural nerves, the amplitudes of the monosynaptic reflexes to GS and to PBST stimulation increased by 16.2 f 7.1 and 9.9 + 3.6% of the controls respectively, and those of the polysynaptic reflexes to PBST and sural nerve stimulation by 34.4 + 8.5 and CONTROL
DPTH
20mg/kg
5 nisec Fig. 2. Effect of 20 mg/kg DPTH on the reflex response and the afferent volley to stimulation of the PBST nerve. Upper tracings: atlerent volley recorded from the dorsal root entry zone L7. Lower tracings: reflex responses recorded from the ventral root L7. The stimulation strength was 4.8 times threshold for the monosynaptic reflex response.
26.2 f 7.1% of the controls respectively. The effect of DPTH had fully developed 10 min after the injection. The latency of the monosynaptic responses remained unchanged after DPTH. Simultaneously with the increase in the reflex amplitudes, the amplitude of the early component of the afferent volley was enhanced by DPTH (Fig. 2). The number of reflex discharges of alpha and gamma motoneurones to dorsal root stimulation was determined in six spinal cats. Before the application of DPTH the number of a discharges amounted to 9.5 i 1.3 per sweep (sweep duration 10msec; the total number of sweeps counted was 68). Ten min after the injection of 20 mg/kg DPTH, the number of a reflex discharges had increased to 14.0 + 0.5 per sweep (the total number of sweeps counted was 63) while the number of y discharges remained unchanged. The
816
W. GROSSMAN,1. JURNAand C. THERES
latency between the stimulus artifact and the first x reflex discharge was not changed by DPTH. DISCUSSION
The results presented show that DPTH exerts an excitatory effect on reflex activity in spinal cats and rats. They are, however, not entirely in accord with the findings obtained with DPH and DPTH by other workers (STILLI:, 1955; ESPLIN. 19.57; RAINESet al., 1971) who, apart from activation, also observed inhibition of spinal reflex activity. This divergence may in part be due to the different type of spinalization employed, because activation as well as inhibition of reflex activity has been observed in this study after the administration of DPTH in unspinalized rats, whereas in spinal rats the drug always increased the reflex activity. Another point deserving consideration is the way of preparing DPTH for an intravenous administration. To dissolve the drug in water a rather high alkalinity (pH 11.5) must be established. Saline of high alkalinity influences spinal reflex activity and thus can mask the effect of the drug dissolved (JURNA and LANZER, 1969). Moreover, it was found in experiments performed with microelectrodes on single spinal motoneurones (GROSSMANN,JLJRNAand RICHTER, 1974) that an injection of saline with a high alkalinity produced depolarization of the ceh membranes which, by a reduction of the action potentials of the motoneurones, must decrease the amplitude of mass reflexes. In the present experiments such interference can be excluded because the medium in which DPTH was suspended did not influence the reflex activity in spinal animals. The increase in reflex activity may be ascribed to an excitatory action of the drug on motoneurones (GROSSMANN, JURNAand RICHTER,1971; GROSSMANN et al., 1974), but also to the excitatory effect on the primary afferent fibres observed in this study. In microelectrode studies, it has been found that not all motoneurones are activated by DPTH (GROSSMANNet al., 1974). In all probability, the motoneurones which are not affected by DPTH are small cells. This would be in accord with the finding that only the reflex discharge from SI but not from y motoneurones was increased by DPTH. A similar result has been obtained in an investigation of the effect of DPH on x and 7 reflex discharges in spinal rats (JURNAand THERES,1969). From the results presented it is concluded that DPTH enhances spinal reflex activity by an effect on CImotoneurones and on afferent fibres. REFERENCES DILL, W. A., KAZENKO, A.. WOLF, L. M. and GLAZKO. A. J. (1956). Studies
on 5.5-diphenylhydantoin (dilantin) in animals and man. J. Phc~,muc. cup. Thw. 118: 27s 279. ESPLIN. D. W. (1957). Effects of diphenylhydantoin on synaptic transmission in cat spinal cord and stellate ganglion. .I. Pharmac. exp. Thu. 120: 301-323. GROSSMANN,W., JURNA,1. and RICHTER, D. (1971). The action of diphenylthiohydantoin on the electrical activity of single spinal motoneurones. NauM~,1-S~h,niedehPrgs Arch. Pharmac. 270: Suppl. R 47. GROSSMANN. W., JURKA. I. and RICHTER. D. (1974). Activating effect of diphenylthiohydantoin on spinal motoneurones. ~Vcu~.o~~/~~~~~f~u~oIo~~~ 13: 803 -8 I I, JLJRNA, 1. and LANZER, G. (1969). Inhibition of the effect of rcserpine on motor control bv. drugs- which influence reserpine rigidity. Naunyn-Sckmirdehc~gs Arch. Pharmac. 262.309-324. JUKNA, I., RuZDI~, N.. NELL. T. and GROSSMANXW. (1972). The effect of x-mcthvl-n-tvrosine and substantia nigra lesions on spinal motor activity in the rat. Eur. J. ‘Pharkac. 20: 341-350. . ’ . JURNA, I. and THERFS, C. (1969). The effect of phenytoin and metamphetamine on spinal motor activity. NuunynSchmivdehergs Arch. Pharmac. 265: 244-259. RAINES, A., SOHN. Y. J. and LEVITT. B. (1971). Spinal excitatory and depressant effects of sodium diphenylthiohydantoin. J. Pharmac. cup. T/w. 177: 35@259.
DPTH and spinal reflex activity
817
SOHN,Y. J., LEVITT,B. and RAINES,A. (1970). Anticon~~ulsant properties of diphenylthiohydantoin. Pha~~~acod~~. T&r. 188: 284-289. STEG,G. (1964). Etferent muscle innervation and rigidity. Acru physiol. stand. 661: Suppl. 225. STILLE,G. (1955). Die Wirkung von Antikonvulsiva auf das Riickenmark. Nuunyn-SchmiedehePgs 226: 172-279.
Archs int.
Arch. Pharmac.
VEREBELY, K., KUTT,H., SOHN,Y. J.. LEVITT.B. and RAINES.A. (1970). Uptakeand distribution of diphenylthiohydantoin. Eur. J. Pharmuc. 10: 106-l 10.