(The) effect of strychnine on membrane properties of spinal motoneurons in the cat

의학과/박사[한글] The Effect of Strychnine en Membrane Properties of Spinal Motoneurons in The Cat Kyu Chang Lee Department of Medical Science The Graduate School, Yonsei University. Max.Piuk-Institut for Brain Research Frankfurt am Main, Federal Republic of Germany (directed by Prof, warred R. Klee and Hun Jae Lee) Introduction The convulsant properties of strychnine have attracted the attention of neuropharmacelogists for over 150 years (Wall et al .,1955). Strychnine has been used as instrument for studies of experimental epilepsy(Dusser de Barenne, 1933 : Chang 1951 : Wall and Horwitz, 1951 : Bremer, 1953 : Purpura and Grundfest. 1957 : Goldring and O'Leary, 1960; Pollen and Sie. 1964), though its precise mode of action has remained obscure. One mechanism of action baa partially clarified in 1954 by the demonstration that subconvulsive doses of strychnine reduce the amplitude of inhibitory postsynaptic potentials (IPSPs) in cat spinal motoneurons (Ecoles et al, 1954). Because of the rapid onset of its action arid the absence of effects upon monosynaptic excitatory postsynaptic potentials (EPSPs), it was proposed that strychnine competed with come unidentified transmitter for inhibitory receptor sites on the postsynaptic membrane (Ecoles, 1957). Electrophoretic application of strychnine is known to block the inhibitory effects of glycine, a likely candidate as an inhibitory transmitter on motoneurons in the cat spinal cord(Werman et al, 1968 ; Curtis et aLl 1967, 1968 a and b). A strychnine-resistant inhibition seems to exist rut only in the higher portions of the central nervous system, but also for the spinal motoneuron (Kellerth 1965 : Klee et al, 1966), Gamma amino butyric acid(GABA) is a candidate for this synaptic transmitter (Curtis et lal., 1971). In experimental epilepsy an increased excitation or excitability of the neurons h7a to exist in order to elicite seizure discharges. This can be due to a reduced resting membrane potential (RMP), to an increase in the synaptic excitation/or reduced inhibition, or finally. in changes in the excitability of the cell membrane due to changes in the characteristics of the electrically excitable ionic channels. In thin study experiments were performed to (1) find the minimal intravenous dose of strychnine which induces seizure in the isolated cerebral cortex and to compare the doses necessary to induce seizure in the cortex with those needed in the apinal cord (2) Under the assumption that strychnine might effect the membrane properties of motoneurons directly, the antidromic action potential and RMP were measured following successive injections of strychine up to 0.85 ne/kg. (3) In order to discover whether the amplitude of the polsynaptic EPSPs were increased because of blockade of an inhibitory component, different methods were u7ed to demonstrate the presistance of a strychnine-resistant inhibitory component in the polysynaptic EPSP. (4) Bicuculline, a known GABA antagonist (Curtis et al, 1968 a, 1971) was injected for the blockade of strychnine-resistant inhibition (5) The GABA-derivative Lioresal was compared with mephenesin (MY 301) in its antagonistic action on seizures induced by strychnine or bicuculline. Materials and Methods Experiments were carried out on 35 cats of both sexes weighing from 1.5 to 3.5 kg under Nembutal anesthesia (30 mg/kg, intraperitoneally). Surgical procedures included tracheotomy. cannulation of the v. jugularis ext. and laminectomy at the level from L3 to S1. Ventral roots L7 & S1 and dorsal roots L7 & S1 or peripheral nerves of the left hindlimb were cut and prepared for stimulation. The animal was fixed in a frame and respired artificially following intravenous injection of Flaxedil (gallamine triethiodide, 2 mg/kg), Bilateral pneumothorax was clone. Exposed tissues were covered with warm paraffin oil. The rectal temperature was maintained at 37±1℃. Intracellular recording of the motoneuron was made with single glass micropipettes filled with 3M KCI having tip diameters ranging from 0.5 to 3 μ. Motoneurons were identified by antidromic activation via the ventral roots. Only cells with a membrane potential greater than 50 mV, a spikeamplitude greater than 60 mV, and a stable resting potential over a period of 20 to 30min were used for analysis. Among 70 neurons penetrated only 28 neurons fullfilled all the requirments noted. Strychnine sulfate was dissolved in Ringer saline arid intravenously administered in consecutive doses of either 0.10 7r 0.15 mg/kg. In some experiments, the interactions between strychnine and GABA-derivative Lioresal (2 mg/kg), HY 301, i. e. guaiacol glyceryl ether (50-100mg/kg) or bicuculline (0.25-0.50 mg/kg) were studied The difference in sensitivity between the cortex and the spinal cord for the generation of spontaneous seizure due to convulsive doses of strychnine was investigated in 4 cats using mass recording technique from the cortical surface and from the dorsal part of the isolated spinal cord after section at the Cl-2 level For these studies consecutive doses of strychnine, 0.10 to 0.50 mg/ kg, up to total of 3.7 mg/kg were administered intravenously. Results Evoked potentials were recorded from the cord dorsum or the ventral root L7 following stimulation of dorsal root L7 or synergistic muscle nerve. Doses of Strychnine inducing spontaneous seizure activity in the spinal cord were in the range of 0.3 to 0.4mg/kg (0.35±0.07mg/kg) in 4 isolated spinal cords. Induced or seizures of the spinal cord were represented by large positive waves. Evoked potentials of the motor cortex were recorded following stimulation of ventralis oralis posterior nucleus of the thalamus. In contrast to the spinal cord, 2.0 to 3.7 mg/kg (2.87±0.97mg/kg) of strychnine was required to elicite seizure in the motor cortex Therefore, the convulsive dose of strychnine is about 8 times higher for the cortex compared to the spinal cord, in the spinalized animal. The effect of strychnine on the RMP of spinal motoneurona was inconsistent. Maximum changes in the RMP within 15 min. following the first injection (0.1-0.15 mg/kg) are as follows : 10 neurons were depolarized by 0.5-10 mV(3.6±1.04 mV) and 5 other neurons were hyperpolarized by 1 to 5mV(2.4± 1.5 mV). Strychnine also had no consistant effect on the antidromic action potential of spinal motoneurons. With subconvulsive doses (0.1-0.2 mg/kg), 9 neurons showed reduction of amplitude by 7-20 % and in 6 cases there were only minimal changes (less than±5 %). In contrast, in 4 neurons the amplitude of the spike was increased by 3-8%. With the doses of strychnine being rome to triter cornvulsion(0.3-0.85 mg/kg) , the amplitude of the action potential was slightly increased, slightly reduced or unchanged. The duration and rate of fall of the action potential was relatively constant. The monosynaptic EPSP elicited by electrical stimulation of the synergistic muscle nerves or the dorsal root was not changed, either with subconvulsive or with convulsive doses of strychnine. Polysynaptic EPSPs elicited by the suprathreshold stimulation of the synergistic muscle nerve or the dorsal root, increased the amplitude by 200-560%, even with subconvulaive fuses of strychnine, and their duration lengthened by about 150 to 200%. The effect occurred with short latency after drug injection and the depolarization often reached firing level, eliciting action potentials. With convulsive theses, the enhanced EPSP gave to multiple bursting discharges. In order to estimate the extent to which the strychnine-dependent increase in polysynaptic EPSP amplitude may be due to the blockade of inhibitory components contaminating the EPSPs, different methods were used to check the EPSPs for IPSP components. Before and following the injection of different amounts of strychnine, polysynaptic EPSPS showed a conductance increase during their time course as detected by a reduction in the amplitude of the antidromic spike, an increase in amplitude when the RMP was increased by current injection, and a temporary increase in amplitude when chloride ions were injected into the cell The strychnine-resistant IPSPS mixed with the EPSPs court be rehuced by intravenous injection of bicuoulline (0.25mg17g). The possibility that these results are 여e to synaptic localization or a different transmitter substance being involved in the generation of the polyaynaptic EPSP can not be ruled out. Intravenous injection of MY 301 (50 mg/kg), a substance chemically similar to mephenesin, resulted in the immediate blocking of discharges induced by strychnine. This drug reduced the amplitude of the polysynaptic EPSPs, which had been enhanced by strychnine. The membrane properties of the motoneurons were unchanged with the doses of MY 301 blocking seizure activity. Intravenous administration of a GABA-derivative, Lioresal (2mg/kg), also resulted in immediate cessation of seizure activity and reduced the amplitude of the polysynaptic EPSP. The RMP and the antidromic action potential were uncharged by doses sufficient to block the effect of strychnine. To conclude, it can be said that there were no consistant changes in the motoneuron action potential which would reflect an action of strychnine upon the motoneuron's membrane properties important to seizure generation. It is still to be resorted whether the increase in polysynaptic EPSP amplitude is due to a strychnine effect upon the membrane properties of excitatory interneurons or to an effect only upon the inhibitory as well as excitatory postsynaptic potentials. [영문] Introduction The convulsant properties of strychnine have attracted the attention of neuropharmacelogists for over 150 years (Wall et al .,1955). Strychnine has been used as instrument for studies of experimental epilepsy(Dusser de Barenne, 1933 : Chang 1951 : Wall and Horwitz, 1951 : Bremer, 1953 : Purpura and Grundfest. 1957 : Goldring and O'Leary, 1960; Pollen and Sie. 1964), though its precise mode of action has remained obscure. One mechanism of action baa partially clarified in 1954 by the demonstration that subconvulsive doses of strychnine reduce the amplitude of inhibitory postsynaptic potentials (IPSPs) in cat spinal motoneurons (Ecoles et al, 1954). Because of the rapid onset of its action arid the absence of effects upon monosynaptic excitatory postsynaptic potentials (EPSPs), it was proposed that strychnine competed with come unidentified transmitter for inhibitory receptor sites on the postsynaptic membrane (Ecoles, 1957). Electrophoretic application of strychnine is known to block the inhibitory effects of glycine, a likely candidate as an inhibitory transmitter on motoneurons in the cat spinal cord(Werman et al, 1968 ; Curtis et aLl 1967, 1968 a and b). A strychnine-resistant inhibition seems to exist rut only in the higher portions of the central nervous system, but also for the spinal motoneuron (Kellerth 1965 : Klee et al, 1966), Gamma amino butyric acid(GABA) is a candidate for this synaptic transmitter (Curtis et lal., 1971). In experimental epilepsy an increased excitation or excitability of the neurons h7a to exist in order to elicite seizure discharges. This can be due to a reduced resting membrane potential (RMP), to an increase in the synaptic excitation/or reduced inhibition, or finally. in changes in the excitability of the cell membrane due to changes in the characteristics of the electrically excitable ionic channels. In thin study experiments were performed to (1) find the minimal intravenous dose of strychnine which induces seizure in the isolated cerebral cortex and to compare the doses necessary to induce seizure in the cortex with those needed in the apinal cord (2) Under the assumption that strychnine might effect the membrane properties of motoneurons directly, the antidromic action potential and RMP were measured following successive injections of strychine up to 0.85 ne/kg. (3) In order to discover whether the amplitude of the polsynaptic EPSPs were increased because of blockade of an inhibitory component, different methods were u7ed to demonstrate the presistance of a strychnine-resistant inhibitory component in the polysynaptic EPSP. (4) Bicuculline, a known GABA antagonist (Curtis et al, 1968 a, 1971) was injected for the blockade of strychnine-resistant inhibition (5) The GABA-derivative Lioresal was compared with mephenesin (MY 301) in its antagonistic action on seizures induced by strychnine or bicuculline. Materials and Methods Experiments were carried out on 35 cats of both sexes weighing from 1.5 to 3.5 kg under Nembutal anesthesia (30 mg/kg, intraperitoneally). Surgical procedures included tracheotomy. cannulation of the v. jugularis ext. and laminectomy at the level from L3 to S1. Ventral roots L7 & S1 and dorsal roots L7 & S1 or peripheral nerves of the left hindlimb were cut and prepared for stimulation. The animal was fixed in a frame and respired artificially following intravenous injection of Flaxedil (gallamine triethiodide, 2 mg/kg), Bilateral pneumothorax was clone. Exposed tissues were covered with warm paraffin oil. The rectal temperature was maintained at 37±1℃. Intracellular recording of the motoneuron was made with single glass micropipettes filled with 3M KCI having tip diameters ranging from 0.5 to 3 μ. Motoneurons were identified by antidromic activation via the ventral roots. Only cells with a membrane potential greater than 50 mV, a spikeamplitude greater than 60 mV, and a stable resting potential over a period of 20 to 30min were used for analysis. Among 70 neurons penetrated only 28 neurons fullfilled all the requirments noted. Strychnine sulfate was dissolved in Ringer saline arid intravenously administered in consecutive doses of either 0.10 7r 0.15 mg/kg. In some experiments, the interactions between strychnine and GABA-derivative Lioresal (2 mg/kg), HY 301, i. e. guaiacol glyceryl ether (50-100mg/kg) or bicuculline (0.25-0.50 mg/kg) were studied The difference in sensitivity between the cortex and the spinal cord for the generation of spontaneous seizure due to convulsive doses of strychnine was investigated in 4 cats using mass recording technique from the cortical surface and from the dorsal part of the isolated spinal cord after section at the Cl-2 level For these studies consecutive doses of strychnine, 0.10 to 0.50 mg/ kg, up to total of 3.7 mg/kg were administered intravenously. Results Evoked potentials were recorded from the cord dorsum or the ventral root L7 following stimulation of dorsal root L7 or synergistic muscle nerve. Doses of Strychnine inducing spontaneous seizure activity in the spinal cord were in the range of 0.3 to 0.4mg/kg (0.35±0.07mg/kg) in 4 isolated spinal cords. Induced or seizures of the spinal cord were represented by large positive waves. Evoked potentials of the motor cortex were recorded following stimulation of ventralis oralis posterior nucleus of the thalamus. In contrast to the spinal cord, 2.0 to 3.7 mg/kg (2.87±0.97mg/kg) of strychnine was required to elicite seizure in the motor cortex Therefore, the convulsive dose of strychnine is about 8 times higher for the cortex compared to the spinal cord, in the spinalized animal. The effect of strychnine on the RMP of spinal motoneurona was inconsistent. Maximum changes in the RMP within 15 min. following the first injection (0.1-0.15 mg/kg) are as follows : 10 neurons were depolarized by 0.5-10 mV(3.6±1.04 mV) and 5 other neurons were hyperpolarized by 1 to 5mV(2.4± 1.5 mV). Strychnine also had no consistant effect on the antidromic action potential of spinal motoneurons. With subconvulsive doses (0.1-0.2 mg/kg), 9 neurons showed reduction of amplitude by 7-20 % and in 6 cases there were only minimal changes (less than±5 %). In contrast, in 4 neurons the amplitude of the spike was increased by 3-8%. With the doses of strychnine being rome to triter cornvulsion(0.3-0.85 mg/kg) , the amplitude of the action potential was slightly increased, slightly reduced or unchanged. The duration and rate of fall of the action potential was relatively constant. The monosynaptic EPSP elicited by electrical stimulation of the synergistic muscle nerves or the dorsal root was not changed, either with subconvulsive or with convulsive doses of strychnine. Polysynaptic EPSPs elicited by the suprathreshold stimulation of the synergistic muscle nerve or the dorsal root, increased the amplitude by 200-560%, even with subconvulaive fuses of strychnine, and their duration lengthened by about 150 to 200%. The effect occurred with short latency after drug injection and the depolarization often reached firing level, eliciting action potentials. With convulsive theses, the enhanced EPSP gave to multiple bursting discharges. In order to estimate the extent to which the strychnine-dependent increase in polysynaptic EPSP amplitude may be due to the blockade of inhibitory components contaminating the EPSPs, different methods were used to check the EPSPs for IPSP components. Before and following the injection of different amounts of strychnine, polysynaptic EPSPS showed a conductance increase during their time course as detected by a reduction in the amplitude of the antidromic spike, an increase in amplitude when the RMP was increased by current injection, and a temporary increase in amplitude when chloride ions were injected into the cell The strychnine-resistant IPSPS mixed with the EPSPs court be rehuced by intravenous injection of bicuoulline (0.25mg17g). The possibility that these results are 여e to synaptic localization or a different transmitter substance being involved in the generation of the polyaynaptic EPSP can not be ruled out. Intravenous injection of MY 301 (50 mg/kg), a substance chemically similar to mephenesin, resulted in the immediate blocking of discharges induced by strychnine. This drug reduced the amplitude of the polysynaptic EPSPs, which had been enhanced by strychnine. The membrane properties of the motoneurons were unchanged with the doses of MY 301 blocking seizure activity. Intravenous administration of a GABA-derivative, Lioresal (2mg/kg), also resulted in immediate cessation of seizure activity and reduced the amplitude of the polysynaptic EPSP. The RMP and the antidromic action potential were uncharged by doses sufficient to block the effect of strychnine. To conclude, it can be said that there were no consistant changes in the motoneuron action potential which would reflect an action of strychnine upon the motoneuron's membrane properties important to seizure generation. It is still to be resorted whether the increase in polysynaptic EPSP amplitude is due to a strychnine effect upon the membrane properties of excitatory interneurons or to an effect only upon the inhibitory as well as excitatory postsynaptic potentials.restrictio