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Electrophysiological studies with the spastic mutant mouse
Brain Research, 234 (1982) 299--308 Elsevier Biomedical Press
299
E L E C T R O P H Y S I O L O G I C A L STUDIES W I T H T H E SPASTIC M U T A N T MOUSE
ALLEN H. HELLER* and MARK HALLETT Department of Neurology, Children's Hospital Medical Center and Section of Neurology, Department of Medicine, Brigham and Women's Hospital, and the Department of Neurology, Harvard Medical School, CHMC, 300 Longwood Avenue, Boston, MA 02115 (U.S.A.)
(Accepted July 30th, 1981) Key words: spastic mutant mouse - - electromyography -- strychnine - - glyeine inhibition - - picrotoxinin - - GABA inhibition
SUMMARY Electromyographic (EMG) studies were carried out with the genetically spastic mouse (spa, autosomal recessive), obtained from matings of B6C3a/a, spa/+ heterozygotes. Spastic homozygotes exhibited high amplitude repetitive E M G bursts during spontaneous activity. Following an electrical stimulus to hindlimb or forelimb, high amplitude stereotyped E M G bursts were recorded from eontralateral limbs in spastic mice, but were not observed in phenotypically unaffected littermates or normal C57BL/6J mice. The timing and latency of this stereotyped response to an electrical stimulus was consistent with the participation of spinal cord neuronal pathways. In normal C57BL/6J mice the administration of strychnine (0.65 mg/kg), but not picrotoxinin (up to convulsant doses), reproduced all of the behavioral and E M G features observed in spastic homozygotes. We hypothesize that the symptoms in the spastic mutant may result from a deficiency of strychnine-sensitive (presumably glycinergic) inhibition in the spinal cord.
INTRODUCTION The 'spastic' mutant mouse (spa, autosomal recessive) exhibits a neuromuscular disorder characterized by episodes of rapid tremor of limbs and tail, lack of flexibility of trunk movements, toe-walking gait, and difficulty in regaining upright posture when placed on its back. When stimulated by shaking of its cage or a sudden loud noise, the * Address for all correspondence: Box 117, Children's Hospital Medical Center, 300 Longwood Avenue, Boston, MA 02115, U.S.A. 0006-8993/82/0000-0000/$02.75 © Elsevier Biomedical Press
300 animal appears to stiffen with an increase in the prominence of the tremor, often falling on its side unable to regain upright posture 5. Although this genetic disorder has been named 'spastic', any relationship to human spasticity is not established. No pathological changes have been found in preliminary histological studies of muscle or central nervous system5. Symptoms in spastic homozygotes are alleviated with the administration of the pyridoxine antagonist aminooxyacetic acid (AOAA) 8, a drug which increases levels of ~,-aminobutyric acid ~GABA) in the central nervous systems. This finding suggested the hypothesis that the primary biochemical defect in this mutant might be a deficiency of GABA inhibition6; however, differences between spastic and normal littermates were not detected in whole brain homogenates assayed for GABA concentration6, or for glutamic acid decarboxylase or GABA-transaminase activityL Furthermore, AOAA is an inhibitor of most if not all transaminases and the transport of many amino acids and amines, and may exert pharmacological effects which are independent of the GABA system ~'~. In order to investigate the physiological mechanism of the neurological symptoms in the spastic mouse and to develop an objective method to assess 'spastic' symptoms in pharmacological experiments, we studied the electromyographic activity in spastic mice. METHODS
Experimental animals Homozygous spastic mice were produced from matings between B6C3a/a,
spa/+ heterozygotes obtained from the Jackson Laboratory (Bar Harbor, ME) and housed in the breeeding colony at Children's Hospital Medical Center. Affected animals were identified at age 4 weeks by assessment of tremor and ability to regain upright posture when turned on their backs. Since mice heterozygous for the spa gene are indistinguishable from homozygous normal mice, unaffected littermates were used as contrc.ls. C57BL./6J mice from the breeding colony at Children's Hospital were also used. Adult animals aged 3-6 months, weighing 20-25 g, were used for all studies. All mice were allowed free access to water and Purina Breeder Chow.
Electromyographic studies In order to study spontaneous electromyographic activity in unrestrained animals, we recorded from pairs of needle electrodes inserted into the flexor muscles of the proximal hindlimb and/or forelimb and secured with tape. In electrical stimulation studies, pairs of needle electrodes were inserted into the flexor muscles of the proximal hindlimb and proximal forelimb in restrained animals. Animals received an electric shock of 0.05 ms duration (0-150 V), and electromyographic activity was recorded in the contralateral muscles as described above, A ground wire was inserted subcutaneously over the abdomen. All recordings were made with a TECA TE-4 etectromyograph. Frequency response of EMG amplifiers was set at 300-3200 Hz (3 dB points).
301
Pharmacological studies Normal mice (C57BL/6J) were injected subcutaneously with strychnine (0.65 mg/kg) or picrotoxinin (0.8 mg/kg) (approximately half the convulsant dose for each drug)L Animals were observed before and after injection, and spontaneous EMG activity and EMG response to electric shock were recorded as described above. In some cases animals received 3.2 mg/kg picrotoxinin (convulsant dose), and studies were carried out until generalized convulsions and death occurred. RESULTS
Electromyographic recordings in unrestrained animals During spontaneous activity spastic homozygotes exhibited high amplitude repetitive EMG bursts at a frequency of 40-50 Hz (Fig. 1A). This activity was not seen in recordings of spontaneous activity in unaffected littermates or C57BL/6J controls (Fig. 1B). In spastic homozygotes, the EMG bursts could be triggered or accentuated by a sudden loud auditory stimulus (e.g. hand clap), by turning the animal on its A
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Fig. 1. EMG activity in hindlimb during walking in a spastic homozygote (A) and control (B). High amplitude repetitive bursts at a frequency of approximately 40 Hz are apparent in the spastic animal. The calibration bar for EMG amplitude represents 200 pV for A and 1000 pV for B.
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Fig. 2. Simultaneous EMG activity in hindlimb (upper trace) and forelimb (lower trace) in spastic homozygote held suspended by its tail. The calibration bar for EMG amplitude represents 400 #V for upper trace and 1000/~V for lower trace. back, or by holding the animal suspended by its tail. These stimuli produced an increase in amplitude of the electrical bursts which persisted for 30 s or more, while the observed tremor of the limbs and tail became more prominent. In simultaneous recordings from hindlimb and forelimb the bursts from hindlimb and forelimb were approximately coincident, the forelimb burst preceding the hindlimb burst by about 2-3 ms. (Fig. 2). Electromyographic response to electric shock In order to study the timing of abnormal E M G bursts following a controlled stimulus, we recorded the E M G response to electric shock. The response to electric shock delivered to hindlimb or forelimb in a typical spastic homozygote is shown in Fig. 3A, B. Shock to the hindlimb (25-50 V) produced a burst of activity in the contralateral hindlimb (latency of approximately 7 ms) followed by a burst in the contralateral forelimb (latency of approximately 8 ms). Shock to the forelimb (25-150 V) produced a burst in the contralateral forelimb (latency of approximately 6 ms) followed by a burst in the contralateral hindlimb (latency of approximately 8 ms). The duration of these abnormal bursts was 5-10 ms, and the amplitude was in the order of 1000/~V. In most spastic animals a second burst lower in amplitude occurred after an interval of about 10 ms in hindlimb and forelimb recordings. In some spastic animals there were two or more additional bursts from hindlimb or forelimb following electric shock (Fig. 4C, D). The E M G response to shock was stereotyped in latency, amplitude and duration in each spastic animal. The stimulus threshold for this E M G response in spastic animals was approximately 25 V in hindlimb or forelimb, and higher voltage electric shock (up to 150 V) produced no change in the latency, amplitude, or duration of the E M G response. Under identical conditions, with electrical shock up to 150 V, there was no comparable response in unaffected Iittermates or C57BL/6J animals tested (Fig. 3C, D). Often there was no short latency response at all. When a short latency response did
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Fig. 3. Simultaneous EMG records from hindlimb (upper trace) and forelimb (lower trace) following an electric stimulus. A: spastic homozygote, hindlimb stimulus. B: spastic homozygote, forelimb stimulus. C: control, hindlimb stimulus. D: control, forelimb stimulus. The electrical stimulus (40-50 V) was applied to the hindlimb or forelimb contralateral to the limbs from which EMG recordings were made, and EMG activity was recorded for the subsequent 45 ms. Calibration bar for EMG amplitude: A, 1000/~V upper and lower trace; B, 2000/~V upper and 5000/~V lower trace; C and D, 200/~V for upper and lower traces. occur in control animals, the response was inconsistent in form and latency, and the amplitude of the response was lower (100-200 /~V) than the response in spastic animals. Hindlimb stimulation occasionally produced a short latency response in the contralateral hindlimb, but produced no short latency response in the contralateral forelimb. Similarly, forelimb stimulation occasionally produced a short-latency response in the contralateral forelimb, but no response in the contralateral hindlimb. The response to electric shock in the spastic animals revealed no evidence to adaptation at stimulus rates from 0.5 to 100 Hz (4-8 stimuli). Any response seen in the control animals adapted rapidly.
Response to strychnine in normal mice Following a subcutaneous dose of 0.65 mg/kg strychnine (approximately half the convulsant dose in mice), normal mice (C57BL/6J) exhibited all the recognizable characteristics of spastic homozygotes including tremor, toe-walking gait, and delay in regaining upright posture when placed on its back. These effects were maximal at about 10 min following a single injection and persisted until about 40 rain after injection.
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Fig. 4. EMG records obtained simultaneously from forelimb (upper trace) and hindlimb (lower trace) following an electrical stimulus in three spastic homozygotcs (A, C and E, hindtimb stimulus; B) D and F, forelimb stimulus). Each record shows 3-5 stimuli at 2-s int~rvais demonstrating the stereotyped nature of the response. Calibration bar for EMO amplitude: A, 1000/~V upper trace and lower trace; B, 2000/~V upper and 5000/~V lower trace; C, 2000/~V upper and lower trace; D, 10,000/zV upper and lower trace; E, 2000/~V upper and lower trace; F, 2000/~V upper and lower trace. In animals treated with strychnine, simultaneous recordings of E M G activity in hindlimb and forelimb flexor muscles during spontaneous activity revealed high amplitude repetitive bursts at a frequency o f 40-50 H z (Fig. 5). In these animals an auditory oz tactile stimulus provoked an iaerease in tremor and the amplitude o f the E M G bursts (as sccn in spastic homozygotes). The response to an electric stimulus before and 30 rain after strychnine injection
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Fig. 5. Simultaneous EMG activity from hindlimb (upper trace) and forelimb (lower trace) in a normal C57BL/6J mouse 15 min after subcutaneous strychnine (0.65 ng/kg). The animal was held suspended by its tail. Note the high-amplitude repetitive bursts. Calibration bar for EMG amplitude represents 500 pV for upper and lower trace.
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Fig. 6. EMG activity obtained simultaneouslyfrom hindlimb (upper trace) and forelimb (lower trace) following an electrical stimulus in a normal C57BL/6J mouse before and 30 rain after subcutaneous strychnine 0.65 mg/kg: A, before strychnine, hindlimb stimulus; B, before strychnine, forelimb stimulus; C, after strychine, hindlimb stimulus; D, after strychnine, forelimb stimulus. The electrical stimulus was applied to the hindlimb or forelimb eontralateral to the limbs from which EMG activity was recorded, and EMG activity was recorded for the subsequent 45 ms. Calibration bar for EMG amplitude: A and B, 1000 pV upper and lower trace; C and D, 500 pV upper and lower trace.
306 is shown in Fig. 6. Following strychnine, shock to the hindlimb produced a burst of activity in the contralateral hindlimb (latency approximately 8 ms) followed by a burst in the contralateral forelimb (latency approximately 9 ms). Shock to the forelimb produced a burst in the contralateral forelimb (latency approximately 6 ms) followed by a burst in the contralateral hindlimb (latency approximately 8 ms). The bursts observed following strychnine injection were similar in duration (5-10 ms) and amplitude (1000 #V) to the abnormal bursts observed in spastic homozygotes. Superimposed tracings at 2 s intervals showed that this EMG response was stereotyped. Response to picrotoxinin in normal mice
Picrotoxinin at 0.80 mg/kg produced no apparent motor effects in normal (C57BL/6J) mice. A subcutaneous dose of 3.2 mg/kg produced generalized convulsions and death within 20 min, but no tremor or other 'spastic' symptoms were observed. No abnormal periodic bursts occurred in EMG recordings during spontaneous activity recorded continuously until the onset of seizure activity. The EMG response to electric shock was monitored before and up to 60 min after the administration of 0.8 mg/kg picrotoxinin, and no drug effect was apparent. This was also true when EMG response to electric shock was monitored continuously until the onset of generalized convulsions following injection of a convulsant dose. DISCUSSION Although the spastic mutation was first described in 1961, the etiology of its neurological features has not been determined. We have demonstrated abnoimal high amplitude regular repetitive EMG bursts in spastic animals during spontaneous activity, corresponding to the observed tremor in these animals. In addition, spastic homozygotes exhibit a characteristic EMG response following electrical stimulation of limb muscles. This 'spastic' response exhibits the following features: (1) the response is stereotyped with respect to latency, duration and amplitude, and is independent of the stimulus voltage (25-150 V); (2) the latency of the response is brief (less than t0 ms); (3) the hindlimb response precedes the forelimb response after electrical stimulation of the contralateral hindlimb; (4) the response shows no evidence of adaptation with repeated electrical stimuli; (5) the response can be elicited at a stimulus frequency as high as 100 Hz. We cannot as yet identify the exact neuronal pathways participating in this stereotyped 'spastic' response; however, the fact that the hindlimb response precedes the forelimb response after electrical stimulation of the eontralateral hindlimb is consistent with a response mediated through spinal cord. The abnormal response might result from several hypothetical mechanisms including abnormal neuronal connections in the spinal cord, failure of normal synaptic inhibition in the spinalcord (from local or descending pathways), or an increase in excitatory input to spinal cord neurons (from local or des~nding pathways). Tremor and other "spastic' symptoms might then result from this abnormal physiological state.
307 In order to further investigate the possibility of alteration in the synaptie activity of spinal cord pathways in spastic mutants, we studied the electrophysiological effects of strychnine and pierotoxinin in normal (C57BL/6J) mice. Strychnine produced all the observable features of spastic mice including the characteristic pattern of EMG activity during spontaneous movement and in response to electrical stimulation. The fact that strychnine produces tremor and the other clinical features of spastic homozygotes as well as the abnormal EMG response to electrical stimulation observed in spastic homozygotes implies that both phenomena may be related as consequences of a single disorder of neuronal function. Strychnine is a known antagonist of glyeine-mediated synaptic inhibition in the spinal cord 8. Strychnine suppresses inhibition by spinal (Ia) inhibitoly interneuronsla, Renshaw cell recurrent inhibition upon spinal motoneurons9, and Renshaw cell inhibition of Ia inhibitory interneurons 17. GABA is probably another major inhibitory transmitter in spinal cord is. Picrotoxinin, a potent convulsant and GABA antagonist~4, did not produce the behavioral or electrical signs seen in spastic animals when administered to normal mice in progressively higher doses up to the convulsant dose. Although alternative explanations are possible, our data suggest that the neurological defect in the spastic mutant may result from a failure of strychninesensitive (presumably glycinergic) inhibition in the spinal cord or an alteration in glycinergic pathways. Although strychnine is known to exert effects on GABA inhibition~2, a deficiency of GABA inhibition in this mutant seems less likely since picrotoxinin did not produce 'spastic' symptoms in subconvulsant doses. Should this hypothesis about glycine be true, the spastic mutant mouse would represent a single gene mutant giving rise to a deficiency in a specific neurotransmitter system. The mechanism of amelioration of symptoms in spastic animals by AOAA remains to be explained. This drug is known to increase levels of GABA in the CNS a, but it exerts other effects as well 14, including effects on glycine metabolism10,16 and glycine uptake in spinal cord 1~. AOAA has been shown to depress segmentally evoked dorsal and ventral root activity in acute spinal cats, but this effect appears to be independent of its effect on GABA levels4. Further delineation of the primary defect in spastic mutants will require pharmacological studies with more specific drugs as well as anatomical and biochemical studies of the spinal cord in these mutants. A final comment concerns the relevance of these findings to human disease. Changes in spinal cord glycine content have been demonstrated with the onset of symptoms of spasticity following spinal cord transectionlL Although the physiological findings in spastic mice are not identical to those of human spasticity, the symptoms in spastic mice (episodic tremor and stiffness provoked by an unexpected auditory or tactile stimulus) are remarkably similar to those described in some cases of startle disease or hyperekplexia1. Whether these genetic illnesses of mice and men share a common physiological or biochemical pathogenesis remains to be determined. ACKNOWLEDGEMENTS We would like to thank Drs. Marc A. Dichter and John Cowen for their
308 c o m m e n t s a n d suggestions, D i s . R i c h a r d S i d m a n a n d W. F r o s t W h i t e for reviewing the m a n u s c r i p t , a n d Ms. D i a n e K i l d a y for secretarial assistance. This w o r k was s u p p o r t e d b y N R S A F32 NS05770 f r o m the N I N C D S (to A . H . H . ) , N S 11237, a n d the C h i l d r e n ' s H o s p i t a l M e n t a l R e t a r d a t i o n C e n t e r Core G r a n t HD06276.
REFERENCES 1 Andermann, F., Keene, D. L., Andermann, E. and Quesney, L. F., Startle disease or hyperekplexia, further delineation of the syndrome, Brain, 103 (1980) 985-997. 2 Barnes, C. D. and Eltherington, L. G., Drug Dosage in Laboratory Animals, A Handbook, Univ. Calif. Press, Berkley, CA, 1973. 3 Baxter, C. F. and Roberts, E., Elevation of 7-aminobutyric acid in brain: selective inhibition of 7-aminobutyric-a-ketoglutaric acid transaminase, J. biol. Chem., 236 (1961) 3287-3294: 4 Bell, J. A. and Anderson, E. G., Dissociation between Amino-oxyacetic acid-induced depression of spinal reflexes and the rise in cord GABA levels, Neuropharmacology, 13 (1974) 885-894i 5 Chai, C. K., Hereditary spasticity in mice, J. Heredity, 52 (1961) 241-243. 6 Chai, C. K., Roberts, E. and Sidman, R. L., Influence of Amino-oxyaceticacid, a gamma-butyrate transaminase inhibitor on hereditary spastic defect in the mouse, Proc. Soc. exp. BioL Med., 109 (1961) 491-495. 7 Chatterjee, S. and Hechtman, P., Gamma-aminobutyric acid metabolism in brain homogenates of the spastic mouse, Biochem. Genet., 15 (1977) 147-151. 8 Curtis, D. R., Duggan, A. W. and Johnston, G. A. R., The specificity of strychnine as a glycine antagonist in the mammalian spinal cord, Exp. Brain Res., 12 (1971) 547-565. 9 Curtis, D., Game, C., Lodge, D. and McCuUoch, R., A pharmacological study of renshaw cell inhibition, J. Physiol. (Lond.), 258 (1976) 227-242. 10 Davies, L. P. and Johnston, G. A. R., Serine hydroxymethyltransferase in the central nervous system: regional and subcellular distribution studies, Brain Research, 54 (1973) 149-156. 11 Hall, P. V., Smith, J. E., Campbell, R. L., Felten, D. L. and Aprison, M. H., Neurochemical correlates of spasticity, Life Sci., 18 (1976) 1467-1472. 12 Hill, R. G., Simmonds, M. A. and Straughan, D. W., Antagonism of 7-aminobutyric acid and glycine by convulsants in the cuneate nucleus of the cat, Brit. J. Pharmacol., 56 (1976) 9-19. 13 Janowska, E. and Lindstrom, S., Morphology of interneurones mediating la reciprocal inhibition of motoneurones in the spinal cord of the cat, J. Physiol. (Lond.), 226 (1972) 805-823. 14 Johnston, G. A. R., Neuropharmacology of amino acid inhibitory transmitters, Ann. Rev. PharmacoL Toxicol., 18 (1978) 269-289. 15 Johnston, G. A. R. and Balcar, V. J., Amino-oxyacetic acid: a relatively non-specific inhibitor of uptake of amino acids and amines by brain and spinal cord, J. Neurochem., 22 (1974) 609-610. 16 Johnston, G. A. R. and Vitali, M. V., Glycine: 2-oxoglutarate transaminase in rat cerebral cortex, Brain Research, 15 (1969) 201-208. 17 Lodge, D., Curtis, D. R, and Brand, S. J., A pharmacological study of ventral group Ia-excited spinal interneurones, Exp. Brain Res., 29 (1977) 97-105. 18 McLaughlin, B., Barber, R., Saito, K., Roberts, E. and Wu, J. Y., lmmunocytochemical localization of glutamate decarboxylase in rat spinal cord, J. comp. Neurol., 164 (1975) 305-321.
299
E L E C T R O P H Y S I O L O G I C A L STUDIES W I T H T H E SPASTIC M U T A N T MOUSE
ALLEN H. HELLER* and MARK HALLETT Department of Neurology, Children's Hospital Medical Center and Section of Neurology, Department of Medicine, Brigham and Women's Hospital, and the Department of Neurology, Harvard Medical School, CHMC, 300 Longwood Avenue, Boston, MA 02115 (U.S.A.)
(Accepted July 30th, 1981) Key words: spastic mutant mouse - - electromyography -- strychnine - - glyeine inhibition - - picrotoxinin - - GABA inhibition
SUMMARY Electromyographic (EMG) studies were carried out with the genetically spastic mouse (spa, autosomal recessive), obtained from matings of B6C3a/a, spa/+ heterozygotes. Spastic homozygotes exhibited high amplitude repetitive E M G bursts during spontaneous activity. Following an electrical stimulus to hindlimb or forelimb, high amplitude stereotyped E M G bursts were recorded from eontralateral limbs in spastic mice, but were not observed in phenotypically unaffected littermates or normal C57BL/6J mice. The timing and latency of this stereotyped response to an electrical stimulus was consistent with the participation of spinal cord neuronal pathways. In normal C57BL/6J mice the administration of strychnine (0.65 mg/kg), but not picrotoxinin (up to convulsant doses), reproduced all of the behavioral and E M G features observed in spastic homozygotes. We hypothesize that the symptoms in the spastic mutant may result from a deficiency of strychnine-sensitive (presumably glycinergic) inhibition in the spinal cord.
INTRODUCTION The 'spastic' mutant mouse (spa, autosomal recessive) exhibits a neuromuscular disorder characterized by episodes of rapid tremor of limbs and tail, lack of flexibility of trunk movements, toe-walking gait, and difficulty in regaining upright posture when placed on its back. When stimulated by shaking of its cage or a sudden loud noise, the * Address for all correspondence: Box 117, Children's Hospital Medical Center, 300 Longwood Avenue, Boston, MA 02115, U.S.A. 0006-8993/82/0000-0000/$02.75 © Elsevier Biomedical Press
300 animal appears to stiffen with an increase in the prominence of the tremor, often falling on its side unable to regain upright posture 5. Although this genetic disorder has been named 'spastic', any relationship to human spasticity is not established. No pathological changes have been found in preliminary histological studies of muscle or central nervous system5. Symptoms in spastic homozygotes are alleviated with the administration of the pyridoxine antagonist aminooxyacetic acid (AOAA) 8, a drug which increases levels of ~,-aminobutyric acid ~GABA) in the central nervous systems. This finding suggested the hypothesis that the primary biochemical defect in this mutant might be a deficiency of GABA inhibition6; however, differences between spastic and normal littermates were not detected in whole brain homogenates assayed for GABA concentration6, or for glutamic acid decarboxylase or GABA-transaminase activityL Furthermore, AOAA is an inhibitor of most if not all transaminases and the transport of many amino acids and amines, and may exert pharmacological effects which are independent of the GABA system ~'~. In order to investigate the physiological mechanism of the neurological symptoms in the spastic mouse and to develop an objective method to assess 'spastic' symptoms in pharmacological experiments, we studied the electromyographic activity in spastic mice. METHODS
Experimental animals Homozygous spastic mice were produced from matings between B6C3a/a,
spa/+ heterozygotes obtained from the Jackson Laboratory (Bar Harbor, ME) and housed in the breeeding colony at Children's Hospital Medical Center. Affected animals were identified at age 4 weeks by assessment of tremor and ability to regain upright posture when turned on their backs. Since mice heterozygous for the spa gene are indistinguishable from homozygous normal mice, unaffected littermates were used as contrc.ls. C57BL./6J mice from the breeding colony at Children's Hospital were also used. Adult animals aged 3-6 months, weighing 20-25 g, were used for all studies. All mice were allowed free access to water and Purina Breeder Chow.
Electromyographic studies In order to study spontaneous electromyographic activity in unrestrained animals, we recorded from pairs of needle electrodes inserted into the flexor muscles of the proximal hindlimb and/or forelimb and secured with tape. In electrical stimulation studies, pairs of needle electrodes were inserted into the flexor muscles of the proximal hindlimb and proximal forelimb in restrained animals. Animals received an electric shock of 0.05 ms duration (0-150 V), and electromyographic activity was recorded in the contralateral muscles as described above, A ground wire was inserted subcutaneously over the abdomen. All recordings were made with a TECA TE-4 etectromyograph. Frequency response of EMG amplifiers was set at 300-3200 Hz (3 dB points).
301
Pharmacological studies Normal mice (C57BL/6J) were injected subcutaneously with strychnine (0.65 mg/kg) or picrotoxinin (0.8 mg/kg) (approximately half the convulsant dose for each drug)L Animals were observed before and after injection, and spontaneous EMG activity and EMG response to electric shock were recorded as described above. In some cases animals received 3.2 mg/kg picrotoxinin (convulsant dose), and studies were carried out until generalized convulsions and death occurred. RESULTS
Electromyographic recordings in unrestrained animals During spontaneous activity spastic homozygotes exhibited high amplitude repetitive EMG bursts at a frequency of 40-50 Hz (Fig. 1A). This activity was not seen in recordings of spontaneous activity in unaffected littermates or C57BL/6J controls (Fig. 1B). In spastic homozygotes, the EMG bursts could be triggered or accentuated by a sudden loud auditory stimulus (e.g. hand clap), by turning the animal on its A
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Fig. 1. EMG activity in hindlimb during walking in a spastic homozygote (A) and control (B). High amplitude repetitive bursts at a frequency of approximately 40 Hz are apparent in the spastic animal. The calibration bar for EMG amplitude represents 200 pV for A and 1000 pV for B.
302
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Fig. 2. Simultaneous EMG activity in hindlimb (upper trace) and forelimb (lower trace) in spastic homozygote held suspended by its tail. The calibration bar for EMG amplitude represents 400 #V for upper trace and 1000/~V for lower trace. back, or by holding the animal suspended by its tail. These stimuli produced an increase in amplitude of the electrical bursts which persisted for 30 s or more, while the observed tremor of the limbs and tail became more prominent. In simultaneous recordings from hindlimb and forelimb the bursts from hindlimb and forelimb were approximately coincident, the forelimb burst preceding the hindlimb burst by about 2-3 ms. (Fig. 2). Electromyographic response to electric shock In order to study the timing of abnormal E M G bursts following a controlled stimulus, we recorded the E M G response to electric shock. The response to electric shock delivered to hindlimb or forelimb in a typical spastic homozygote is shown in Fig. 3A, B. Shock to the hindlimb (25-50 V) produced a burst of activity in the contralateral hindlimb (latency of approximately 7 ms) followed by a burst in the contralateral forelimb (latency of approximately 8 ms). Shock to the forelimb (25-150 V) produced a burst in the contralateral forelimb (latency of approximately 6 ms) followed by a burst in the contralateral hindlimb (latency of approximately 8 ms). The duration of these abnormal bursts was 5-10 ms, and the amplitude was in the order of 1000/~V. In most spastic animals a second burst lower in amplitude occurred after an interval of about 10 ms in hindlimb and forelimb recordings. In some spastic animals there were two or more additional bursts from hindlimb or forelimb following electric shock (Fig. 4C, D). The E M G response to shock was stereotyped in latency, amplitude and duration in each spastic animal. The stimulus threshold for this E M G response in spastic animals was approximately 25 V in hindlimb or forelimb, and higher voltage electric shock (up to 150 V) produced no change in the latency, amplitude, or duration of the E M G response. Under identical conditions, with electrical shock up to 150 V, there was no comparable response in unaffected Iittermates or C57BL/6J animals tested (Fig. 3C, D). Often there was no short latency response at all. When a short latency response did
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Fig. 3. Simultaneous EMG records from hindlimb (upper trace) and forelimb (lower trace) following an electric stimulus. A: spastic homozygote, hindlimb stimulus. B: spastic homozygote, forelimb stimulus. C: control, hindlimb stimulus. D: control, forelimb stimulus. The electrical stimulus (40-50 V) was applied to the hindlimb or forelimb contralateral to the limbs from which EMG recordings were made, and EMG activity was recorded for the subsequent 45 ms. Calibration bar for EMG amplitude: A, 1000/~V upper and lower trace; B, 2000/~V upper and 5000/~V lower trace; C and D, 200/~V for upper and lower traces. occur in control animals, the response was inconsistent in form and latency, and the amplitude of the response was lower (100-200 /~V) than the response in spastic animals. Hindlimb stimulation occasionally produced a short latency response in the contralateral hindlimb, but produced no short latency response in the contralateral forelimb. Similarly, forelimb stimulation occasionally produced a short-latency response in the contralateral forelimb, but no response in the contralateral hindlimb. The response to electric shock in the spastic animals revealed no evidence to adaptation at stimulus rates from 0.5 to 100 Hz (4-8 stimuli). Any response seen in the control animals adapted rapidly.
Response to strychnine in normal mice Following a subcutaneous dose of 0.65 mg/kg strychnine (approximately half the convulsant dose in mice), normal mice (C57BL/6J) exhibited all the recognizable characteristics of spastic homozygotes including tremor, toe-walking gait, and delay in regaining upright posture when placed on its back. These effects were maximal at about 10 min following a single injection and persisted until about 40 rain after injection.
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Fig. 4. EMG records obtained simultaneously from forelimb (upper trace) and hindlimb (lower trace) following an electrical stimulus in three spastic homozygotcs (A, C and E, hindtimb stimulus; B) D and F, forelimb stimulus). Each record shows 3-5 stimuli at 2-s int~rvais demonstrating the stereotyped nature of the response. Calibration bar for EMO amplitude: A, 1000/~V upper trace and lower trace; B, 2000/~V upper and 5000/~V lower trace; C, 2000/~V upper and lower trace; D, 10,000/zV upper and lower trace; E, 2000/~V upper and lower trace; F, 2000/~V upper and lower trace. In animals treated with strychnine, simultaneous recordings of E M G activity in hindlimb and forelimb flexor muscles during spontaneous activity revealed high amplitude repetitive bursts at a frequency o f 40-50 H z (Fig. 5). In these animals an auditory oz tactile stimulus provoked an iaerease in tremor and the amplitude o f the E M G bursts (as sccn in spastic homozygotes). The response to an electric stimulus before and 30 rain after strychnine injection
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Fig. 5. Simultaneous EMG activity from hindlimb (upper trace) and forelimb (lower trace) in a normal C57BL/6J mouse 15 min after subcutaneous strychnine (0.65 ng/kg). The animal was held suspended by its tail. Note the high-amplitude repetitive bursts. Calibration bar for EMG amplitude represents 500 pV for upper and lower trace.
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Fig. 6. EMG activity obtained simultaneouslyfrom hindlimb (upper trace) and forelimb (lower trace) following an electrical stimulus in a normal C57BL/6J mouse before and 30 rain after subcutaneous strychnine 0.65 mg/kg: A, before strychnine, hindlimb stimulus; B, before strychnine, forelimb stimulus; C, after strychine, hindlimb stimulus; D, after strychnine, forelimb stimulus. The electrical stimulus was applied to the hindlimb or forelimb eontralateral to the limbs from which EMG activity was recorded, and EMG activity was recorded for the subsequent 45 ms. Calibration bar for EMG amplitude: A and B, 1000 pV upper and lower trace; C and D, 500 pV upper and lower trace.
306 is shown in Fig. 6. Following strychnine, shock to the hindlimb produced a burst of activity in the contralateral hindlimb (latency approximately 8 ms) followed by a burst in the contralateral forelimb (latency approximately 9 ms). Shock to the forelimb produced a burst in the contralateral forelimb (latency approximately 6 ms) followed by a burst in the contralateral hindlimb (latency approximately 8 ms). The bursts observed following strychnine injection were similar in duration (5-10 ms) and amplitude (1000 #V) to the abnormal bursts observed in spastic homozygotes. Superimposed tracings at 2 s intervals showed that this EMG response was stereotyped. Response to picrotoxinin in normal mice
Picrotoxinin at 0.80 mg/kg produced no apparent motor effects in normal (C57BL/6J) mice. A subcutaneous dose of 3.2 mg/kg produced generalized convulsions and death within 20 min, but no tremor or other 'spastic' symptoms were observed. No abnormal periodic bursts occurred in EMG recordings during spontaneous activity recorded continuously until the onset of seizure activity. The EMG response to electric shock was monitored before and up to 60 min after the administration of 0.8 mg/kg picrotoxinin, and no drug effect was apparent. This was also true when EMG response to electric shock was monitored continuously until the onset of generalized convulsions following injection of a convulsant dose. DISCUSSION Although the spastic mutation was first described in 1961, the etiology of its neurological features has not been determined. We have demonstrated abnoimal high amplitude regular repetitive EMG bursts in spastic animals during spontaneous activity, corresponding to the observed tremor in these animals. In addition, spastic homozygotes exhibit a characteristic EMG response following electrical stimulation of limb muscles. This 'spastic' response exhibits the following features: (1) the response is stereotyped with respect to latency, duration and amplitude, and is independent of the stimulus voltage (25-150 V); (2) the latency of the response is brief (less than t0 ms); (3) the hindlimb response precedes the forelimb response after electrical stimulation of the contralateral hindlimb; (4) the response shows no evidence of adaptation with repeated electrical stimuli; (5) the response can be elicited at a stimulus frequency as high as 100 Hz. We cannot as yet identify the exact neuronal pathways participating in this stereotyped 'spastic' response; however, the fact that the hindlimb response precedes the forelimb response after electrical stimulation of the eontralateral hindlimb is consistent with a response mediated through spinal cord. The abnormal response might result from several hypothetical mechanisms including abnormal neuronal connections in the spinal cord, failure of normal synaptic inhibition in the spinalcord (from local or descending pathways), or an increase in excitatory input to spinal cord neurons (from local or des~nding pathways). Tremor and other "spastic' symptoms might then result from this abnormal physiological state.
307 In order to further investigate the possibility of alteration in the synaptie activity of spinal cord pathways in spastic mutants, we studied the electrophysiological effects of strychnine and pierotoxinin in normal (C57BL/6J) mice. Strychnine produced all the observable features of spastic mice including the characteristic pattern of EMG activity during spontaneous movement and in response to electrical stimulation. The fact that strychnine produces tremor and the other clinical features of spastic homozygotes as well as the abnormal EMG response to electrical stimulation observed in spastic homozygotes implies that both phenomena may be related as consequences of a single disorder of neuronal function. Strychnine is a known antagonist of glyeine-mediated synaptic inhibition in the spinal cord 8. Strychnine suppresses inhibition by spinal (Ia) inhibitoly interneuronsla, Renshaw cell recurrent inhibition upon spinal motoneurons9, and Renshaw cell inhibition of Ia inhibitory interneurons 17. GABA is probably another major inhibitory transmitter in spinal cord is. Picrotoxinin, a potent convulsant and GABA antagonist~4, did not produce the behavioral or electrical signs seen in spastic animals when administered to normal mice in progressively higher doses up to the convulsant dose. Although alternative explanations are possible, our data suggest that the neurological defect in the spastic mutant may result from a failure of strychninesensitive (presumably glycinergic) inhibition in the spinal cord or an alteration in glycinergic pathways. Although strychnine is known to exert effects on GABA inhibition~2, a deficiency of GABA inhibition in this mutant seems less likely since picrotoxinin did not produce 'spastic' symptoms in subconvulsant doses. Should this hypothesis about glycine be true, the spastic mutant mouse would represent a single gene mutant giving rise to a deficiency in a specific neurotransmitter system. The mechanism of amelioration of symptoms in spastic animals by AOAA remains to be explained. This drug is known to increase levels of GABA in the CNS a, but it exerts other effects as well 14, including effects on glycine metabolism10,16 and glycine uptake in spinal cord 1~. AOAA has been shown to depress segmentally evoked dorsal and ventral root activity in acute spinal cats, but this effect appears to be independent of its effect on GABA levels4. Further delineation of the primary defect in spastic mutants will require pharmacological studies with more specific drugs as well as anatomical and biochemical studies of the spinal cord in these mutants. A final comment concerns the relevance of these findings to human disease. Changes in spinal cord glycine content have been demonstrated with the onset of symptoms of spasticity following spinal cord transectionlL Although the physiological findings in spastic mice are not identical to those of human spasticity, the symptoms in spastic mice (episodic tremor and stiffness provoked by an unexpected auditory or tactile stimulus) are remarkably similar to those described in some cases of startle disease or hyperekplexia1. Whether these genetic illnesses of mice and men share a common physiological or biochemical pathogenesis remains to be determined. ACKNOWLEDGEMENTS We would like to thank Drs. Marc A. Dichter and John Cowen for their
308 c o m m e n t s a n d suggestions, D i s . R i c h a r d S i d m a n a n d W. F r o s t W h i t e for reviewing the m a n u s c r i p t , a n d Ms. D i a n e K i l d a y for secretarial assistance. This w o r k was s u p p o r t e d b y N R S A F32 NS05770 f r o m the N I N C D S (to A . H . H . ) , N S 11237, a n d the C h i l d r e n ' s H o s p i t a l M e n t a l R e t a r d a t i o n C e n t e r Core G r a n t HD06276.
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