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An analysis of the myoclonic jerks produced by 1,2-dihydroxybenzene in the rat
Electroencephalography and Clinical Neurophysiology, 1973, 35:58%601 (c ~Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands
AN ANALYSIS
OF THE
MYOCLONIC
B Y 1, 2 - D I H Y D R O X Y B E N Z E N E
JERKS
589
PRODUCED
IN THE RAT
A. ANGELAND R. N. LEMON Department of Physiolooy, The University, Western Bank, Sheffield $10 2TN (Great Britain) (Accepted for publication: June 6, 1973)
The characteristic action of 1,2 dihydroxy- were used. In the acute experiments, each animal benzene (catechol) and other hydroxybenzenes was immobilized with ether and then given an in the anaesthetized animal is to produce intraperitoneal (i.p.) injection of urethane (25~o "spontaneous" convulsive jerks and twitches in in 0.9~ saline in a 1.0 ml per 200 g body weight the absence of any intentional stimulus (Brieger primary dose). The final anaesthetic dose was 1879; Bacq 1936; Angel and Lemon 1973). In adjusted so that reflex withdrawal of the limbs to addition to this type of convulsive activity, Angel a strong pinch was just abolished, and this and Dawson (1964) observed in rats an- usually required between 1.3 and 1.5 g/kg aesthetized with urethane that after an intrave- urethane. Procedures for fixation, stimulation, nous injection of catechol, an external stimulus recording of electrical and mechanical responses produced a brief myoclonic jerk. The stimulus of the limb muscles, and for chronic spinal could be either in the form of a sharp auditory transection have been fully described in a click or a brief electrical shock applied to one of previous paper (Angel and Lemon 1973). It the limbs ; the type of stimulus was not critical. should be restated, since it is an important point, Angel (1969) has shown that the stimulus- that stimulation and recording were made on sensitive state induced by catechol is associated the same limb. with a greatly increased transmission through The spinal column was rigidly fixed and spinal the dorsal column medial lemniscal sensory laminectomy performed from L4 to S1 ; the dura pathway. Thus in the rat, electrical responses was reflected and the dorsal and ventral roots recorded from the thalamic sensory relay nucleus giving rise to the sciatic trunk (Green 1935; and primary cortical receiving area in response Kaizawa and Takahashi 1970a) were prepared to a test stimulus applied to the periphery were for recording. Where necessary spinal transection greatly increased in size after catechol injection, was performed between T10 and T12. A pool was and remained so for the entire convulsive period. constructed from the cut skin edges and filled with These results bear an interesting resemblance to liquid paraffin B.P. (equilibrated with physiothe clinical studies of patients suffering from logical saline) maintained at 37°C. In the hind certain types of sensory myoclonus, where there limb, the sural, peroneal and final nerves were is an abnormal motor jerk to a sensory stimulus exposed and separated, as were the median and (Dawson 1946, 1947; Halliday, 1967). ulnar nerves in the forelimb. Mechanical stimulaThis paper presents a study of the muscle tion was obtained with a modified loudspeaker responses evoked by a variety of stimuli in with a probe attached to the coil. This gave a anaesthetized rats given catechol ; a preliminary monophasic output with a time to peak of account of the work lias already been published 0.4 msec, a duration of 1.4 msec and an excursion (Angel et al. 1968). of the tip approximately of 80 # with no following oscillation. METHODS The experiments were performed on animals One hundred and three female albino rats given i.p. injections of 60 mg/kg catechol, and
590 this dose was sufficient to produce convulsions in all the animals studied. In no case did this dose prove lethal. Intravenous (i.v.) injections were made via a polythene cannula in the femoral or jugular vein. The i.v. dose varied between 3 and 8 mg/kg (Angel 1969). Catechol (1,2 dihydroxybenzene; British Drug Houses) was made up in a solution of 10 mg/ml in 0.9~ saline (w/v) at 37°C. RESULTS
a. Responses in the limb muscles to electrical stimulation at the periphery In animals deeply anaesthetized with urethane, electrical stimulation of the fore- and hind paws through lint pads soaked in 3 M NaC1 produced no electromyographic (EMG) response in any of the limb muscles studied (Fig. 1, A, C) and no motor volley could be recorded in either the appropriate ventral root or peripheral nerve. In contrast, shortly after an i.v. injection of catechol (4-6 mg/kg), a complex EMG response with a duration of 50-70 msec was produced by each stimulus, and this response was always associated with a sharp contraction of the muscles of the limb to which the stimulus had been applied. The complex consisted of three distinct components (Fig. 1, B, D) and the mean latencies of these components were 4.3, 13.4 and 40.0 msec in the forelimb, and 8.1, 19.4 and 51.4 msec in the hindlimb (see Table I). The duration of this effect was relatively short lived (Angel 1969); the peak-to-peak voltage of the responses reached a maximum 30-45 sec after injection, then declined and had usually disappeared within 5 min. Since the convulsive effects of catechol are more prolonged after i.p. administration, injection of catechol by this route was employed for a more detailed study of the muscular responses. Fig. 2, A - H shows a typical series of records from the biceps brachii muscle of the rat at different times after an i.p. injection. The three responses all appeared about 1 min after injection (Fig. 2, B) and reached maximum size at approximately 3 min (Fig. 2, C), after which there was a rapid decline in the amplitude of the responses (Fig. 2, D-G). At 20 min the effect had disappeared (Fig. 2, H). The mean amplitude of each of the three
A. ANGEL AND R. N. LEMON
responses recorded from the rat biceps brachii muscle was measured in 12 rats and plotted against the time after injection (Fig. 2, J). Because the amplitude of the response varied considerably from experiment to experiment, the results have been expressed graphically as a percentage of the maximum peak-to-peak voltage recorded. The pattern shown in Fig. 2 was much the same for the hind limb muscle responses, although these often persisted for up to 25 or 30 rain after injection (Fig. 5). In Fig. 2, K the
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591
MYOCLONIC JERKS BY 1,2-DIHYDROXYBENZENE TABLE I Latency, amplitude and probability of occurrence of the three evoked muscle responses. 1st
2nd
3rd
A. Electrical stimulation
Forelimb : (N = 53)
Triceps brachii Latency (msec) Probability Relative amplitude
4.30( + 0.6) 0.97( _+0.05) 1.00( ± 0.04)
13.40(+ 1.6) 0.59( + 0.09) 0.40( + 0.02)
40.00( + 7.0) 0.17( + 0.10) 0.17( +_0.02)
Hind limb : (N = 53)
Gastrocnemius Latency (msec) Probability Relative amplitude
8.10( + 0.9) 0.91 ( + 0.05) 1.00(+0.05)
19.40( + 3.7) 0.35( + 0.08) 0.15(+0.03)
51.40( + 7.3) 0.14( + 0.08) 0.12(+0.03)
Hind limb : (N = 13)
Tibialis anterior Latency (msec) Probability Relative amplitude
8.30( _+ 1.1) 0.90( + 0.09) 1.00( + 0.20)
20.00( ± 2.4) 0.57( + 0.09) 0.60( + 0.20)
m
B. Mechanical stimulation
Forelimb : (N = 8)
Triceps brachii Latency (msec)
5.5( + 0.9)
15.8( + 2.5)
44.8( + 5.4)
Hind limb : (N = 8)
Gastrocnemius Latency (msec)
8.9( + 1.4)
21.6( _+2.5)
48.1( + 3.5)
A. E•ectrica• stimu•ati•n. Data are sh•wn f•r resp•nses re••rd•d
in the triceps brachii t• e•ectrica• stimu•ati•n
•f the ipsi•atera•
forepaw, and in the gastrocnemius and tibialis anterior to electrical stimulation of the ipsilateral hind paw. The latency and probability of occurrence of each of the three responses was determined together with the maximum recorded voltage of each response. The results are the means from the number (N) of experiments given, together with the standard deviation from the mean (in brackets), except in the case of the amplitude, where the mean is given with the standard error from the mean. The amplitudes of the three responses in each muscle have been expressed in relative terms, taking the mean maximum amplitude of the first response as unity. All the results are derived from observations made on the E M G responses recorded between 2 and 10 min after an i.p. injection of catechol (60 mg/kg). B. Mechanical stimulation. The table shows the mean latency (together with the standard deviation in brackets) of the three responses evoked by mechanical stimulation of the fore- or hind paw in the ipsilateral triceps brachii and gastrocnemius respectively.
amplitude of the first response recorded from the triceps brachii of a rat had been plotted against time for two injections separated by 1 h. The time course of the responses recorded after each injection was practically identical, and follows the same pattern as the averaged results shown in Fig. 2, J. The mean latency, amplitude and probability of occurrence for the three responses recorded from the triceps brachii and from the gastrocnemius and tibialis anterior are shown in Table I. The first response was always the largest, had the least latency scatter and the highest probability
of occurrence. The probability of occurrence of each of the three components was determined during the time that catechol exerted its maximum effect, i.e., 2-10 min after i.p. injection. The probability was taken as unity if the component occurred to each of 20 consecutive stimuli (1/sec) applied in batches at 1 min intervals throughout this time. The second and third responses were smaller in voltage and probability of occurrence, and had a greater latency scatter. The second response was more easily obtained from the forelimb muscles; in the hind limb it was more frequently observed in the flexor (tibialis anterior)
592
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Fig. 2. Time course of the E M G responses in the rat. EMG response of the triceps brachii muscle to stimulation of the ipsilateral forepaw before (A) and at 1 (B), 3 (C), 6 (D), 9 (E), 12 (F), 15 (G) and 20 (H) min after an i.p. injection of catechol (60 mg/kg) in a rat anaesthetized with urethane. Body temperature 37°C. Calibrations : 250/~V and 10 msec. J : The amplitudes of the first (O), second (©) and third (A) responses recorded from the triceps brachii muscle to stimulation of the forepaw, have been plotted against the time after an i.p. injection of catechol (60 mg/kg). Each point represents the amplitude of the response expressed as a percentage of the maximum amplitude recorded. The results are the mean from 12 different animals. K : The amplitude time course of the first response recorded from the triceps brachii in an anaesthetized rat. The response amplitude (in mV) recorded after two identical injections of catechol, the second ((3) given 1 h after the first (O), has been plotted against the time after each injection. Body temperature 37°C.
than in the extensor (gastrocnemius). The amplitude of these evoked responses was of the same order as that of the "spontaneous" responses recorded from the same muscle (Angel and Lemon 1973), although dose/response experiments revealed that the evoked jerks could be obtained at lower doses than the spontaneous variety. In anaesthetized rats the i.p. EDs0 (dose required to produce an effect in 50~o of the animals studied) determined by the method of Weil (1952), was 30 mg/kg for myoclonic jerks evoked by peripheral electrical stimulation compared with 49 mg/kg for spontaneous jerks. Simultaneous recordings of the mechanical and electrical response from the triceps brachii are shown in Fig. 3, A - C . The first evoked response was always associated with a "twitchlike" contraction of the muscle (Fig. 3, A). These longer-latency second and third responses were usually smaller than the first response and produced tension changes which summated with the first response twitch (Fig. 3, B, C).
The evoked muscle responses could be recorded from direct antagonist muscles at similar latencies and with the same probability of occurrence. Because of the possibility of "crosstalk" existing in the recording system, where, owing to the spread of bio-electric activity, the EMG response of an active muscle could be recorded in a remote inactive muscle, experiments were performed in which the hind leg was completely denervated sparing only the sural (sensory) nerve and the muscle nerve supplying either the gastrocnemius or the tibialis anterior. These experiments demonstrated that
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Fig. 3. A - C : Tension changes during the evoked muscle response. Simultaneous single-sweep oscilloscope records of the tension changes in the rat triceps brachii (upper trace in each record) and the E M G of the same muscle (lower trace). The records, taken from 3 to 10 min after injection ofcatechol (60 mg/kg i.p.) show that a single electrical shock applied to the ipsilateral forepaw produced a typical early muscle response either alone (A), or with the third response (B), or with both the second and third responses (C). Each spike in the EMG was associated with a change in muscle tension, developing from a single twitch in A to a summated response in B and C. Tension calibration : 100 g. D-F : E M G response of the gastrocnemius (upper trace in each record) and tibialis anterior (lower trace) in the hind limb of the rat to peripheral electrical stimulation after catechol injection. The early (first) response could be recorded from both muscles at similar latencies (D). After section of the peroneal nerve, the amplitude of the tibialis response was greatly reduced and its shape completely altered (E). The remaining "activity" demonstrated that there was a small amount of electronic spread between the two muscles. Section of the nerve supply to the gastrocnemius abolished both responses (F). Calibration: 250 pV.
MYOCLONIC JERKS BY
1,2-DIHYDROXYBENZENE
crosstalk between the calf muscles only accounts for a small fraction of the response in the intact preparation (Fig. 3, D-F). Additionally comparison of simultaneous records taken from the gastrocnemius and tibialis anterior showed that one muscle could be active while the other was silent. b. Limb muscle responses to other types of stimuli Most of the experiments on the evoked muscle responses were performed using electrical
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Fig. 4. Muscle responses to mechanical and auditory stimuli after catechol. A and B : Single sweep record of the E M G response of triceps brachii (A) to a light mechanical tap applied to the tactile pads of the ipsilateral forepaw after injection of catechol (60 mg/kg i.p.). The response in the gastrocnemius to a tap applied to the hind paw is shown in B. C and D: Simultaneous single sweep records of the response in the triceps (C) and gastrocnemius (D) to a binaural auditory stimulus after injection of catechol. A sharp (0.1 msec) click was delivered through a loudspeaker at the beginning of the sweep. Voltage calibration : 250/~V. Time: 1, 5 and 10 msec.
593 stimulation, but it was established that similar types of muscle response could be evoked by physiological stimuli. As shown in Fig. 4, A, B, a light mechanical tap applied to the sole of the paw produced muscle responses similar to those evoked by electrical stimulation, although the mean latencies were slightly greater (Table I). A binaural auditory stimulus, in the form of a sharp click, frequently produced a generalized body convulsion after injection of catechol. An early response in the forelimb with a mean latency of 5.7 (SD __+_0.7) msec and in the hind limb at 8.4 (_+0.8) msec could be recorded, and there'was also a later response in both limbs, at 19.3 (+3.5) and 21.8 (+3.2) msec respectively (Fig. 4, C, D). Visual stimuli were not effective in producing a generalized body convulsion; only small localized jerks were occasionally observed and these were usually difficult to distinguish from any on-going spontaneous activity. c. Limb muscle responses in spinal and pithed animals After acute (12 animals) or chronic (8 day; 13 animals) section of the spinal cord at the level T10-T 12, the gastrocnemius response to stimulation of the hind paw after catechol consisted of the first component only (7-9 msec). The second and third components of the muscle response were never observed in the hind limb muscles of these rats (Fig. 5, O), although the forelimb muscles remained unaffected (Fig. 5, B). In the acute preparations, comparison of the responses evoked before and 1 h after spinal section showed that the amplitude of the first response was always greater in the spinal animal. In addition, the time course of the remaining first response was prolonged and lasted for up to 40-45 min, compared with 20-25 min in the intact animal (Fig. 5). The evoked muscle responses were completely abolished by denervation of the appropriate muscles or by destruction of the spinal cord. d. Reflex pathway of the evoked muscle response Recordings from the L5 dorsal root entry zone showed that threshold stimulation of the sural nerve (cutaneous) at the knee and supramaximal stimulation at the ankle (supramaximal to overcome the anomalous effect of stimulating nerve trunks through the skin with a diffuse
594
A. ANGEL AND R. N. LEMON
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recorded from the ventral root or muscle nerve (see also Fig. 9, A). The latency of the L5 ventral root volley corresponding to the first muscleevoked response was recorded in 15 experiments and the results are presented in Fig. 6. Thrbshold stimulation of the tibial nerve produced a first response of consistent short latency and large amplitude (Fig. 6, D). The mean latency of this response at the ventral root was 3.18 (S.D. _+ 0.31) msec and the spinal delay (from L5 dorsal root to L5 ventral root) was 2.18 (_+0.21) msec. The response followed stimulation rates of 20/sec. These observations suggest that the ventral root response was monosynaptic, and this was confirmed by comparison with the reflex elicited by Group Ia stimulation in untreated, unanaesthetized decerebrate rats (Fig. 7,
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Fig. 5. Effect of acute spinal section on the evoked muscle responses. The records A - D show the response in the rat triceps brachii (A and B) and in the gastrocnemius (C and D) to electrical stimulation of the fore- and hind paws respectively. All records were taken 3-5 rain after catechol injection (60 mg/kg i.p.), before (A and C) and 1 h after (B and O) spinal transection at T12. The amplitude of the gastrocnemius first response expressed as a percentage of the maximum recorded amplitude before (O) and after (O) spinal section has been plotted against the time in minutes after catechol injection.
cathode, Angel and Brown (1967)) showed that fibres having conduction velocities between 45 and 50 m/sec were excited. Threshold stimulation of the tibial nerve, at the knee, excited fibres with conduction velocities of 70-80 m/sec. The distributions of dorsal root responses to each of these three stimulating sites are shown in histogram form in Fig. 6. The latency difference between stimulation at the ankle and the sural nerve can be ascribed to the increased conduction distance in the former. The latency, form and amplitude of the ascending volley were unaffected by the injection of catechol (Fig. 6). In deeply anaesthetized rats there was no ventral root response to electrical stimulation of the tibial nerve (Fig. 6, A), sural nerve (Fig. 6, B) or foot (Fig. 6, C). After injection of catechol (6 mg/kg i.v.) volleys corresponding to each of the three evoked muscle responses could be
Stimulation of muscle nerves with suprathreshold shocks (exciting fibres with slower conduction velocities, 45-50 m/sec) produced a further ventral root response with a more variable latency and smaller amplitude (Fig. 7, D) ; this response failed to follow stimulation rates above 3/sec. This response closely resembled that obtained by stimulation of the sural nerve or foot (Fig. 6 , - ~ , F). The spinal delay 3.38 (_+0.44) msec indicates a di- or tri-synaptic reflex. The latency and form of this response resembled that of the polysynaptic reflex obtained from unanaesthetized decerebrate rats (Fig. 7, A). Mono- and polysynaptic reflex responses were recorded from the L5 ventral root, to supra-threshold stimulation of the tibial nerve in unanaesthetized decerebrate rats. Both reflex components were increased by i.v. catechol (4 mg/kg) (Fig. 7, A, C). Since the ventral root responses were not symmetrical they have been integrated, and the integral of the response size has been plotted against time after injection in Fig. 7. e. Behaviour of the motoneurone pool Analysis of single sweep records from the limb muscles showed that the second, and to a lesser extent the third, responses occurred more often when the first response was small or absent. Conversely, when the first response was very large, the two later responses were rarely seen.
595
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Fig. 6. A - F : Superimposed records taken from the L5 dorsal root entry zone (upper trace in each record) and from the ventral root (lower trace) of an anaesthetized rat to threshold stimulation of the ipsilateral tibial nerve CAand D), sural nerve (B and E) and hind paw (C and F). The records were taken before (A, B, C) and 1 min after (D, E,/7) intravenous injection of catechol (6 mg/kg). Calibrations: dorsal root 500 pV, ventral root 250 #V. In this figure and Fig. 7 ventral root responses displayed such that negativity at the proximal recording electrode gives an upward deflection. The histograms show the latency distribution of responses recorded from the L5 dorsal root entry zone Cclearcolumns) and ventral root (black columns) to threshold stimulation of the tibial nerve (TN), sural nerve (SN) and to maximal stimulation of the hind paw (FT). The histograms were constructed from the results obtained from 15 anaesthetized rats 1-2 min after injection of catechol (6 mg/kg i.v.) and show the probability of occurrence (p) of the afferent volleys at the dorsal root, and the motor volleys at the ventral root that would normally precede the first evoked muscle response. The histograms have been aligned for the meantime of entry at the dorsal root, by moving the ordinate to the left in SN and FT.
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Fig. 7. Effect of catechol on mono- and polysynaptic reflexes in the rat. A D: Simultaneous records obtained from the L5 dorsal root entry zone (upper trace) and from the ventral root Clowertrace) to supra-threshold stimulation of the tibial nerve in an unanaesthetized, decerebrate rat (A and C) and in an intact, anaesthetized rat (B and D). Records were taken before CA and B) and 2 min after (C and D) i.v. injection of catechol. Calibrations: dorsal root 500/~V, ventral root 250/tV. The graph shows the time course of the potentiation of spinal reflexes in decerebrate rat by i.v. catechol (6 mg/kg). The voltage-time integral of the monosynaptic ( • ) and polysynaptic (O) components of the ventral root response are plotted against the time in seconds after catechol injection at time zero. The points to the left of zero time are the mean response sizes recorded over 2.5 min control periods prior to injection.
596 It was also noticed that the first response itself was either reduced in amplitude or abolished if the stimulus at the periphery was applied during or immediately after a large, spontaneous discharge had occurred in the muscle. This diminished responsiveness of the motoneurone pool to later inputs was clearly seen when maximal shocks were applied to the nerves or to the foot through the skin, in order to evoke a large first response. In 6 animals experiments were carried out in which the amplitudes of the first and second responses recorded from the forelimb median nerve were measured in response to stimulation of the ulnar nerve with strengths from threshold to supramaximal. All measurements were made betwee.n 2 and 10 min after injection of catechol (60 mg/kg i.p.). Between 6 and 10 responses were recorded at each stimulus setting, and the mean amplitude of the 2 responses determined. The amplitude of the ulnar nerve volley was determined for each setting. As shown in Fig. 8, A and B, there was a positive correlation between the amplitude of the ulnar nerve volley and that of the first response in the median nerve, but a negative correlation with the second response amplitude. The second response was maximal when the stimulus strength was just above threshold and when the first response was small or absent. With progressively stronger stimulation, the first response amplitude increased while that of the second response fell. Maximal shocks to the nerve produced a large consistent first response while the second response became greatly reduced. If these results are a true demonstration of the refractory nature of some units in the gastrocnemius motoneurone pool, a single pool could account for all three responses, and the diminished responsiveness of the pool after the first discharge could be one of the reasons for the low probability of occurrence found for the two later responses. Experiments were performed to establish whether a single motoneurone pool was subjected to three phasic excitatory inputs, or whether the three components of the evoked muscle response reflected three different groups of motoneurones which responded at different times after a peripheral stimulus. Recordings
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Fig. 8. Behaviour of the m o t o n e u r o n e pool. The graphs show the change in amplitudes of the first (A) and second (B) responses recorded from the median nerve to increasing stimulation of the ipsilateral ulnar nerve. The amplitude of the responses, expressed as a percentage of the m a x i m u m recorded amplitude have been plotted against the amplitude of the afferent volley recorded from the ulnar nerve at the elbow, again expressed as a percentage of the maximum. The results are taken from 6 experiments.
were made from small filaments teased from the central stump of the lateral gastrocnemius nerve. In Fig. 9, C the latency distribution of 53 units recorded from 6 rats is shown. In agreement with the records made e n m a s s e from the trunk of the nerve or from the muscle, the discharge latency of units responding to peripheral stimuli fell into a pattern of three distinct components. The discharges which occurred during the earliest component (latency scatter 5 12 msec) had
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Fig. 9. Behaviour of motor units in the tibial nerve. A and B: Either superimposed (A) or single (B) records were obtained from small filaments of the lateral branch of the tibial nerve. Supra-threshold stimuli were applied to the ipsilateral sural nerve below the knee. No responses were recorded to sural nerve stimulation before catechol injection (A,1 ; B,1). The discharge pattern of units in the nerve after the injection of catechol (6 mg/kg i.v.) showed three distinct components (A2), and units responding with the two later components generally had a smaller recorded voltage than those responding with only the first response. Records B2-B6 show the variation in the discharge pattern of a single unit after sural stimulation. The second, smaller unit included in record B3, discharged three times within the latency limits of the second response. Calibrations: 250 /~V and 5 msec. C: Latency distribution of tibial nerve motor units. The mean probability of occurrence (P) for discharges recorded from 53 units of 6 rats has been plotted against the latency of the discharges in milliseconds. D : The distribution of units with respect to their recorded amplitude has been expressed in histogram form by plotting the number of units (N) against their recorded voltage (in mV). The histogram shows that after an injection of catechol, those units that responded to stimulation of the sural nerve with only the first early component (unhatched columns) had a greater amplitude than those that responded with the later components as well (hatched columns).
597 predictably higher probabilities than any of the other discharges. However, as shown in Fig. 9, B, some of the units had a very flexible pattern, and during the 3-4 min period after an i.v. catechol injection (6 mg/kg) any combination of firing pattern could be re~orded to supra-threshold stimulation of the sural nerve. Thus a single unit might give the first response, either alone (Fig. 9, B, 2), or with the second response (Fig. 9, B, 6) or with the third (Fig. 9, B, 4). In most of the experiments it was found that the units having the largest recorded voltage (and presumably recorded from large diameter axons with large parent nerve cells) only responded to a peripheral stimulus with the first component. This is shown in Fig. 9, D, where the distribution of units with respect to their discharge amplitude has been plotted. The figure shows that units responding with all three components (hatched area) had a smaller recorded amplitude. A typical example of this can be seen in the superimposed record from a multifilament preparation shown in Fig. 9, A. The smaller voltage units only fired once during the latency limits of the first response, but often discharged repetitively within the limits of the second and third responses (Fig. 9, B, 3). DISCUSSION
In animals sufficiently deeply anaesthetized with urethane to prevent reflex withdrawal the administration of catechol produces brief muscular movements to a variety of sensory stimuli. After electrical or mechanical stimulation a series of three jerks is produced in the muscles. The first is not abolished by spinal transection and corresponds to the early reflex response observed in a number of studies on the effects of convulsant agents (Alvord and Fuortes 1954, chloralose and strychnine; Ascher 1965, chloralose; Osuide 1968, picrotoxin and leptazol). It has a short consistent latency and a high probability of occurrence, but fails to follow stimulation rates in excess of 3/sec. Records of the first response taken from the central end of the cut ventral root show that it is similar in form and latency to the polysynaptic reflex found in the untreated, unanaesthetized decerebrate rat (Fig. 7). Thus it would seem reasonable that the first muscle
598 response represents a polysynaptic reflex in response to stimulation of cutaneous afferents. The fastest afferent fibres excited by stimulation at the ankle or by direct stimulation of the sural nerve had conduction velocities ranging from 45 to 50 m/sec. In all experiments, the latency, form and amplitude of the dorsal root response to stimulation of the foot, or of muscle or cutaneous nerves was unchanged by the injection of catechol. It would appear, therefore, that catechol has no significant action on peripheral afferent pathways (Angel 1969). This early polysynaptic reflex does not seem to be simple since it can be recorded from both extensor and flexor muscles at the same latency (Fig. 3, D), an effect not due to "crosstalk" between adjacent flexor and extensor muscles (Fig. 3). Alvord and Fuortes (1954) originally reported simultaneous reflex activity in both muscle groups in their study of the chloralose jerk, but later investigators (Ascher 1965; Devanandan et al. 1969a, b) found that stimulation of cutaneous nerves excited flexors, and that extensors were often inhibited. The response in the gastrocnemius (ankle extensor) produced by stimulation through the skin of the hind paw, may represent an extensor thrust reaction (Barton 1934) to stimulation of the tactile pad; the response in the tibialis anterior (ankle flexor) on the other hand, may follow excitation of flexor reflex afferents. Preliminary experiments with direct stimulation of the sural nerve show that selective excitation of fast cutaneous afferents (45-50 m/sec) evokes larger first responses in extensor muscles than in flexors. The opposite is true for percutaneous stimulation of the hind paw in which the slower afferents are excited. It has also been found that threshold mechanical stimuli applied to different parts of the paw produce large first responses in the extensors while none were observed in the flexors. Threshold stimulation of muscle nerves in the anaesthetized rat after injection of catechol produced a consistent short-latency response in the cut ventral root which was similar to the monosynaptic response obtained in the u n anaesthetized, decerebrate animal, and able to follow stimulation rates of up to 20/sec. The mean spinal delay of the monosynap'tic response at
A. A N G E L A N D R. N. L E M O N
the fifth lumbar segment was found to be 2.18 msec. A number of different factors probably contribute to this rather lengthy delay. The conduction velocity over the long oblique pathways in the lumbar spinal cord of the rat is unlikely to be very fast since not many dorsal root fibres have diameters in excess of 12 p (Kaizawa and Takahashi 1970a). The monosynaptic excitatory postynaptic potential (EPS P) in the rat has a slower rise time than in the cat and this has been attributed to the poor functional connection between primary afferents and motoneurones (Kaizawa and Takahashi 1970b). Apart from being able to revive both monoand polysynaptic reflexes in the anaesthetized animal, catechol markedly potentiates the amplitude and duration of both types of reflex in the unanaesthetized, decerebrate animal. There have been some reports that hyperventilation will facilitate spinal reflexes (Kirstein 1951; K itahata et al. 1969) and though it is possible that the hyperventilation caused by catechol (Angel and Lemon 1973) may play some part in its action it is not a significant one, since reflex facilitation can still be observed in animals breathing 95 7o O2 and 5 ~ C O 2. There are a number of possible mechanisms whereby a convulsant such as catechol could facilitate reflex activity and reverse the depressive effect of anaesthetics on spinal pathways. Both segmental and supraspinal influences are involved in re-setting the overall balance between inhibition and excitation. At the segmental level, most reports have concentrated on the excitability created by convulsants due to a reduction of inhibition (Eccles et al. 1963; Devanandan et al. 1964, 1965a, b; Shimamura et al. 1968; Barnes and Pompeiano 1970; Davidoff 1972). Widespread synaptic facilitation by a convulsant would also be expected to bring about elevated levels of excitation and inhibition and this process may explain the mixed effects observed with some convulsants. Banna and Jabbur (1970) have recently reported that catechol increased both pre- and postsynaptic inhibition. Further studies on inhibition and excitation in the spinal cord may reveal the action of catechol in more detail, and in particular the significance of the proposed ability of this substance to potentiate transmitter release (Otsuka and Nonomura 1962).
599
MYOCLON1C JERKS BY 1,2-DIHYDROXYBENZENE
The polysynaptic response evoked by hind paw stimulation is potentiated and prolonged in spinal animals, whereas the spontaneous convulsions produced by catechol are reduced below the level of section (Angel and Lemon 1972). Changes in the level of local reflex excitability associated with supraspinal activation have been reported for a number of drugs (Alvord and Fuortes 1954; Eidelberg and Buchwald 1960; Kawai and Sasaki 1964; Osuide 1968). The paradoxical effects of spinal section on the spontaneous and evoked muscle jerks suggest that catechol may act in different ways on the spinal cord. The studies on single motor units in the tibial nerve show that after catechol, some motoneurones will discharge within the latency limits of al! three responses to any one stimulus. It thus seems unlikely that different groups of motoneurones are responsible for different components of the reflex jerk, but that the whole motoneurone pool receives three phasic excitatory inputs. The units that responded to all three inputs usually had a small recorded voltage, and exhibited signs of multiple discharge within the latency limits of the later responses. The units with a large recorded voltage usually responded to each stimulus with a single discharge at a latency that corresponded with the first jerk. Henneman and Olson (1965) and Henneman et al. (1965) have shown that large motoneurones generally innervate fast, twitch-type motor units, while smaller motoneurones innervate slow units, which are associated with tonic muscular contraction. Multiple discharge of the smaller units would help to explain the larger latency scatter found for both the second and third responses. However, a less synchronous input than that producing the first response and the Renshaw activity that succeeds this response may also influence the latency of the later responses. The probability of occurrence for the first, spinal response was always high (Table I) except when the peripheral stimulus was delivered during or immediately after a large spontaneous response had occurred in the muscle. It seems that after a large discharge, the motoneurone pool is in a state of decreased excitability. This is reflected in the probabilities of the second and third responses, which were always lower than
that of the spinal response, except when the latter was small or absent. This may account for the low probability of occurrence for the second response when nerve muscles were stimulated, since this type of stimulation always evoked a large first response. There are several possible explanations for the hypoexcitability of the motoneurone pool after a large discharge. A discharge involving a significant fraction of the pool, and the subsequent contraction of the muscle, would result in increased Renshaw and autogenic inhibition and decreased spindle excitation. It is interesting to note that the chloralose jerks studied by Devanandan et al. (1969a), were associated with an EPSP in the flexor motoneurones that was succeeded by a period of hyperpolarisation lasting for several hundred milliseconds. These authors concluded that this was not an inhibitory postsynaptic potential generated across the neuronal membrane by inhibitory synapses, but since there was a reduction in synaptic "noise" after the jerks, they attributed it to a general reduction in background excitatory impulses, i.e., dis-facilitation. SUMMARY
In rats deeply anaesthetized with urethane, injection of 1,2 dihydroxybenzene (catechol; 60 mg/kg i.p.) produces a stimulus-sensitive state in which a variety of sensory stimuli, e.g., a brief electrical shock or mechanical tap applied to the periphery, or a binaural click, produce brief (myoclonic) muscular jerks. These jerks have been recorded electromyographically in the forelimb from the biceps and triceps brachii and in the hind limb from gastrocnemius and tibialis anterior. The myoclonic jerks produced in these muscles by electrical or mechanical stimulation of the corresponding paw consisted of three distinct components. The early, first response to electrical stimulation has a short consistent latency (4.3 _+ 0.6 msec in the forelimb ; 8.1 _+ 0.9 msec in the hind limb), large amplitude and high probability of occurrence. This first response represents a polysynaptic reflex elicited by stimulation of cutaneous afferents. It is not, however, a simple reflex since it could be recorded in direct antagonists at a similar
600 latency. The first response persisted in the hind limb muscles with both chronic and acute spinal transection at T10-T12. The second response (13.4 -+ 1.6 msec in the forelimb; 19.4 + 3.7 msec in the hind limb) and the third response (40.0 _+ 7.3 msec and 51.4 _ 7.3 msec respectively) were both abolished in the hind limb muscles by spinal transection. Records of single motor units in the tibial nerve showed that discharge of a single motoneurone can account for all three components-of the reflex jerk. The second, and to a lesser extent, the third response were always reduced in amplitude when the first response was large. Conversely, large second responses were only recorded when the first response was small or absent. Catechol had no effect on the amplitude or latency of the afferent volley recorded at the dorsal root entry zone, but markedly potentiated both mono- and polysynaptic reflexes. RESUME ANALYSE DES SECOUSSES MYOCLONIQUES PROVOQUEES PAR LE 1, 2-DIHYDROXYBENZENE CHEZ LE
A. ANGEL AND R. N. LEMON
deuxi6me composante (13, 4 + 1,6 msec en avant; 19,4_+3,7 msec en arri6re) et la troisi6me (respectivement 40,0_+7,3 msec et 51,4+7,3 msec) furent toutes deux abolies dans les membres post6rieurs par la transsection spinale. En enregistrant "fi partir d'unit6s isol6es du nerf tibial, il est montr6 qu'un motoneurone donn6 peut participer aux trois composantes successives de la secousse. La seconde et, dans une certaine mesure, la troisi6me composante~ sent toujours r6duites lorsque l'amplitude de la premi&e est importante; inversement d'amples secondes composantes ne s'obtiennent que lorsque la premi6re est r6duite ou absente. Le catdchol n'affecte pas l'amplitude ou la latence de la volde aff6rente (recueillie au niveau d'entr6e de la racine dorsale), mais potentialise de faqon marqu6e les r6flexes mono- et polysynaptiques. Most of the work was performed whilst R.N.L. was in receipt of an M.R.C. Scholarship. A.A. wishes to acknowledge the financial assistance of the Medical Research Council for this work.
RAT
Chez le rat en anesth6sie profonde ~tl'ur6thane, l'injection de 1-2-dihydroxybenz6ne (cat6chol; 60 mg/kg i.p.), d6termine une sensibilisation aux incitations p6riph6riques en sorte que des stimulus tels un choc bref 61ectrique ou m6canique ~ la p6riph6rie ou un clic auditifbinaural d6terminent une secousse musculaire br6ve (myoclonique). Celle-ci a 6t6 enregistr6e 61ectromyographiquement dans le biceps et le triceps brachii, pour le membre ant6rieur et le gastrocn6mien et le tibialis ant6rieur pour le membre post&ieur. Les secousses induites dans ces muscles par stimulation (61ectrique ou m6canique) du membre correspondant ont comport6 trois composantes. Pour la stimulation 61ectrique, la premi6re composante, h latence br6ve et stable (4,3_+ 0,6 msec dans le membre ant6rieur; 8,1 _+0,9 msec dans le membre post6rieur) est ample et offre une probabilit6 61ev6e d'apparition. I1 s'agit d'un r6flexe polysynaptique dfi ~t la stimulation des aff6rents cutan6s, encore que complexe puisqu'il peut int6resser des antagonistes, simultan6ment et avec la m6me latence. I1 persiste dans les membres post6rieurs, apr6s transsection spinale, aigu6 ou chronique, ~t Tlo-Tx2. En revanche la
REFERENCES ALVORD, E. C. and FUORTES, M. G. F. A comparison of generalised reflex myoclonic reactions elicitable in cats under chloralose anaesthesia and under strychnine. Amer. J. Physiol., 1954, 176:253 261. Axc~l E, A. An analysis of the effect of 1,2-dihydroxybenzene on transmission through the dorsal column sensory pathway. Electroenceph. olin. Neurophysiol.,1969, 27: 392-403. ANGEL, A. and BROWN, B. H. Some effects of stimulating nervetrunks through the skin. J. Physiol. (Lend).), 1967, 194:57 59P. ANGEL, A. and DAWSON, G. D. The effects of 1,2-dihydroxybenzene on transmission in the sensory pathway of the rat. Electroenceph. olin. Neurophysiol., 1964, lC~: 312. ANGEL, A. and LEMON, R. N. The convulsive action of catechol in the anaesthetised rat. J. Physiol. (Lend.), 1972, 225:56 57P. ANGEl,, A. and LEMON, R. N. The convulsive action of ],2dihydroxybenzene in the anaesthetised rat. Eh'ctroenceph. clin. Neurophysiol., 1973, 34:369 378. ANGEL, A., LEMON, R. N. and ORPWOOD, R. The muscular jerks produced by catechol (1,2-dihydroxybenzene) in the anaesthetised rat. J. Physiol. (Lend.), 1968, 198: 10-11P. ASCHER, P. La r~action de sursaut du chat anaesthesia; au chloralose. Th6se Dec. Sci., Gap: Louis-Jean, Paris, 1965. BACQ, Z. M. Recherches sur la physiologie et la pharmacologie du syst6me nerveux autonome. XX. Sensibililisation l'adr6naline et l'excitation des nerfs adr6nergiques
MYOCLONIC JERKS BY 1,2-DIHYDROXYBENZENE par les antioxygenes. Arch. int. Physiol., 1936, 42: 340 366. BANNA, N. R. and JABBUR, S. J. Increased transmitter release induced by convulsant phenols. Brains Res., 1970, 2(1:471 473. BARNES, C. D. and POMPE1ANO.O. Presynaptic inhibition of extensor monosynaptic reflex by Ia afferents from flexors. Brain Res., 1970, 18:380 383. BARRON, D. H. The results of unilateral pyramidal section in the rat. J. ¢omp. Neurol., 1934, 60:45 55. BRIE(;ER, L. Uber das physiologische Benehmen von Katechol Hydroquinone und Resorkinol und ihre Formation im tierischen K6rper. Dubois Arch. Physiol., 1879, Suppl. 61. DAVIDOFE, R. A. Gamma-aminobutyric acid antagonism and presynaptic inhibition in the frog spinal cord. Science, 1972, 175: 331-333. DAWSON, G. D. The relation between the electroencephalogram and muscle action potentials in certain convulsive states. J. Neurol. Neurosurg. Psyehiat., 1946, 9 : 5 22. D,~WSON, G. D. Investigations on a patient subject to myoclonic seizures after sensory stimulation. J. Neurol. Neurosurg. Psychiat., 1947, 10: 141-162. DF.VANANDAN, M. S., ECCLES, R. M., LEWIS, D. M. and STEN,OUSE, D. Responses of flexor alpha-motoneurones in cats anaesthetised with chloralose. Exp. Brain Res., 1969a, 8:163 176. DEVANANDAN.M. S., ECCLES, R. M., LEWIS, D. M. and STENItOUSE, D, Responses of extensor alpha-motoneurones in cats anaesthetised with chloralose. Exp. Brain Res., 1969b,8:177 189. DI \.\NANDAN, M. S., ECCLES, R. M. and YOKOTA, T. Presynaptic inhibition induced by muscle stretch. Nature (Lond.), 1964, 204:996 998. DE'~ANANDAN, M. S., ECCLES, R. M. and YOKOTA, T. Depolarisation of afferent terminals evoked by muscle stretch. J. Physiol. (Lond.;, 1965a. 179: 417M.29. DEVANANDAN,M. S., ECCLES, R. M. and YOKOTA, T. Muscle stretch and the presynaptic inhibition of the group la pathway to motoneurones. J. Physiol. (Lond.), 1965b, 179: 430-441.
601 ECCLES,J. C., Schmidt, R. F. and WILLIS,W. D. Pharmacological studies on presynaptic inhibition. J. Physiol. (Lond.), 1963, 168: 50(~530. EIDELBERG, E. and BUCHWALD, N. A. Effects of thiosemicarbazide on spinal reflex activity. Neurolo~ly (Minneap.), 1960, 10:267 270. GREEN, E. C. The anatomy of the rat. Trans. Amer. Philosoph. Soc., 1935, Vol. 27. HALLIDAY, A. M. The electrophysiological study of myoclonus in man. Brain, 1967, 90:241 284. HENNEMAN, E. and OesoY, C. B. Relations between structure and function in the design of skeletal muscles.J. Neurophysiol., 1965, 28:581-598. HENNEMAN,E., SOMJEN,G. and CARPENTER,D. O. Functional significance of cell size in spinal motoneurones. J. Neurophysiol., 1965, 28:560 580. KAlZAWA, J. and TAKAHASHI,I. Fiber analysis of the lumbar spinal roots and their sciatic branches in rats. Tohuku J. exp. Med., 1970a, 100:61 74. KAIZAWA, J. and TAKAHASHI, I. The monosynaptic origin of the fast synchronous wave in rat spinal reflex. Tohuku J. exp. Med., 1970b, 100: 115-125. KAWAI, I. and SASAKI, K. Effects of strychnine on supraspinal inhibition. Jap. J. Pysiol., 1964, 14:309 318. KtRSTEIN, L. Early effects of oxygen lack and carbon dioxide excess on spinal reflexes. Acta physiol, seand., 1951, 23, Suppl. 80: 1- 54. KITAHATA, L. M., TAUB, A. and SATO, I. Hyperventilation and spinal reflexes. Anesthesiolooy, 1969, 31:321 326. Osuide, G. Effects of some centrally-acting drugs in conscious chicks and on spinal reflexes. Europ. J. Pharmacol., 1968.3:283 293. OTSUKA, M. and NONOMURA, Y. The action of phenolic substances on motor nerve endings. J. Pharmacol. e.vp. Ther., 1962, 140:41 ~5. SHIMAMURA, M., YAMAUfm, T. and AOR1, M. Effects of chloralose anaesthesia on spinal reflexes. Jap. J. Phl'siol., 1968, 18:788 797. WEn., C. S. Tables for convenient calculation of medianeffective dose (LDs0 or ED50) and instructions in their use. Biometrics, 1952, 8 : 2 4 9 262.
AN ANALYSIS
OF THE
MYOCLONIC
B Y 1, 2 - D I H Y D R O X Y B E N Z E N E
JERKS
589
PRODUCED
IN THE RAT
A. ANGELAND R. N. LEMON Department of Physiolooy, The University, Western Bank, Sheffield $10 2TN (Great Britain) (Accepted for publication: June 6, 1973)
The characteristic action of 1,2 dihydroxy- were used. In the acute experiments, each animal benzene (catechol) and other hydroxybenzenes was immobilized with ether and then given an in the anaesthetized animal is to produce intraperitoneal (i.p.) injection of urethane (25~o "spontaneous" convulsive jerks and twitches in in 0.9~ saline in a 1.0 ml per 200 g body weight the absence of any intentional stimulus (Brieger primary dose). The final anaesthetic dose was 1879; Bacq 1936; Angel and Lemon 1973). In adjusted so that reflex withdrawal of the limbs to addition to this type of convulsive activity, Angel a strong pinch was just abolished, and this and Dawson (1964) observed in rats an- usually required between 1.3 and 1.5 g/kg aesthetized with urethane that after an intrave- urethane. Procedures for fixation, stimulation, nous injection of catechol, an external stimulus recording of electrical and mechanical responses produced a brief myoclonic jerk. The stimulus of the limb muscles, and for chronic spinal could be either in the form of a sharp auditory transection have been fully described in a click or a brief electrical shock applied to one of previous paper (Angel and Lemon 1973). It the limbs ; the type of stimulus was not critical. should be restated, since it is an important point, Angel (1969) has shown that the stimulus- that stimulation and recording were made on sensitive state induced by catechol is associated the same limb. with a greatly increased transmission through The spinal column was rigidly fixed and spinal the dorsal column medial lemniscal sensory laminectomy performed from L4 to S1 ; the dura pathway. Thus in the rat, electrical responses was reflected and the dorsal and ventral roots recorded from the thalamic sensory relay nucleus giving rise to the sciatic trunk (Green 1935; and primary cortical receiving area in response Kaizawa and Takahashi 1970a) were prepared to a test stimulus applied to the periphery were for recording. Where necessary spinal transection greatly increased in size after catechol injection, was performed between T10 and T12. A pool was and remained so for the entire convulsive period. constructed from the cut skin edges and filled with These results bear an interesting resemblance to liquid paraffin B.P. (equilibrated with physiothe clinical studies of patients suffering from logical saline) maintained at 37°C. In the hind certain types of sensory myoclonus, where there limb, the sural, peroneal and final nerves were is an abnormal motor jerk to a sensory stimulus exposed and separated, as were the median and (Dawson 1946, 1947; Halliday, 1967). ulnar nerves in the forelimb. Mechanical stimulaThis paper presents a study of the muscle tion was obtained with a modified loudspeaker responses evoked by a variety of stimuli in with a probe attached to the coil. This gave a anaesthetized rats given catechol ; a preliminary monophasic output with a time to peak of account of the work lias already been published 0.4 msec, a duration of 1.4 msec and an excursion (Angel et al. 1968). of the tip approximately of 80 # with no following oscillation. METHODS The experiments were performed on animals One hundred and three female albino rats given i.p. injections of 60 mg/kg catechol, and
590 this dose was sufficient to produce convulsions in all the animals studied. In no case did this dose prove lethal. Intravenous (i.v.) injections were made via a polythene cannula in the femoral or jugular vein. The i.v. dose varied between 3 and 8 mg/kg (Angel 1969). Catechol (1,2 dihydroxybenzene; British Drug Houses) was made up in a solution of 10 mg/ml in 0.9~ saline (w/v) at 37°C. RESULTS
a. Responses in the limb muscles to electrical stimulation at the periphery In animals deeply anaesthetized with urethane, electrical stimulation of the fore- and hind paws through lint pads soaked in 3 M NaC1 produced no electromyographic (EMG) response in any of the limb muscles studied (Fig. 1, A, C) and no motor volley could be recorded in either the appropriate ventral root or peripheral nerve. In contrast, shortly after an i.v. injection of catechol (4-6 mg/kg), a complex EMG response with a duration of 50-70 msec was produced by each stimulus, and this response was always associated with a sharp contraction of the muscles of the limb to which the stimulus had been applied. The complex consisted of three distinct components (Fig. 1, B, D) and the mean latencies of these components were 4.3, 13.4 and 40.0 msec in the forelimb, and 8.1, 19.4 and 51.4 msec in the hindlimb (see Table I). The duration of this effect was relatively short lived (Angel 1969); the peak-to-peak voltage of the responses reached a maximum 30-45 sec after injection, then declined and had usually disappeared within 5 min. Since the convulsive effects of catechol are more prolonged after i.p. administration, injection of catechol by this route was employed for a more detailed study of the muscular responses. Fig. 2, A - H shows a typical series of records from the biceps brachii muscle of the rat at different times after an i.p. injection. The three responses all appeared about 1 min after injection (Fig. 2, B) and reached maximum size at approximately 3 min (Fig. 2, C), after which there was a rapid decline in the amplitude of the responses (Fig. 2, D-G). At 20 min the effect had disappeared (Fig. 2, H). The mean amplitude of each of the three
A. ANGEL AND R. N. LEMON
responses recorded from the rat biceps brachii muscle was measured in 12 rats and plotted against the time after injection (Fig. 2, J). Because the amplitude of the response varied considerably from experiment to experiment, the results have been expressed graphically as a percentage of the maximum peak-to-peak voltage recorded. The pattern shown in Fig. 2 was much the same for the hind limb muscle responses, although these often persisted for up to 25 or 30 rain after injection (Fig. 5). In Fig. 2, K the
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5m$ Fig. 1. EMG response from the forelimb (triceps brachii, A and B) and the hind limb (gastrocnemius, C and D) in an anaesthetized rat to electrical stimulation of the respectiye ipsilateral paw before (A and C) and 3 rain after (B and D) an i.p. injection of catechol (60 mg/kg). Voltage calibration 250 gV. Unless otherwise stated, in this and all succeeding figures, each record is of 20 consecutive responses supe/'imposed.
591
MYOCLONIC JERKS BY 1,2-DIHYDROXYBENZENE TABLE I Latency, amplitude and probability of occurrence of the three evoked muscle responses. 1st
2nd
3rd
A. Electrical stimulation
Forelimb : (N = 53)
Triceps brachii Latency (msec) Probability Relative amplitude
4.30( + 0.6) 0.97( _+0.05) 1.00( ± 0.04)
13.40(+ 1.6) 0.59( + 0.09) 0.40( + 0.02)
40.00( + 7.0) 0.17( + 0.10) 0.17( +_0.02)
Hind limb : (N = 53)
Gastrocnemius Latency (msec) Probability Relative amplitude
8.10( + 0.9) 0.91 ( + 0.05) 1.00(+0.05)
19.40( + 3.7) 0.35( + 0.08) 0.15(+0.03)
51.40( + 7.3) 0.14( + 0.08) 0.12(+0.03)
Hind limb : (N = 13)
Tibialis anterior Latency (msec) Probability Relative amplitude
8.30( _+ 1.1) 0.90( + 0.09) 1.00( + 0.20)
20.00( ± 2.4) 0.57( + 0.09) 0.60( + 0.20)
m
B. Mechanical stimulation
Forelimb : (N = 8)
Triceps brachii Latency (msec)
5.5( + 0.9)
15.8( + 2.5)
44.8( + 5.4)
Hind limb : (N = 8)
Gastrocnemius Latency (msec)
8.9( + 1.4)
21.6( _+2.5)
48.1( + 3.5)
A. E•ectrica• stimu•ati•n. Data are sh•wn f•r resp•nses re••rd•d
in the triceps brachii t• e•ectrica• stimu•ati•n
•f the ipsi•atera•
forepaw, and in the gastrocnemius and tibialis anterior to electrical stimulation of the ipsilateral hind paw. The latency and probability of occurrence of each of the three responses was determined together with the maximum recorded voltage of each response. The results are the means from the number (N) of experiments given, together with the standard deviation from the mean (in brackets), except in the case of the amplitude, where the mean is given with the standard error from the mean. The amplitudes of the three responses in each muscle have been expressed in relative terms, taking the mean maximum amplitude of the first response as unity. All the results are derived from observations made on the E M G responses recorded between 2 and 10 min after an i.p. injection of catechol (60 mg/kg). B. Mechanical stimulation. The table shows the mean latency (together with the standard deviation in brackets) of the three responses evoked by mechanical stimulation of the fore- or hind paw in the ipsilateral triceps brachii and gastrocnemius respectively.
amplitude of the first response recorded from the triceps brachii of a rat had been plotted against time for two injections separated by 1 h. The time course of the responses recorded after each injection was practically identical, and follows the same pattern as the averaged results shown in Fig. 2, J. The mean latency, amplitude and probability of occurrence for the three responses recorded from the triceps brachii and from the gastrocnemius and tibialis anterior are shown in Table I. The first response was always the largest, had the least latency scatter and the highest probability
of occurrence. The probability of occurrence of each of the three components was determined during the time that catechol exerted its maximum effect, i.e., 2-10 min after i.p. injection. The probability was taken as unity if the component occurred to each of 20 consecutive stimuli (1/sec) applied in batches at 1 min intervals throughout this time. The second and third responses were smaller in voltage and probability of occurrence, and had a greater latency scatter. The second response was more easily obtained from the forelimb muscles; in the hind limb it was more frequently observed in the flexor (tibialis anterior)
592
A. ANGEL AND R. N. LEMON 1OO~
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Fig. 2. Time course of the E M G responses in the rat. EMG response of the triceps brachii muscle to stimulation of the ipsilateral forepaw before (A) and at 1 (B), 3 (C), 6 (D), 9 (E), 12 (F), 15 (G) and 20 (H) min after an i.p. injection of catechol (60 mg/kg) in a rat anaesthetized with urethane. Body temperature 37°C. Calibrations : 250/~V and 10 msec. J : The amplitudes of the first (O), second (©) and third (A) responses recorded from the triceps brachii muscle to stimulation of the forepaw, have been plotted against the time after an i.p. injection of catechol (60 mg/kg). Each point represents the amplitude of the response expressed as a percentage of the maximum amplitude recorded. The results are the mean from 12 different animals. K : The amplitude time course of the first response recorded from the triceps brachii in an anaesthetized rat. The response amplitude (in mV) recorded after two identical injections of catechol, the second ((3) given 1 h after the first (O), has been plotted against the time after each injection. Body temperature 37°C.
than in the extensor (gastrocnemius). The amplitude of these evoked responses was of the same order as that of the "spontaneous" responses recorded from the same muscle (Angel and Lemon 1973), although dose/response experiments revealed that the evoked jerks could be obtained at lower doses than the spontaneous variety. In anaesthetized rats the i.p. EDs0 (dose required to produce an effect in 50~o of the animals studied) determined by the method of Weil (1952), was 30 mg/kg for myoclonic jerks evoked by peripheral electrical stimulation compared with 49 mg/kg for spontaneous jerks. Simultaneous recordings of the mechanical and electrical response from the triceps brachii are shown in Fig. 3, A - C . The first evoked response was always associated with a "twitchlike" contraction of the muscle (Fig. 3, A). These longer-latency second and third responses were usually smaller than the first response and produced tension changes which summated with the first response twitch (Fig. 3, B, C).
The evoked muscle responses could be recorded from direct antagonist muscles at similar latencies and with the same probability of occurrence. Because of the possibility of "crosstalk" existing in the recording system, where, owing to the spread of bio-electric activity, the EMG response of an active muscle could be recorded in a remote inactive muscle, experiments were performed in which the hind leg was completely denervated sparing only the sural (sensory) nerve and the muscle nerve supplying either the gastrocnemius or the tibialis anterior. These experiments demonstrated that
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Fig. 3. A - C : Tension changes during the evoked muscle response. Simultaneous single-sweep oscilloscope records of the tension changes in the rat triceps brachii (upper trace in each record) and the E M G of the same muscle (lower trace). The records, taken from 3 to 10 min after injection ofcatechol (60 mg/kg i.p.) show that a single electrical shock applied to the ipsilateral forepaw produced a typical early muscle response either alone (A), or with the third response (B), or with both the second and third responses (C). Each spike in the EMG was associated with a change in muscle tension, developing from a single twitch in A to a summated response in B and C. Tension calibration : 100 g. D-F : E M G response of the gastrocnemius (upper trace in each record) and tibialis anterior (lower trace) in the hind limb of the rat to peripheral electrical stimulation after catechol injection. The early (first) response could be recorded from both muscles at similar latencies (D). After section of the peroneal nerve, the amplitude of the tibialis response was greatly reduced and its shape completely altered (E). The remaining "activity" demonstrated that there was a small amount of electronic spread between the two muscles. Section of the nerve supply to the gastrocnemius abolished both responses (F). Calibration: 250 pV.
MYOCLONIC JERKS BY
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crosstalk between the calf muscles only accounts for a small fraction of the response in the intact preparation (Fig. 3, D-F). Additionally comparison of simultaneous records taken from the gastrocnemius and tibialis anterior showed that one muscle could be active while the other was silent. b. Limb muscle responses to other types of stimuli Most of the experiments on the evoked muscle responses were performed using electrical
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593 stimulation, but it was established that similar types of muscle response could be evoked by physiological stimuli. As shown in Fig. 4, A, B, a light mechanical tap applied to the sole of the paw produced muscle responses similar to those evoked by electrical stimulation, although the mean latencies were slightly greater (Table I). A binaural auditory stimulus, in the form of a sharp click, frequently produced a generalized body convulsion after injection of catechol. An early response in the forelimb with a mean latency of 5.7 (SD __+_0.7) msec and in the hind limb at 8.4 (_+0.8) msec could be recorded, and there'was also a later response in both limbs, at 19.3 (+3.5) and 21.8 (+3.2) msec respectively (Fig. 4, C, D). Visual stimuli were not effective in producing a generalized body convulsion; only small localized jerks were occasionally observed and these were usually difficult to distinguish from any on-going spontaneous activity. c. Limb muscle responses in spinal and pithed animals After acute (12 animals) or chronic (8 day; 13 animals) section of the spinal cord at the level T10-T 12, the gastrocnemius response to stimulation of the hind paw after catechol consisted of the first component only (7-9 msec). The second and third components of the muscle response were never observed in the hind limb muscles of these rats (Fig. 5, O), although the forelimb muscles remained unaffected (Fig. 5, B). In the acute preparations, comparison of the responses evoked before and 1 h after spinal section showed that the amplitude of the first response was always greater in the spinal animal. In addition, the time course of the remaining first response was prolonged and lasted for up to 40-45 min, compared with 20-25 min in the intact animal (Fig. 5). The evoked muscle responses were completely abolished by denervation of the appropriate muscles or by destruction of the spinal cord. d. Reflex pathway of the evoked muscle response Recordings from the L5 dorsal root entry zone showed that threshold stimulation of the sural nerve (cutaneous) at the knee and supramaximal stimulation at the ankle (supramaximal to overcome the anomalous effect of stimulating nerve trunks through the skin with a diffuse
594
A. ANGEL AND R. N. LEMON
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recorded from the ventral root or muscle nerve (see also Fig. 9, A). The latency of the L5 ventral root volley corresponding to the first muscleevoked response was recorded in 15 experiments and the results are presented in Fig. 6. Thrbshold stimulation of the tibial nerve produced a first response of consistent short latency and large amplitude (Fig. 6, D). The mean latency of this response at the ventral root was 3.18 (S.D. _+ 0.31) msec and the spinal delay (from L5 dorsal root to L5 ventral root) was 2.18 (_+0.21) msec. The response followed stimulation rates of 20/sec. These observations suggest that the ventral root response was monosynaptic, and this was confirmed by comparison with the reflex elicited by Group Ia stimulation in untreated, unanaesthetized decerebrate rats (Fig. 7,
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Fig. 5. Effect of acute spinal section on the evoked muscle responses. The records A - D show the response in the rat triceps brachii (A and B) and in the gastrocnemius (C and D) to electrical stimulation of the fore- and hind paws respectively. All records were taken 3-5 rain after catechol injection (60 mg/kg i.p.), before (A and C) and 1 h after (B and O) spinal transection at T12. The amplitude of the gastrocnemius first response expressed as a percentage of the maximum recorded amplitude before (O) and after (O) spinal section has been plotted against the time in minutes after catechol injection.
cathode, Angel and Brown (1967)) showed that fibres having conduction velocities between 45 and 50 m/sec were excited. Threshold stimulation of the tibial nerve, at the knee, excited fibres with conduction velocities of 70-80 m/sec. The distributions of dorsal root responses to each of these three stimulating sites are shown in histogram form in Fig. 6. The latency difference between stimulation at the ankle and the sural nerve can be ascribed to the increased conduction distance in the former. The latency, form and amplitude of the ascending volley were unaffected by the injection of catechol (Fig. 6). In deeply anaesthetized rats there was no ventral root response to electrical stimulation of the tibial nerve (Fig. 6, A), sural nerve (Fig. 6, B) or foot (Fig. 6, C). After injection of catechol (6 mg/kg i.v.) volleys corresponding to each of the three evoked muscle responses could be
Stimulation of muscle nerves with suprathreshold shocks (exciting fibres with slower conduction velocities, 45-50 m/sec) produced a further ventral root response with a more variable latency and smaller amplitude (Fig. 7, D) ; this response failed to follow stimulation rates above 3/sec. This response closely resembled that obtained by stimulation of the sural nerve or foot (Fig. 6 , - ~ , F). The spinal delay 3.38 (_+0.44) msec indicates a di- or tri-synaptic reflex. The latency and form of this response resembled that of the polysynaptic reflex obtained from unanaesthetized decerebrate rats (Fig. 7, A). Mono- and polysynaptic reflex responses were recorded from the L5 ventral root, to supra-threshold stimulation of the tibial nerve in unanaesthetized decerebrate rats. Both reflex components were increased by i.v. catechol (4 mg/kg) (Fig. 7, A, C). Since the ventral root responses were not symmetrical they have been integrated, and the integral of the response size has been plotted against time after injection in Fig. 7. e. Behaviour of the motoneurone pool Analysis of single sweep records from the limb muscles showed that the second, and to a lesser extent the third, responses occurred more often when the first response was small or absent. Conversely, when the first response was very large, the two later responses were rarely seen.
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Fig. 6. A - F : Superimposed records taken from the L5 dorsal root entry zone (upper trace in each record) and from the ventral root (lower trace) of an anaesthetized rat to threshold stimulation of the ipsilateral tibial nerve CAand D), sural nerve (B and E) and hind paw (C and F). The records were taken before (A, B, C) and 1 min after (D, E,/7) intravenous injection of catechol (6 mg/kg). Calibrations: dorsal root 500 pV, ventral root 250 #V. In this figure and Fig. 7 ventral root responses displayed such that negativity at the proximal recording electrode gives an upward deflection. The histograms show the latency distribution of responses recorded from the L5 dorsal root entry zone Cclearcolumns) and ventral root (black columns) to threshold stimulation of the tibial nerve (TN), sural nerve (SN) and to maximal stimulation of the hind paw (FT). The histograms were constructed from the results obtained from 15 anaesthetized rats 1-2 min after injection of catechol (6 mg/kg i.v.) and show the probability of occurrence (p) of the afferent volleys at the dorsal root, and the motor volleys at the ventral root that would normally precede the first evoked muscle response. The histograms have been aligned for the meantime of entry at the dorsal root, by moving the ordinate to the left in SN and FT.
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Fig. 7. Effect of catechol on mono- and polysynaptic reflexes in the rat. A D: Simultaneous records obtained from the L5 dorsal root entry zone (upper trace) and from the ventral root Clowertrace) to supra-threshold stimulation of the tibial nerve in an unanaesthetized, decerebrate rat (A and C) and in an intact, anaesthetized rat (B and D). Records were taken before CA and B) and 2 min after (C and D) i.v. injection of catechol. Calibrations: dorsal root 500/~V, ventral root 250/tV. The graph shows the time course of the potentiation of spinal reflexes in decerebrate rat by i.v. catechol (6 mg/kg). The voltage-time integral of the monosynaptic ( • ) and polysynaptic (O) components of the ventral root response are plotted against the time in seconds after catechol injection at time zero. The points to the left of zero time are the mean response sizes recorded over 2.5 min control periods prior to injection.
596 It was also noticed that the first response itself was either reduced in amplitude or abolished if the stimulus at the periphery was applied during or immediately after a large, spontaneous discharge had occurred in the muscle. This diminished responsiveness of the motoneurone pool to later inputs was clearly seen when maximal shocks were applied to the nerves or to the foot through the skin, in order to evoke a large first response. In 6 animals experiments were carried out in which the amplitudes of the first and second responses recorded from the forelimb median nerve were measured in response to stimulation of the ulnar nerve with strengths from threshold to supramaximal. All measurements were made betwee.n 2 and 10 min after injection of catechol (60 mg/kg i.p.). Between 6 and 10 responses were recorded at each stimulus setting, and the mean amplitude of the 2 responses determined. The amplitude of the ulnar nerve volley was determined for each setting. As shown in Fig. 8, A and B, there was a positive correlation between the amplitude of the ulnar nerve volley and that of the first response in the median nerve, but a negative correlation with the second response amplitude. The second response was maximal when the stimulus strength was just above threshold and when the first response was small or absent. With progressively stronger stimulation, the first response amplitude increased while that of the second response fell. Maximal shocks to the nerve produced a large consistent first response while the second response became greatly reduced. If these results are a true demonstration of the refractory nature of some units in the gastrocnemius motoneurone pool, a single pool could account for all three responses, and the diminished responsiveness of the pool after the first discharge could be one of the reasons for the low probability of occurrence found for the two later responses. Experiments were performed to establish whether a single motoneurone pool was subjected to three phasic excitatory inputs, or whether the three components of the evoked muscle response reflected three different groups of motoneurones which responded at different times after a peripheral stimulus. Recordings
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were made from small filaments teased from the central stump of the lateral gastrocnemius nerve. In Fig. 9, C the latency distribution of 53 units recorded from 6 rats is shown. In agreement with the records made e n m a s s e from the trunk of the nerve or from the muscle, the discharge latency of units responding to peripheral stimuli fell into a pattern of three distinct components. The discharges which occurred during the earliest component (latency scatter 5 12 msec) had
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Fig. 9. Behaviour of motor units in the tibial nerve. A and B: Either superimposed (A) or single (B) records were obtained from small filaments of the lateral branch of the tibial nerve. Supra-threshold stimuli were applied to the ipsilateral sural nerve below the knee. No responses were recorded to sural nerve stimulation before catechol injection (A,1 ; B,1). The discharge pattern of units in the nerve after the injection of catechol (6 mg/kg i.v.) showed three distinct components (A2), and units responding with the two later components generally had a smaller recorded voltage than those responding with only the first response. Records B2-B6 show the variation in the discharge pattern of a single unit after sural stimulation. The second, smaller unit included in record B3, discharged three times within the latency limits of the second response. Calibrations: 250 /~V and 5 msec. C: Latency distribution of tibial nerve motor units. The mean probability of occurrence (P) for discharges recorded from 53 units of 6 rats has been plotted against the latency of the discharges in milliseconds. D : The distribution of units with respect to their recorded amplitude has been expressed in histogram form by plotting the number of units (N) against their recorded voltage (in mV). The histogram shows that after an injection of catechol, those units that responded to stimulation of the sural nerve with only the first early component (unhatched columns) had a greater amplitude than those that responded with the later components as well (hatched columns).
597 predictably higher probabilities than any of the other discharges. However, as shown in Fig. 9, B, some of the units had a very flexible pattern, and during the 3-4 min period after an i.v. catechol injection (6 mg/kg) any combination of firing pattern could be re~orded to supra-threshold stimulation of the sural nerve. Thus a single unit might give the first response, either alone (Fig. 9, B, 2), or with the second response (Fig. 9, B, 6) or with the third (Fig. 9, B, 4). In most of the experiments it was found that the units having the largest recorded voltage (and presumably recorded from large diameter axons with large parent nerve cells) only responded to a peripheral stimulus with the first component. This is shown in Fig. 9, D, where the distribution of units with respect to their discharge amplitude has been plotted. The figure shows that units responding with all three components (hatched area) had a smaller recorded amplitude. A typical example of this can be seen in the superimposed record from a multifilament preparation shown in Fig. 9, A. The smaller voltage units only fired once during the latency limits of the first response, but often discharged repetitively within the limits of the second and third responses (Fig. 9, B, 3). DISCUSSION
In animals sufficiently deeply anaesthetized with urethane to prevent reflex withdrawal the administration of catechol produces brief muscular movements to a variety of sensory stimuli. After electrical or mechanical stimulation a series of three jerks is produced in the muscles. The first is not abolished by spinal transection and corresponds to the early reflex response observed in a number of studies on the effects of convulsant agents (Alvord and Fuortes 1954, chloralose and strychnine; Ascher 1965, chloralose; Osuide 1968, picrotoxin and leptazol). It has a short consistent latency and a high probability of occurrence, but fails to follow stimulation rates in excess of 3/sec. Records of the first response taken from the central end of the cut ventral root show that it is similar in form and latency to the polysynaptic reflex found in the untreated, unanaesthetized decerebrate rat (Fig. 7). Thus it would seem reasonable that the first muscle
598 response represents a polysynaptic reflex in response to stimulation of cutaneous afferents. The fastest afferent fibres excited by stimulation at the ankle or by direct stimulation of the sural nerve had conduction velocities ranging from 45 to 50 m/sec. In all experiments, the latency, form and amplitude of the dorsal root response to stimulation of the foot, or of muscle or cutaneous nerves was unchanged by the injection of catechol. It would appear, therefore, that catechol has no significant action on peripheral afferent pathways (Angel 1969). This early polysynaptic reflex does not seem to be simple since it can be recorded from both extensor and flexor muscles at the same latency (Fig. 3, D), an effect not due to "crosstalk" between adjacent flexor and extensor muscles (Fig. 3). Alvord and Fuortes (1954) originally reported simultaneous reflex activity in both muscle groups in their study of the chloralose jerk, but later investigators (Ascher 1965; Devanandan et al. 1969a, b) found that stimulation of cutaneous nerves excited flexors, and that extensors were often inhibited. The response in the gastrocnemius (ankle extensor) produced by stimulation through the skin of the hind paw, may represent an extensor thrust reaction (Barton 1934) to stimulation of the tactile pad; the response in the tibialis anterior (ankle flexor) on the other hand, may follow excitation of flexor reflex afferents. Preliminary experiments with direct stimulation of the sural nerve show that selective excitation of fast cutaneous afferents (45-50 m/sec) evokes larger first responses in extensor muscles than in flexors. The opposite is true for percutaneous stimulation of the hind paw in which the slower afferents are excited. It has also been found that threshold mechanical stimuli applied to different parts of the paw produce large first responses in the extensors while none were observed in the flexors. Threshold stimulation of muscle nerves in the anaesthetized rat after injection of catechol produced a consistent short-latency response in the cut ventral root which was similar to the monosynaptic response obtained in the u n anaesthetized, decerebrate animal, and able to follow stimulation rates of up to 20/sec. The mean spinal delay of the monosynap'tic response at
A. A N G E L A N D R. N. L E M O N
the fifth lumbar segment was found to be 2.18 msec. A number of different factors probably contribute to this rather lengthy delay. The conduction velocity over the long oblique pathways in the lumbar spinal cord of the rat is unlikely to be very fast since not many dorsal root fibres have diameters in excess of 12 p (Kaizawa and Takahashi 1970a). The monosynaptic excitatory postynaptic potential (EPS P) in the rat has a slower rise time than in the cat and this has been attributed to the poor functional connection between primary afferents and motoneurones (Kaizawa and Takahashi 1970b). Apart from being able to revive both monoand polysynaptic reflexes in the anaesthetized animal, catechol markedly potentiates the amplitude and duration of both types of reflex in the unanaesthetized, decerebrate animal. There have been some reports that hyperventilation will facilitate spinal reflexes (Kirstein 1951; K itahata et al. 1969) and though it is possible that the hyperventilation caused by catechol (Angel and Lemon 1973) may play some part in its action it is not a significant one, since reflex facilitation can still be observed in animals breathing 95 7o O2 and 5 ~ C O 2. There are a number of possible mechanisms whereby a convulsant such as catechol could facilitate reflex activity and reverse the depressive effect of anaesthetics on spinal pathways. Both segmental and supraspinal influences are involved in re-setting the overall balance between inhibition and excitation. At the segmental level, most reports have concentrated on the excitability created by convulsants due to a reduction of inhibition (Eccles et al. 1963; Devanandan et al. 1964, 1965a, b; Shimamura et al. 1968; Barnes and Pompeiano 1970; Davidoff 1972). Widespread synaptic facilitation by a convulsant would also be expected to bring about elevated levels of excitation and inhibition and this process may explain the mixed effects observed with some convulsants. Banna and Jabbur (1970) have recently reported that catechol increased both pre- and postsynaptic inhibition. Further studies on inhibition and excitation in the spinal cord may reveal the action of catechol in more detail, and in particular the significance of the proposed ability of this substance to potentiate transmitter release (Otsuka and Nonomura 1962).
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MYOCLON1C JERKS BY 1,2-DIHYDROXYBENZENE
The polysynaptic response evoked by hind paw stimulation is potentiated and prolonged in spinal animals, whereas the spontaneous convulsions produced by catechol are reduced below the level of section (Angel and Lemon 1972). Changes in the level of local reflex excitability associated with supraspinal activation have been reported for a number of drugs (Alvord and Fuortes 1954; Eidelberg and Buchwald 1960; Kawai and Sasaki 1964; Osuide 1968). The paradoxical effects of spinal section on the spontaneous and evoked muscle jerks suggest that catechol may act in different ways on the spinal cord. The studies on single motor units in the tibial nerve show that after catechol, some motoneurones will discharge within the latency limits of al! three responses to any one stimulus. It thus seems unlikely that different groups of motoneurones are responsible for different components of the reflex jerk, but that the whole motoneurone pool receives three phasic excitatory inputs. The units that responded to all three inputs usually had a small recorded voltage, and exhibited signs of multiple discharge within the latency limits of the later responses. The units with a large recorded voltage usually responded to each stimulus with a single discharge at a latency that corresponded with the first jerk. Henneman and Olson (1965) and Henneman et al. (1965) have shown that large motoneurones generally innervate fast, twitch-type motor units, while smaller motoneurones innervate slow units, which are associated with tonic muscular contraction. Multiple discharge of the smaller units would help to explain the larger latency scatter found for both the second and third responses. However, a less synchronous input than that producing the first response and the Renshaw activity that succeeds this response may also influence the latency of the later responses. The probability of occurrence for the first, spinal response was always high (Table I) except when the peripheral stimulus was delivered during or immediately after a large spontaneous response had occurred in the muscle. It seems that after a large discharge, the motoneurone pool is in a state of decreased excitability. This is reflected in the probabilities of the second and third responses, which were always lower than
that of the spinal response, except when the latter was small or absent. This may account for the low probability of occurrence for the second response when nerve muscles were stimulated, since this type of stimulation always evoked a large first response. There are several possible explanations for the hypoexcitability of the motoneurone pool after a large discharge. A discharge involving a significant fraction of the pool, and the subsequent contraction of the muscle, would result in increased Renshaw and autogenic inhibition and decreased spindle excitation. It is interesting to note that the chloralose jerks studied by Devanandan et al. (1969a), were associated with an EPSP in the flexor motoneurones that was succeeded by a period of hyperpolarisation lasting for several hundred milliseconds. These authors concluded that this was not an inhibitory postsynaptic potential generated across the neuronal membrane by inhibitory synapses, but since there was a reduction in synaptic "noise" after the jerks, they attributed it to a general reduction in background excitatory impulses, i.e., dis-facilitation. SUMMARY
In rats deeply anaesthetized with urethane, injection of 1,2 dihydroxybenzene (catechol; 60 mg/kg i.p.) produces a stimulus-sensitive state in which a variety of sensory stimuli, e.g., a brief electrical shock or mechanical tap applied to the periphery, or a binaural click, produce brief (myoclonic) muscular jerks. These jerks have been recorded electromyographically in the forelimb from the biceps and triceps brachii and in the hind limb from gastrocnemius and tibialis anterior. The myoclonic jerks produced in these muscles by electrical or mechanical stimulation of the corresponding paw consisted of three distinct components. The early, first response to electrical stimulation has a short consistent latency (4.3 _+ 0.6 msec in the forelimb ; 8.1 _+ 0.9 msec in the hind limb), large amplitude and high probability of occurrence. This first response represents a polysynaptic reflex elicited by stimulation of cutaneous afferents. It is not, however, a simple reflex since it could be recorded in direct antagonists at a similar
600 latency. The first response persisted in the hind limb muscles with both chronic and acute spinal transection at T10-T12. The second response (13.4 -+ 1.6 msec in the forelimb; 19.4 + 3.7 msec in the hind limb) and the third response (40.0 _+ 7.3 msec and 51.4 _ 7.3 msec respectively) were both abolished in the hind limb muscles by spinal transection. Records of single motor units in the tibial nerve showed that discharge of a single motoneurone can account for all three components-of the reflex jerk. The second, and to a lesser extent, the third response were always reduced in amplitude when the first response was large. Conversely, large second responses were only recorded when the first response was small or absent. Catechol had no effect on the amplitude or latency of the afferent volley recorded at the dorsal root entry zone, but markedly potentiated both mono- and polysynaptic reflexes. RESUME ANALYSE DES SECOUSSES MYOCLONIQUES PROVOQUEES PAR LE 1, 2-DIHYDROXYBENZENE CHEZ LE
A. ANGEL AND R. N. LEMON
deuxi6me composante (13, 4 + 1,6 msec en avant; 19,4_+3,7 msec en arri6re) et la troisi6me (respectivement 40,0_+7,3 msec et 51,4+7,3 msec) furent toutes deux abolies dans les membres post6rieurs par la transsection spinale. En enregistrant "fi partir d'unit6s isol6es du nerf tibial, il est montr6 qu'un motoneurone donn6 peut participer aux trois composantes successives de la secousse. La seconde et, dans une certaine mesure, la troisi6me composante~ sent toujours r6duites lorsque l'amplitude de la premi&e est importante; inversement d'amples secondes composantes ne s'obtiennent que lorsque la premi6re est r6duite ou absente. Le catdchol n'affecte pas l'amplitude ou la latence de la volde aff6rente (recueillie au niveau d'entr6e de la racine dorsale), mais potentialise de faqon marqu6e les r6flexes mono- et polysynaptiques. Most of the work was performed whilst R.N.L. was in receipt of an M.R.C. Scholarship. A.A. wishes to acknowledge the financial assistance of the Medical Research Council for this work.
RAT
Chez le rat en anesth6sie profonde ~tl'ur6thane, l'injection de 1-2-dihydroxybenz6ne (cat6chol; 60 mg/kg i.p.), d6termine une sensibilisation aux incitations p6riph6riques en sorte que des stimulus tels un choc bref 61ectrique ou m6canique ~ la p6riph6rie ou un clic auditifbinaural d6terminent une secousse musculaire br6ve (myoclonique). Celle-ci a 6t6 enregistr6e 61ectromyographiquement dans le biceps et le triceps brachii, pour le membre ant6rieur et le gastrocn6mien et le tibialis ant6rieur pour le membre post&ieur. Les secousses induites dans ces muscles par stimulation (61ectrique ou m6canique) du membre correspondant ont comport6 trois composantes. Pour la stimulation 61ectrique, la premi6re composante, h latence br6ve et stable (4,3_+ 0,6 msec dans le membre ant6rieur; 8,1 _+0,9 msec dans le membre post6rieur) est ample et offre une probabilit6 61ev6e d'apparition. I1 s'agit d'un r6flexe polysynaptique dfi ~t la stimulation des aff6rents cutan6s, encore que complexe puisqu'il peut int6resser des antagonistes, simultan6ment et avec la m6me latence. I1 persiste dans les membres post6rieurs, apr6s transsection spinale, aigu6 ou chronique, ~t Tlo-Tx2. En revanche la
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