Exp. Brain Res. 25, 35-43 (1976)
Experimental Brain Research 9 by Springer-Verlag1976
Effect of Oxotremorine on the Response of Antidromically Activated Renshaw Cells in Decerebrate Cats D.K. Ganguly 1, H.-G. Ross, J. Haase and S. Cleveland Physiologisches Institut (Lehrstuhl II) der Universit/it Diisseldorf, Moorenstr. 5, D - 4000 Dtisseldorf (FRG)
Summary. In intercollicular decerebrate cats, some of which were made spinal in addition, the effect of oxotremorine (intravenous injection, 10-30 ~g/kg) was tested on antidromically activated Renshaw cells. Methylatropine premedication prevented the otherwise often fatal drop in blood pressure; ipsilateral dorsal roots L6-S1 and contralateral hindlimb nerves were cut to exclude segmental receptor interference. During supramaximal stimulation of ventral root L7 or the gastrocnemius nerves, an increase of activity ranging from 10-110 % was observed. The drug occasionally evoked spontaneous discharges in Renshaw cells, or enhanced activity already present. Alpha motoneuron activity decreased in most cases. The interaction of oxotremorine with atropine and eserine was also investigated on Renshaw cells. Our results suggest that one of the effects of oxotremorine may be a disbalance between motor output and recurrent inhibition.
Key words: Tremorogenic agents - Oxotremorine - Renshaw cells - Alpha motoneurons.
Parkinson's disease is characterized by rigidity, hypokinesia and tremor at rest (4-7 Hz). Of these symptoms, tremor can be imitated to a certain extent in animal experiments by the application of tremorogenic agents, which, it is hoped, may facilitate the analysis of the mechanisms by which tremor originates. Tremorine or its active metabolite oxotremorine [l(2-oxopyrrolidono)-4-pyrrolidono-butyne-2] have been reported to cause Parkinson-like symptoms in homeothermic vertebrates, including the cat (Everett, Blockus, Shepperd and Toman, 1956; Nash and Emerson, 1959; Kaelber and Hamel, 1960; George, Haslett and Jenden, 1966), the dog (Chalmers and Yim, 1963; Everett, Blockus and Shepperd, 1956; Everett, Blockus, Shepperd and Toman, 1956), the rat (Chalmers and Yim, 1962; Everett, Blockus and Shepperd, 1956; Everett, Blockus, Shepperd 1 Present address: Indian Institute of Experimental Medicine, Calcutta-32, India; Dr. Ganguly received support from the CSIR-DAAD Exchange of Scientists Programme. 3*
36
D.K. Ganguly et al.
and T o m a n , 1956; Jurna, Nell and Schreyer, 1970; G e o r g e , Haslett and Jenden, 1966; T a s k e r and Kertesz, 1965), and the m o n k e y (Everett, Blockus and Shepperd, 1956; Everett, Blockus, S h e p p e r d and T o m a n , 1956). T h e r e have b e e n m a n y suggestions as to the site of action of these drugs, but as yet no general a g r e e m e n t has b e e n r e a c h e d on this point. I n particular, there is a divergence of opinion a m o n g the a b o v e - m e n t i o n e d authors about w h e t h e r or not supraspinal structures are necessary for the genesis of the m o t o r disturbances caused by these drugs. H o w e v e r , there is considerable evidence that o x o t r e m o r i n e leads to increased levels of acetylcholine in the brain (Holmstedt, L u n d g r e n and Sundvall, 1963; H o l m s t e d t and L u n d g r e n , 1964; G a n g u l y and Saha, 1969). Further, using a nerve-muscle preparation, G a n g u l y and C h a u d h u r i (1970) have d e m o n s t r a t e d enh a n c e d acetylcholine release at m o t o r end-plates. In the light of these findings it seems plausible that o x o t r e m o r i n e develops its effects not by acting at o n e specific site, but rather by influencing a variety of cholinergic junctions. O f the central cholinergic synapses, those f r o m m o t o r axon collaterals onto R e n s h a w cells are not only easily accessible, but have also b e e n thor o u g h l y investigated (cf. Haase, Cleveland and Ross, 1975, for ref.). W e have therefore sought evidence of an effect of o x o t r e m o r i n e at this site. A n u m b e r of ancillary experiments were p e r f o r m e d to check the effect of oxot r e m o r i n e on alpha m o t o n e u r o n excitability.
Methods General
Experiments were carried out on 24 intercollicular decerebrate cats, which were immobilized by intravenous injection ofN,N'-diallylnortoxferinium-dichloride (Alloferin| 0.25 mg/kg) and artificially respired. In three experiments, the spinal cord was also transected at the low thoracic level. Blood pressure was monitored throughout the experiments by means of a cannula in the common carotid artery. Body temperature was kept between 37 and 39~ C. All measurements were performed at least 2 b after decerebration and removal of anesthesia. The right (contralateral) hind limb was denervated as completely as possible and the left (ipsilateral) dorsal roots L6 to S 1 were cut intradurally. In experiments in which single, orthodromically activated alpha motoneurons were recorded the ipsilateral dorsal roots L7 and S1 remained intact.
Premedication and Application of Oxotremorine
Cho, Haslett and Jenden (1962) have shown that as little as 0.06 ~g/kg oxotremorine noticeably lowers blood pressure. In order to prevent this effect methylatropine (Eumydrin| 0.5-1.0 mg/kg), which does not cross the blood-brain barrier, was administered intravenously some 20 rain before recording began. Whenever blood pressure fell below 80 mm Hg despite this precaution, the experiment was discarded. After the control period, oxotremorine sesquifumarate (Aldrich Chem. Corp.) was applied either by slow automatic infusion or by injection into the femoral vein. Maximal doses ranged between 10 and 30 ~tg/kg; in one case (interaction with eserine) the maximal dose was 40 ~tg/kg.
Oxotremorine and Renshaw Cells Fig. 1. Renshaw cell discharge in decerebrate cat: effect of oxotremorine infusion (20 ~g/kg maximum). Number of spikes (N) per supramaximal antidromic stimulus to ventral root L7 plotted against time (averages of 10 bursts, plus-minus standard deviation). Heavy bar: period of drug infusion
37
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Identification and Activation of Renshaw Cells After laminectomy, Renshaw cells located in the segment L7 were recorded extracellularly by means of conventional glass micropipettes filled with 3M KC1 and identified by their monosynaptic burst response to single antidromic volleys. Due to the slow inactivation rate of oxotremorine, only one cell per cat could be studied. Throughout the experiments the Renshaw cells were excited by antidromic stimulation of either the central end of the cut ipsilateral ventral root L7, or the ipsilateral gastrocnemius nerves. Stimuli 0.2 msec wide and supramaximal for alpha fibers were presented every 2 sec, in some cases every second. When near-threshold excitation was desired, this was achieved by stimulation of small filaments of the ventral root L7 at strengths supramaximal for alpha fibers. The response of a given Renshaw cell was measured by counting the number of action potentials during the first 100 msec after a stimulus (burst length).
Recording of Monosynaptic Reflexes and Single Motor Axons Monosynaptic reflexes were elicited by single shocks applied to the central end of the cut dorsal root L7 or S1 and recorded from the gastrocnemins or anterior tibial nerves; their height was taken as a convenient measure of synchronous motor activity. Stimuli were presented every 5 sec and were submaximal as judged by the reflex amplitude during the control period. In another series of experiments single alpha fibers which exhibited tonic activity during tetanic stimulation of the ipsilateral gastrocnemius nerves were functionally isolated from the ventral root L7 or S1; their discharges were recorded with the help of an interval meter, as well as on film. Every 30 or 60 sec a train of stimulus pulses (0.2 msec wide, 125 Hz) lasting 15 sec was applied.
Results
R e n s h a w Cells O f 14 R e n s h a w cells w h i c h w e h a v e i n v e s t i g a t e d , 11 r e s p o n d e d to o x o t r e m o r i n e w i t h a s i g n i f i c a n t i n c r e a s e in t h e n u m b e r o f d i s c h a r g e s p r o d u c e d b y a n a n t i d r o m i c s t i m u l u s , w h i l e t h e d r u g h a d n o e f f e c t o n t h e r e m a i n i n g t h r e e cells. T y p i c a l f o r t h e i n c r e a s e o f R e n s h a w cell e x c i t a b i l i t y d u r i n g i n f u s i o n o f o x o t r e m o r i n e is t h e e x p e r i m e n t s h o w n in Fig. 1. S t i m u l a t i o n o f t h e c e n t r a l e n d o f t h e c u t v e n t r a l r o o t L 7 r e s u l t e d in a b o u t 9 s p i k e s p e r b u r s t b e f o r e a p p l i c a t i o n o f t h e d r u g . D u r i n g i n f u s i o n (20 ~ g / k g t o t a l ) , this r e s p o n s e i n c r e a s e d to a m a x i m u m o f m o r e t h a n 2 0 s p i k e s p e r burst.
38
D.K. Oanguly et al. s N [%]
N
Fig. 2. Summary of oxotremorine (10-30 ~tg/kg)effects on 11 Renshaw cells (decerebrate and spinal cats). Stimuli applied to either ventral root L7 or gastrocnemius nerves. Left diagram: number N of Renshaw cell discharges per stimulus (plus-minus standard deviation) during control period (open circles) and at time of maximal oxotremorine effect (filled circles). Right diagram: percent increase AN in Renshaw cell excitability at maximum of drug effect (closed circles) relative to control value (open circle)
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Fig. 3. Comparing the effect of oxotremorine rejection (25 ~tg/kg at arrow) on the response of a Renshaw cell m supramaximal (open circles) and near-threshold (filled circles) stimuli to ventral root L7 (spinalized decerebrate cat). Points are average number of spikes in 5 bursts, plus-minus standard deviation. Original records: responses to supramaximal (upper row) and near-threshold (lower row) stimulibefore (a) and after (b, c) oxotremorine injection
Oxotremorine and Renshaw Cells Fig. 4. Showingconcomitantincrease in burst response and spontaneous activity of a Renshaw cell (decerebrate cat). Upper row: control (a: burst response, b: spontaneous activity); lowerrow: correspondingrecords (c and d) after 15 ~tg/kgoxotremorine
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The effect of oxotremorine on 11 cells is summarized in Fig. 2, the left half of which portrays the average responses to supramaximal antidromic stimulation before the drug was administered and at the time of maximal oxotremorine effect. Shown in the right diagram is the increase in spikes per burst relative to the control response. This increase ranged from 10 to 110 %. The results of the three experiments on spinalized cats are included in the figure, as this procedure caused no detectable change in the effect of oxotremorine. An interesting effect occurred in two spinal cats in which not only supramaximal, but also near-threshold stimuli were employed (Fig. 3). Near-threshold excitation of Renshaw cells was achieved by using small filaments of the ventral root (see Methods). While oxotremorine resulted in the usual increase in response to whole-root stimulation, the number of spikes elicited by near-threshold stimulation vacilated strongly; this is reflected in the size of the standard deviations. The possibility that these fluctuations may have resulted from a periodic change in Renshaw cell excitability at characteristic tremor frequencies (8-12 Hz; Friedman and Everett, 1964) could not be tested by our method, since in this experiment stimuli were presented only every 2 sec. The effect of oxotremorine on the spontaneous activity of a Renshaw cell is shown in Fig. 4. Although spontaneously active Renshaw cells are rare in preparations in which the segmental afferent input is cut off, a clear increase in spontaneous discharge rate could be seen in two cells, one of which is depicted in the figure. The spontaneous activity of the other cell took the form of bursts occurring at an average rate of 3.8/sec, which were not present during the control period.
Motoneuron Activity Since increased Renshaw cell excitability, especially the augmented spontaneous activity, might conceivably follow from enhanced alpha motoneuron activity, it became necessary to check this possibility directly. Chalmers and Yim (1963) have already demonstrated that tremorine either reduces the height of monosynaptic reflexes in the spinal dog or else leaves them unaffected. In four of seven experiments employing monosynaptic extensor reflexes in our decerebrate preparation, injection of oxotremorine also diminished reflex height; no effect occurred in the remaining three experiments. This result is corroborated by the experiments on functionally isolated alpha axons, which showed that single units become less excitable following application of the drug.
40
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Fig. 5. Effect of oxotremorine (20 ~g/kg) on motoneuron excitability (decerebrate cat). Top: discharges of functionally isolated alpha axon (ventral root L7); bar below c indicates first and final 3.5 sec of the 15 sec period of tetanic stimulation of the medial gastrocnemius nerve (80 mV, 125 Hz). Bottom: corresponding records of interspike interval vs. time (measurement range: 0-200 msec), a: control recordings before oxotremorine; b and c: recordings 7 and 20 rain, respectively, after drug injection
This decreased excitability is reflected in the fact that (1) the f r e q u e n c y of tonic discharge is reduced, and that (2) the threshold for m o t o n e u r o n firing is raised or that (3) the initially tonic response to o r t h o d r o m i c tetanization b e c o m e s irregular or even phasic, as illustrated in Fig. 5. In no case did the alpha m o t o n e u r o n s investigated b e c o m e s p o n t a n e o u s l y active u n d e r the o x o t r e m o r i n e dosages used in the R e n s h a w cell experiments. Since Jurna, Nell and Schreyer (1970) have reported that in anesthesized, non-deafferented rats large doses of oxotremorine (up to 600 ~tg/kg) increase alpha motoneuron activity, an additional dose of 100 ~tg/kgwas applied in one experiment in which the discharge rate of an alpha motoneuron had dropped as a result of an injection of 30 ~tg/kgof the drug. Spontaneous activity appeared for a few seconds immediately after the additional injection and the rate of tonic discharge was increased.
Drug Interaction
T h e interaction of o x o t r e m o r i n e with atropine, which is k n o w n to ameliorate the s y m p t o m s o f Parkinsonism, and eserine, which has the opposite effect (Duvoisin, 1967), m a y be studied at the junction of m o t o r axon collaterals and R e n s h a w cells, as the actions o f the latter drugs on R e n s h a w cells have b e e n described in considerable detail (Eccles, Fatt and Koketsu, 1954; Curtis and Ryall, 1966). A t r o p i n e intravenously applied depresses the response of R e n s h a w cells to antidromic stimulation, as illustrated in Fig. 6 and first described by Eccles et al. (1954). L a t e r injection of o x o t r e m o r i n e clearly antagonizes this effect. O n the o t h e r hand, the expected synergism of eserin and o x o t r e m o r i n e is m o r e difficult to demonstrate, since eserine alone potentiates R e n s h a w cell activity to such an extent that a further increase due to o x o t r e m o r i n e is not likely to oc-
Oxotremorine and Renshaw Cells
41
Fig. 6. Antagonistic effects of atropine (2 mg/kg; open arrow) and oxotremorine (30 ~g/kg; filled arrow). Number of spikes (N) per supramaximal antidromic stimulus to ventral root L7 plotted against time (averages of 5 bursts, plus-minus standard deviation)
sp~kes,N
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Fig. 7. Synergistic effects of oxotremorine (filled arrows) and eserine (open arrow). Number of spikes (N) per supramaximal antidromic stimulus to ventral root L7 plotted against time (averages of 50-100 bursts, plus-minus standard deviation). Original records a-e correspond to letters in graph. Drug dosages given in text
cur. Nevertheless, synergism may be demonstrated by making use of the observation that oxotremorine increases the Renshaw cell response only up to a certain point, after which multiple applications of the drug have no further effect. This is illustrated by the first three filled arrows in Fig. 7, which correspond to oxotremorine doses of 5, 5 and 10 ~g/kg, respectively. If, however, a new dose of oxotremorine is given during the declining phase of the effect of eserine (open arrow; 100 ~g/kg), a renewed increase comparable to that produced by the first injection is obtained (fourth filled arrow; oxotremorine; 20 ~tg/kg). This is taken as evidence that the remaining concentration of eserine suffices to facilitate the action of oxotremorine
42
D.K. Ganguly et al.
Discussion The use of antidromic stimulation is the simplest means of testing the effect of oxotremorine on the chemical transmission from motor axon collaterals to Renshaw cells, because only one synaptic junction is involved. In addition, the fact that the cats used in our experiments were decerebrated or spinalized and deafferented eliminated most of the extraneous influences on the cells tested. We have shown that in most cases the drug facilitates the synaptic transmission to a considerable degree. Thus, the idea (Stern, 1963, p. 88) that " . . . Renshaw cells in rest tremor are either inactive all the time or even destructed" cannot be the explanation of the oxotremorine effects on motor activity in experimental animals. The constant motor output imitated by our method of stimulation of the axons of alpha motoneurons is associated with augmented Renshaw cell activity, which presumably means that transmission in the recurrent pathway is more potent. The total effect need not be simple, however; in particular, it should be emphasized that this increased potency will affect not only the agonist motoneurons, but also those of the antagonist via the recurrent control of reciprocal inhibition (cf. Hultborn, 1972). This is indeed of importance, as tremor is associated with alternating contractions of antagonistic muscles (cf. Kaada, 1963). The irregularity of the threshold Renshaw cell response occasionally observed under oxotremorine (see Fig. 4) will also perturb muscle activity. Clearly the oxotremorine-induced changes in Renshaw cell excitability must affect motor performance, independently of the effects of the drug on muscle receptors and higher centers of the central nervous system. Since augmented m o t o n e u r o n excitability was not observed in our preparation, this is unlikely to be the explanation for the increased spontaneous firing of some Renshaw cells seen following oxotremorine. A more probable cause is the activation of muscarinic acetylcholine receptors at the Renshaw cell membrane (Curtis and Ryall, 1966). Some symptoms of Parkinsonism have been suggested to be the result of a disbalance of transmitter substances in the basal ganglia (Barbeau, 1962; Steg, 1969), with a dominance of acetylcholine. We would submit that a similar dominance of acetylcholine ought to exist in the oxotremorine model of Parkinson's disease and that this notion of disbalance must be extended to include cholinergic synapses in the spinal cord. Acknowledgement. We thank I. Lippitschfor skilfultechnicalassistance.
References Barbeau, A.: The pathogenesis of Parkinson's disease: A new hypothesis. Canad. reed. Ass. J. 87, 802-807 (1962) Chalmers, R.K., Yim, G.K.W.: Tremorine tremor in chronic spinal rats. Proc. Soc. exp. Biol. (N.Y.) 109, 202-205 (1962) Chalmers, R.K., Yim, G.K.W.: Spinal actions of tremorine in the dog. Arch. int. Pharmacodyn.145, 322-333 (1963) Cho, A.K., Haslett, W.L., Jenden, D.J.: The peripheral actions of oxotremorine, a metabolite of tremorine. J. Pharmacol. exp. Ther. 138, 249-257 (1962)
Oxotremorine and Renshaw Cells
43
Curtis, D.R., Ryall, R.W.: The synaptic excitation of Renshaw cells. Exp. Brain Res. 2, 81-96 (1966) Duvoisin, R.C.: Cholinergic-anticholinergic antagonism in Parkinsonism. Arch. Neurol. (Chic.) 17, 124-136 (1967) Eccles, J.C., Fatt, P., Koketsu, K.: Cholinergic and inhibitory synapses in a pathway from motor-axon collaterals to motoneurones. J. Physiol. (Lond.) 126, 524-562 (1954) Everett, G.M., Blockus, L.E., Shepperd, I.M.: Tremor induced by tremorine and its antagonism by anti-Parkinson drugs. Science 124, 79 (1956) Everett, G.M., Blockus, L.E., Shepperd, I.M., Toman, J.E.P.: Production of tremor and a Parkinsonlike syndrmne by 1-4-dipyrrolidono-2-butyne, "Tremorine." Fed. Proc. 15, 420-421 (1956) Friedman, A.H., Everett, G.H.: Pharmacological aspects of Parkinsonism. Advanc. Pharmacol. 3, 83-127 (1964) Ganguly, D.K., Chaudhuri, S.K.: Neuromuscular pharmacology of oxotremorine. Europ. J. Pharmacol. 11, 84-89 (1970) Ganguly, D.K., Saha, L.: Effect of oxotremorine on acetylcholine content of brain and peripheral tissues in rats. Ind. J. exp. Biol. 7, 176-177 (1969) George, R., Haslett, W.L., Jenden, D.J.: The production of tremor by cholinergic drugs: central sites of action. Int. J. Pharmacol. 5, 27-34 (1966) Haase, J., Cleveland, S., Ross, H.-G.: Problems of postsynaptic autogenous and recurrent inhibition in the mammalian spinal cord. Rev. Physiol. Biochem. Pharmacol. 73, 73-129 (1975) Holmstedt, B., Lundgren, G.: Tremorigenetic agents and brain acetylcholine. Oxford: Pergamon Press 1964 Holmstedt, B., Lundgren, G., Sundvall, A.: Tremorine and atropine effects on brain acetylcholine. Life Sci. 2, 731-736 (1963) Hultborn, H.: Convergence on interneurones in the reciprocal Ia inhibitory pathway to motoneurones. Acta physiol, scan& Suppl. 375, 3-42 (1972) Jurna, I., Nell, T., Schreyer, I.: Motor disturbance induced by tremorine and oxotremorine. NaunynSchmiedebergs Arch. Pharmak. 267, 80-98 (1970) Kaada, B.R.: The pathophysiology of Parkinsonian tremor, rigidity, and hypokinesia. Acta neurol. scand. Suppl. 39, 41-51 (1963) Kaelber, W.W., Hamel, E.G.: Drug (tremorine)-induced tremor in the cat. Arch. Neurol. (Chic.) 2, 338-340 (1960) Nash, J.B., Emerson, G.A.: Studies on the mode of action of tremorine, (1,4-dipyrrolidone-2-butyne). Fed. Proc. 18, 426 (1959) Steg, G.: Striatal cell activity during systematic administration of monaminergic and cholinergic drugs. In: Third Symposium on Parkinson's disease. (Ed. F.J. Gillingham and I.M.L. Donaldson), pp. 26-29. Edinburgh: E. & S. Livingstone 1969 Stern, P.: Contribution on the pathophysiology of intentional tremor. In: Biochemical and Neurophysiological Correlation of Centrally Acting Drugs. (Ed. E. Trabucchi et al.), pp. 81-91. Oxford: Pergamon Press 1963 Tasker, R., Kertesz, A.: The physiology of tremorine-induced tremor. J. Neurosurg. 22, 4 4 9 4 5 6 (1965)
Received September 5, 1975
Experimental Brain Research 9 by Springer-Verlag1976
Effect of Oxotremorine on the Response of Antidromically Activated Renshaw Cells in Decerebrate Cats D.K. Ganguly 1, H.-G. Ross, J. Haase and S. Cleveland Physiologisches Institut (Lehrstuhl II) der Universit/it Diisseldorf, Moorenstr. 5, D - 4000 Dtisseldorf (FRG)
Summary. In intercollicular decerebrate cats, some of which were made spinal in addition, the effect of oxotremorine (intravenous injection, 10-30 ~g/kg) was tested on antidromically activated Renshaw cells. Methylatropine premedication prevented the otherwise often fatal drop in blood pressure; ipsilateral dorsal roots L6-S1 and contralateral hindlimb nerves were cut to exclude segmental receptor interference. During supramaximal stimulation of ventral root L7 or the gastrocnemius nerves, an increase of activity ranging from 10-110 % was observed. The drug occasionally evoked spontaneous discharges in Renshaw cells, or enhanced activity already present. Alpha motoneuron activity decreased in most cases. The interaction of oxotremorine with atropine and eserine was also investigated on Renshaw cells. Our results suggest that one of the effects of oxotremorine may be a disbalance between motor output and recurrent inhibition.
Key words: Tremorogenic agents - Oxotremorine - Renshaw cells - Alpha motoneurons.
Parkinson's disease is characterized by rigidity, hypokinesia and tremor at rest (4-7 Hz). Of these symptoms, tremor can be imitated to a certain extent in animal experiments by the application of tremorogenic agents, which, it is hoped, may facilitate the analysis of the mechanisms by which tremor originates. Tremorine or its active metabolite oxotremorine [l(2-oxopyrrolidono)-4-pyrrolidono-butyne-2] have been reported to cause Parkinson-like symptoms in homeothermic vertebrates, including the cat (Everett, Blockus, Shepperd and Toman, 1956; Nash and Emerson, 1959; Kaelber and Hamel, 1960; George, Haslett and Jenden, 1966), the dog (Chalmers and Yim, 1963; Everett, Blockus and Shepperd, 1956; Everett, Blockus, Shepperd and Toman, 1956), the rat (Chalmers and Yim, 1962; Everett, Blockus and Shepperd, 1956; Everett, Blockus, Shepperd 1 Present address: Indian Institute of Experimental Medicine, Calcutta-32, India; Dr. Ganguly received support from the CSIR-DAAD Exchange of Scientists Programme. 3*
36
D.K. Ganguly et al.
and T o m a n , 1956; Jurna, Nell and Schreyer, 1970; G e o r g e , Haslett and Jenden, 1966; T a s k e r and Kertesz, 1965), and the m o n k e y (Everett, Blockus and Shepperd, 1956; Everett, Blockus, S h e p p e r d and T o m a n , 1956). T h e r e have b e e n m a n y suggestions as to the site of action of these drugs, but as yet no general a g r e e m e n t has b e e n r e a c h e d on this point. I n particular, there is a divergence of opinion a m o n g the a b o v e - m e n t i o n e d authors about w h e t h e r or not supraspinal structures are necessary for the genesis of the m o t o r disturbances caused by these drugs. H o w e v e r , there is considerable evidence that o x o t r e m o r i n e leads to increased levels of acetylcholine in the brain (Holmstedt, L u n d g r e n and Sundvall, 1963; H o l m s t e d t and L u n d g r e n , 1964; G a n g u l y and Saha, 1969). Further, using a nerve-muscle preparation, G a n g u l y and C h a u d h u r i (1970) have d e m o n s t r a t e d enh a n c e d acetylcholine release at m o t o r end-plates. In the light of these findings it seems plausible that o x o t r e m o r i n e develops its effects not by acting at o n e specific site, but rather by influencing a variety of cholinergic junctions. O f the central cholinergic synapses, those f r o m m o t o r axon collaterals onto R e n s h a w cells are not only easily accessible, but have also b e e n thor o u g h l y investigated (cf. Haase, Cleveland and Ross, 1975, for ref.). W e have therefore sought evidence of an effect of o x o t r e m o r i n e at this site. A n u m b e r of ancillary experiments were p e r f o r m e d to check the effect of oxot r e m o r i n e on alpha m o t o n e u r o n excitability.
Methods General
Experiments were carried out on 24 intercollicular decerebrate cats, which were immobilized by intravenous injection ofN,N'-diallylnortoxferinium-dichloride (Alloferin| 0.25 mg/kg) and artificially respired. In three experiments, the spinal cord was also transected at the low thoracic level. Blood pressure was monitored throughout the experiments by means of a cannula in the common carotid artery. Body temperature was kept between 37 and 39~ C. All measurements were performed at least 2 b after decerebration and removal of anesthesia. The right (contralateral) hind limb was denervated as completely as possible and the left (ipsilateral) dorsal roots L6 to S 1 were cut intradurally. In experiments in which single, orthodromically activated alpha motoneurons were recorded the ipsilateral dorsal roots L7 and S1 remained intact.
Premedication and Application of Oxotremorine
Cho, Haslett and Jenden (1962) have shown that as little as 0.06 ~g/kg oxotremorine noticeably lowers blood pressure. In order to prevent this effect methylatropine (Eumydrin| 0.5-1.0 mg/kg), which does not cross the blood-brain barrier, was administered intravenously some 20 rain before recording began. Whenever blood pressure fell below 80 mm Hg despite this precaution, the experiment was discarded. After the control period, oxotremorine sesquifumarate (Aldrich Chem. Corp.) was applied either by slow automatic infusion or by injection into the femoral vein. Maximal doses ranged between 10 and 30 ~tg/kg; in one case (interaction with eserine) the maximal dose was 40 ~tg/kg.
Oxotremorine and Renshaw Cells Fig. 1. Renshaw cell discharge in decerebrate cat: effect of oxotremorine infusion (20 ~g/kg maximum). Number of spikes (N) per supramaximal antidromic stimulus to ventral root L7 plotted against time (averages of 10 bursts, plus-minus standard deviation). Heavy bar: period of drug infusion
37
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Identification and Activation of Renshaw Cells After laminectomy, Renshaw cells located in the segment L7 were recorded extracellularly by means of conventional glass micropipettes filled with 3M KC1 and identified by their monosynaptic burst response to single antidromic volleys. Due to the slow inactivation rate of oxotremorine, only one cell per cat could be studied. Throughout the experiments the Renshaw cells were excited by antidromic stimulation of either the central end of the cut ipsilateral ventral root L7, or the ipsilateral gastrocnemius nerves. Stimuli 0.2 msec wide and supramaximal for alpha fibers were presented every 2 sec, in some cases every second. When near-threshold excitation was desired, this was achieved by stimulation of small filaments of the ventral root L7 at strengths supramaximal for alpha fibers. The response of a given Renshaw cell was measured by counting the number of action potentials during the first 100 msec after a stimulus (burst length).
Recording of Monosynaptic Reflexes and Single Motor Axons Monosynaptic reflexes were elicited by single shocks applied to the central end of the cut dorsal root L7 or S1 and recorded from the gastrocnemins or anterior tibial nerves; their height was taken as a convenient measure of synchronous motor activity. Stimuli were presented every 5 sec and were submaximal as judged by the reflex amplitude during the control period. In another series of experiments single alpha fibers which exhibited tonic activity during tetanic stimulation of the ipsilateral gastrocnemius nerves were functionally isolated from the ventral root L7 or S1; their discharges were recorded with the help of an interval meter, as well as on film. Every 30 or 60 sec a train of stimulus pulses (0.2 msec wide, 125 Hz) lasting 15 sec was applied.
Results
R e n s h a w Cells O f 14 R e n s h a w cells w h i c h w e h a v e i n v e s t i g a t e d , 11 r e s p o n d e d to o x o t r e m o r i n e w i t h a s i g n i f i c a n t i n c r e a s e in t h e n u m b e r o f d i s c h a r g e s p r o d u c e d b y a n a n t i d r o m i c s t i m u l u s , w h i l e t h e d r u g h a d n o e f f e c t o n t h e r e m a i n i n g t h r e e cells. T y p i c a l f o r t h e i n c r e a s e o f R e n s h a w cell e x c i t a b i l i t y d u r i n g i n f u s i o n o f o x o t r e m o r i n e is t h e e x p e r i m e n t s h o w n in Fig. 1. S t i m u l a t i o n o f t h e c e n t r a l e n d o f t h e c u t v e n t r a l r o o t L 7 r e s u l t e d in a b o u t 9 s p i k e s p e r b u r s t b e f o r e a p p l i c a t i o n o f t h e d r u g . D u r i n g i n f u s i o n (20 ~ g / k g t o t a l ) , this r e s p o n s e i n c r e a s e d to a m a x i m u m o f m o r e t h a n 2 0 s p i k e s p e r burst.
38
D.K. Oanguly et al. s N [%]
N
Fig. 2. Summary of oxotremorine (10-30 ~tg/kg)effects on 11 Renshaw cells (decerebrate and spinal cats). Stimuli applied to either ventral root L7 or gastrocnemius nerves. Left diagram: number N of Renshaw cell discharges per stimulus (plus-minus standard deviation) during control period (open circles) and at time of maximal oxotremorine effect (filled circles). Right diagram: percent increase AN in Renshaw cell excitability at maximum of drug effect (closed circles) relative to control value (open circle)
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Fig. 3. Comparing the effect of oxotremorine rejection (25 ~tg/kg at arrow) on the response of a Renshaw cell m supramaximal (open circles) and near-threshold (filled circles) stimuli to ventral root L7 (spinalized decerebrate cat). Points are average number of spikes in 5 bursts, plus-minus standard deviation. Original records: responses to supramaximal (upper row) and near-threshold (lower row) stimulibefore (a) and after (b, c) oxotremorine injection
Oxotremorine and Renshaw Cells Fig. 4. Showingconcomitantincrease in burst response and spontaneous activity of a Renshaw cell (decerebrate cat). Upper row: control (a: burst response, b: spontaneous activity); lowerrow: correspondingrecords (c and d) after 15 ~tg/kgoxotremorine
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The effect of oxotremorine on 11 cells is summarized in Fig. 2, the left half of which portrays the average responses to supramaximal antidromic stimulation before the drug was administered and at the time of maximal oxotremorine effect. Shown in the right diagram is the increase in spikes per burst relative to the control response. This increase ranged from 10 to 110 %. The results of the three experiments on spinalized cats are included in the figure, as this procedure caused no detectable change in the effect of oxotremorine. An interesting effect occurred in two spinal cats in which not only supramaximal, but also near-threshold stimuli were employed (Fig. 3). Near-threshold excitation of Renshaw cells was achieved by using small filaments of the ventral root (see Methods). While oxotremorine resulted in the usual increase in response to whole-root stimulation, the number of spikes elicited by near-threshold stimulation vacilated strongly; this is reflected in the size of the standard deviations. The possibility that these fluctuations may have resulted from a periodic change in Renshaw cell excitability at characteristic tremor frequencies (8-12 Hz; Friedman and Everett, 1964) could not be tested by our method, since in this experiment stimuli were presented only every 2 sec. The effect of oxotremorine on the spontaneous activity of a Renshaw cell is shown in Fig. 4. Although spontaneously active Renshaw cells are rare in preparations in which the segmental afferent input is cut off, a clear increase in spontaneous discharge rate could be seen in two cells, one of which is depicted in the figure. The spontaneous activity of the other cell took the form of bursts occurring at an average rate of 3.8/sec, which were not present during the control period.
Motoneuron Activity Since increased Renshaw cell excitability, especially the augmented spontaneous activity, might conceivably follow from enhanced alpha motoneuron activity, it became necessary to check this possibility directly. Chalmers and Yim (1963) have already demonstrated that tremorine either reduces the height of monosynaptic reflexes in the spinal dog or else leaves them unaffected. In four of seven experiments employing monosynaptic extensor reflexes in our decerebrate preparation, injection of oxotremorine also diminished reflex height; no effect occurred in the remaining three experiments. This result is corroborated by the experiments on functionally isolated alpha axons, which showed that single units become less excitable following application of the drug.
40
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Fig. 5. Effect of oxotremorine (20 ~g/kg) on motoneuron excitability (decerebrate cat). Top: discharges of functionally isolated alpha axon (ventral root L7); bar below c indicates first and final 3.5 sec of the 15 sec period of tetanic stimulation of the medial gastrocnemius nerve (80 mV, 125 Hz). Bottom: corresponding records of interspike interval vs. time (measurement range: 0-200 msec), a: control recordings before oxotremorine; b and c: recordings 7 and 20 rain, respectively, after drug injection
This decreased excitability is reflected in the fact that (1) the f r e q u e n c y of tonic discharge is reduced, and that (2) the threshold for m o t o n e u r o n firing is raised or that (3) the initially tonic response to o r t h o d r o m i c tetanization b e c o m e s irregular or even phasic, as illustrated in Fig. 5. In no case did the alpha m o t o n e u r o n s investigated b e c o m e s p o n t a n e o u s l y active u n d e r the o x o t r e m o r i n e dosages used in the R e n s h a w cell experiments. Since Jurna, Nell and Schreyer (1970) have reported that in anesthesized, non-deafferented rats large doses of oxotremorine (up to 600 ~tg/kg) increase alpha motoneuron activity, an additional dose of 100 ~tg/kgwas applied in one experiment in which the discharge rate of an alpha motoneuron had dropped as a result of an injection of 30 ~tg/kgof the drug. Spontaneous activity appeared for a few seconds immediately after the additional injection and the rate of tonic discharge was increased.
Drug Interaction
T h e interaction of o x o t r e m o r i n e with atropine, which is k n o w n to ameliorate the s y m p t o m s o f Parkinsonism, and eserine, which has the opposite effect (Duvoisin, 1967), m a y be studied at the junction of m o t o r axon collaterals and R e n s h a w cells, as the actions o f the latter drugs on R e n s h a w cells have b e e n described in considerable detail (Eccles, Fatt and Koketsu, 1954; Curtis and Ryall, 1966). A t r o p i n e intravenously applied depresses the response of R e n s h a w cells to antidromic stimulation, as illustrated in Fig. 6 and first described by Eccles et al. (1954). L a t e r injection of o x o t r e m o r i n e clearly antagonizes this effect. O n the o t h e r hand, the expected synergism of eserin and o x o t r e m o r i n e is m o r e difficult to demonstrate, since eserine alone potentiates R e n s h a w cell activity to such an extent that a further increase due to o x o t r e m o r i n e is not likely to oc-
Oxotremorine and Renshaw Cells
41
Fig. 6. Antagonistic effects of atropine (2 mg/kg; open arrow) and oxotremorine (30 ~g/kg; filled arrow). Number of spikes (N) per supramaximal antidromic stimulus to ventral root L7 plotted against time (averages of 5 bursts, plus-minus standard deviation)
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Fig. 7. Synergistic effects of oxotremorine (filled arrows) and eserine (open arrow). Number of spikes (N) per supramaximal antidromic stimulus to ventral root L7 plotted against time (averages of 50-100 bursts, plus-minus standard deviation). Original records a-e correspond to letters in graph. Drug dosages given in text
cur. Nevertheless, synergism may be demonstrated by making use of the observation that oxotremorine increases the Renshaw cell response only up to a certain point, after which multiple applications of the drug have no further effect. This is illustrated by the first three filled arrows in Fig. 7, which correspond to oxotremorine doses of 5, 5 and 10 ~g/kg, respectively. If, however, a new dose of oxotremorine is given during the declining phase of the effect of eserine (open arrow; 100 ~g/kg), a renewed increase comparable to that produced by the first injection is obtained (fourth filled arrow; oxotremorine; 20 ~tg/kg). This is taken as evidence that the remaining concentration of eserine suffices to facilitate the action of oxotremorine
42
D.K. Ganguly et al.
Discussion The use of antidromic stimulation is the simplest means of testing the effect of oxotremorine on the chemical transmission from motor axon collaterals to Renshaw cells, because only one synaptic junction is involved. In addition, the fact that the cats used in our experiments were decerebrated or spinalized and deafferented eliminated most of the extraneous influences on the cells tested. We have shown that in most cases the drug facilitates the synaptic transmission to a considerable degree. Thus, the idea (Stern, 1963, p. 88) that " . . . Renshaw cells in rest tremor are either inactive all the time or even destructed" cannot be the explanation of the oxotremorine effects on motor activity in experimental animals. The constant motor output imitated by our method of stimulation of the axons of alpha motoneurons is associated with augmented Renshaw cell activity, which presumably means that transmission in the recurrent pathway is more potent. The total effect need not be simple, however; in particular, it should be emphasized that this increased potency will affect not only the agonist motoneurons, but also those of the antagonist via the recurrent control of reciprocal inhibition (cf. Hultborn, 1972). This is indeed of importance, as tremor is associated with alternating contractions of antagonistic muscles (cf. Kaada, 1963). The irregularity of the threshold Renshaw cell response occasionally observed under oxotremorine (see Fig. 4) will also perturb muscle activity. Clearly the oxotremorine-induced changes in Renshaw cell excitability must affect motor performance, independently of the effects of the drug on muscle receptors and higher centers of the central nervous system. Since augmented m o t o n e u r o n excitability was not observed in our preparation, this is unlikely to be the explanation for the increased spontaneous firing of some Renshaw cells seen following oxotremorine. A more probable cause is the activation of muscarinic acetylcholine receptors at the Renshaw cell membrane (Curtis and Ryall, 1966). Some symptoms of Parkinsonism have been suggested to be the result of a disbalance of transmitter substances in the basal ganglia (Barbeau, 1962; Steg, 1969), with a dominance of acetylcholine. We would submit that a similar dominance of acetylcholine ought to exist in the oxotremorine model of Parkinson's disease and that this notion of disbalance must be extended to include cholinergic synapses in the spinal cord. Acknowledgement. We thank I. Lippitschfor skilfultechnicalassistance.
References Barbeau, A.: The pathogenesis of Parkinson's disease: A new hypothesis. Canad. reed. Ass. J. 87, 802-807 (1962) Chalmers, R.K., Yim, G.K.W.: Tremorine tremor in chronic spinal rats. Proc. Soc. exp. Biol. (N.Y.) 109, 202-205 (1962) Chalmers, R.K., Yim, G.K.W.: Spinal actions of tremorine in the dog. Arch. int. Pharmacodyn.145, 322-333 (1963) Cho, A.K., Haslett, W.L., Jenden, D.J.: The peripheral actions of oxotremorine, a metabolite of tremorine. J. Pharmacol. exp. Ther. 138, 249-257 (1962)
Oxotremorine and Renshaw Cells
43
Curtis, D.R., Ryall, R.W.: The synaptic excitation of Renshaw cells. Exp. Brain Res. 2, 81-96 (1966) Duvoisin, R.C.: Cholinergic-anticholinergic antagonism in Parkinsonism. Arch. Neurol. (Chic.) 17, 124-136 (1967) Eccles, J.C., Fatt, P., Koketsu, K.: Cholinergic and inhibitory synapses in a pathway from motor-axon collaterals to motoneurones. J. Physiol. (Lond.) 126, 524-562 (1954) Everett, G.M., Blockus, L.E., Shepperd, I.M.: Tremor induced by tremorine and its antagonism by anti-Parkinson drugs. Science 124, 79 (1956) Everett, G.M., Blockus, L.E., Shepperd, I.M., Toman, J.E.P.: Production of tremor and a Parkinsonlike syndrmne by 1-4-dipyrrolidono-2-butyne, "Tremorine." Fed. Proc. 15, 420-421 (1956) Friedman, A.H., Everett, G.H.: Pharmacological aspects of Parkinsonism. Advanc. Pharmacol. 3, 83-127 (1964) Ganguly, D.K., Chaudhuri, S.K.: Neuromuscular pharmacology of oxotremorine. Europ. J. Pharmacol. 11, 84-89 (1970) Ganguly, D.K., Saha, L.: Effect of oxotremorine on acetylcholine content of brain and peripheral tissues in rats. Ind. J. exp. Biol. 7, 176-177 (1969) George, R., Haslett, W.L., Jenden, D.J.: The production of tremor by cholinergic drugs: central sites of action. Int. J. Pharmacol. 5, 27-34 (1966) Haase, J., Cleveland, S., Ross, H.-G.: Problems of postsynaptic autogenous and recurrent inhibition in the mammalian spinal cord. Rev. Physiol. Biochem. Pharmacol. 73, 73-129 (1975) Holmstedt, B., Lundgren, G.: Tremorigenetic agents and brain acetylcholine. Oxford: Pergamon Press 1964 Holmstedt, B., Lundgren, G., Sundvall, A.: Tremorine and atropine effects on brain acetylcholine. Life Sci. 2, 731-736 (1963) Hultborn, H.: Convergence on interneurones in the reciprocal Ia inhibitory pathway to motoneurones. Acta physiol, scan& Suppl. 375, 3-42 (1972) Jurna, I., Nell, T., Schreyer, I.: Motor disturbance induced by tremorine and oxotremorine. NaunynSchmiedebergs Arch. Pharmak. 267, 80-98 (1970) Kaada, B.R.: The pathophysiology of Parkinsonian tremor, rigidity, and hypokinesia. Acta neurol. scand. Suppl. 39, 41-51 (1963) Kaelber, W.W., Hamel, E.G.: Drug (tremorine)-induced tremor in the cat. Arch. Neurol. (Chic.) 2, 338-340 (1960) Nash, J.B., Emerson, G.A.: Studies on the mode of action of tremorine, (1,4-dipyrrolidone-2-butyne). Fed. Proc. 18, 426 (1959) Steg, G.: Striatal cell activity during systematic administration of monaminergic and cholinergic drugs. In: Third Symposium on Parkinson's disease. (Ed. F.J. Gillingham and I.M.L. Donaldson), pp. 26-29. Edinburgh: E. & S. Livingstone 1969 Stern, P.: Contribution on the pathophysiology of intentional tremor. In: Biochemical and Neurophysiological Correlation of Centrally Acting Drugs. (Ed. E. Trabucchi et al.), pp. 81-91. Oxford: Pergamon Press 1963 Tasker, R., Kertesz, A.: The physiology of tremorine-induced tremor. J. Neurosurg. 22, 4 4 9 4 5 6 (1965)
Received September 5, 1975
