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Presynaptic inhibition of the monosynaptic reflex produced by injections of nicotine or eserine in the spinal cat
ESPERlAIEKTAL
Presynaptic
Inhibition
Injections
I(.
( 1976)
of the Monosynaptic
of Nicotine J.
Dr~ar.twrut
50, 736-707
KEUI:OI.OGU
ENGELHARDT
of Anatomy, School Uttiuersity of California, Rereined
or Eserine AND
E.
Reflex in the Spinal
E. DECI~~A
by
Cat
l
of Alcdicirlc, and Brain Rcscarch Los A~rgelcs, Califorrtia 90024 October
Produced
Institute,
6,19ij
In completely deafferented cats with spinal cord transected and paralyzed with gallamine, the close arterial injection of nicotine (5 to 33 fig) into the spinal cord circulation or the intravenous injection of eserine (2 mg/kg) produced a transient increase in excitability of the central terminals of primary afferent fibers. Continuous d-c records from dorsal roots during nicotine injections indicated that a depolarization of the terminals was probably responsible for the observed excitability increase. Mecamylamine prcvented both the depolarization and the increase in excitability of primary afferent central terminals whereas atropine and gallamine did not. The monosynaptic reflex was always depressed following injections of nicotine or eserine, but the excitability of the motorpool was found to he increased by these drugs. It was concluded that the monosynaptic reflex depression following injections of nicotine or eserine had a significant presynaptic inhibitory component as a consequence of a drug-induced depolarization of the central terminals of Group la afferent fibers.
IKTRODUCTTON Since the work of Schweitzer and Wright ( lS), nicotine has been known to inhibit the monosynaptic reflex by a central action. Eccles and coworkers have established that drugs active at nicotinic receptors have a dramatic effect on Renshaw interneurons, producing a prolonged increase in the 1 The work presented partial fulfillment of Physiology. This work GMOO448/8-12. It is a Bevan and C. Su while Grinnell in preparation Brink of Merch, Sharp of Miss S. Henriksson
represents portions of a dissertation submitted by J. K. E. in requirements for the degree of Doctor of Philosophy in was supported by USPHS Grants NB 07154 and S-TOlpleasure to acknowledge some helpful discussion with Drs. J. A. this work was in progress, and the valuable criticism of Dr. A. of the manuscript. We also wish to think Dr. Norman G. & Dohme for providing mecamylamine. The technical assistance is gratefully acknowledged.
the
786 Copyright All rights
0 1976 by Academic Press, Inc. of reproduction in any form resewed.
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spontaneous activity of these cells at dosages which inhibit the monosynaptic reflex (6, 8). These same investigators advanced the hypothesis that cholinergic drugs inhibit the monosynaptic reflex by first exciting Renshaw interneurons. The hyperactive Renshaw cells would then inhibit the reflex by means of their inhibitory synapses on motoneurons (10). A recent report by MPszjros (15) that cholinergic drugs influence the excitability of presynaptic terminals in the lateral geniculate nucleus of cats raises the possibility that these drugs might have a similar effect on presynaptic terminals in the cat spinal cord. If cholinergic drugs were found to depolarize Group Ia afferent terminals, then presynaptic inhibition would be a reasonable alternative mechanism for the effect of cholinergic drugs on the monosynaptic reflex. The research reported here was specifically designed to examine this possibility. The results indicated that nicotine and eserine produce primary afferent depolarization by a central mechanism, METHODS Observations were made on 16 adult cats weighing 2.2 to 4.3 kg. Under ether anesthesia a tracheotomy was performed. The spinal cord was then cut at the level of C-l and the brain destroyed by interruption of its blood supply. After this procedure, anesthesia was discontinued and the animal was put on artificial respiration. A cannula was placed in the carotid artery to monitor blood pressure, and a second cannula was placed in a femoral vein for intravenous injections. Twelve cats were prepared for close arterial injection of drugs into the spinal cord circulation. The abdomen was opened in these preparations, and an Intramedic polyethylene cannula (Clay Adams, Inc.; i.d., 0.086 cm; o.d., 0.127 cm) was placed in the abdominal aorta via one external iliac artery with the tip just below the level of the renal artery. All branches of the abdominal aorta below the renal arteries were ligated except the lumbar arteries. This is a modification of the close arterial injection technique described by Curtis et al. (6). In one cat, a bilateral adrenalectomy was performed at this time. After the abdominal aorta cannula was in place, the laparotomy was closed. A lumbar laminectomy was then performed in these animals (as well as in those used for iv injection). Following the laminectomy, four cats had an additional spinal sectioning at segment L-l. Three of these low-spinal cats were completely deafferented by severing all dorsal roots on both sides that entered the cbrd below segment L-l. All deafferented cats were prepared for close arterial injections. After the cord was exposed, cats were mounted in a rigid metal frame, the dura opened and the cord covered with warm mineral oil. The rectal and cord pool temperature were maintained near 38.5 C by a heating pad under the body and a second heating element in the mineral oil which covered the cord. Ventral and/or dorsal roots (L-7 or S-l) were cut and
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proximal or distal stumps were placed on bipolar silver hook electrodes. When d-c recordings were to be made, nonpolarizable Ag-AgCl electrodes were used. The electrical activity picked up by the recording electrodes was differentially amplified by high input impedance amplifiers and stored on magnetic tape (Sanborn 3917A tape recorder). After each experiment, the tape was replayed into a Tektronix 565 oscilloscope and responses were photographed with a Grass kymographic camera. Prior to injection of nicotine or eserine, the cat was prepared with intravenous injections of 2 mg/kg atropine to prevent cardiac arrest and 5 to 10 mg/kg gallamine to block cholinergic effects at the neuromuscular junction. Before a close arterial injection, a sufficient amount of the nicotine solution was slowly injected to prefill the cannula in the abdominal aorta. Within 5 min of the preparatory drugs, 1 ml test solution was injected by hand in approximately 1 sec. Injections consisted of the appropriate amount of nicotine dissolved in 0.9% NaCl buffered to pH 7.4 with 5 mM phosphate buffer. All eserine injections were intravenous and consisted of 2 mg/ml eserine sulfate in 0.9% NaCl. Control injections of the carrier solution (0.9% NaCl) given from time to time, had no effect. The following drugs were used : atropine sulfate (Ruger Chemical Company) ; gallamine triethiodide (Flaxedil, American Cyanamid Company) ; eserine sulfate (Nutritional Biochemical Corporation) ; nicotine bitartrate (K and K Laboratories) ; nicotine alkaloid (Nutritional Biochemical Corporation) ; and Mecamylamine hydrochloride (Merck). The doses of the drugs administered are expressed in terms of the weight of the salt used, except those of nicotine, which are all given in terms of the base. The parameters routinely monitored during a drug injection were the excitability of primary afferent presynaptic terminals, the monosynaptic reflex, and the blood pressure. The excitability of presynaptic terminals was monitored by Wall’s technique (22). Current pulses were delivered through a glass micropipette filled with 0.9% NaCl (tip diameter lo-30 pm, resistance 3-10 MO). The tip of the pipette was sited near a pool of motoneurons by using the criterion of the antidromic field-potential recorded by the pipette after stimulus of a ventral root (3). After the pipette was in place, it was switched from the recording to a stimulating circuit, and the response in the appropriate dorsal root was observed at various stimulus intensities to insure that the experiment would be conducted in the linear portion of the stimulus-response curve. As long as the response remained in the linear portion of the stimulus-response curve and the stimulus remained constant, a drug-induced increase in the response could be interpreted as a proportional increase in excitability of the presynaptic terminals under study (22).
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Although various recording and stimulation arrangements were used and will be illustrated when the results are presented, stimuli were always delivered with a standard temporal relation. The first stimulus in a cycle was given to a dorsal root to elicit the monosynaptic reflex in the corresponding ventral root. The pulse used to test the excitability of presynaptic terminals (Wall’s test) always followed the monosynaptic reflex test stimulus by 500 msec. This cycle of monosynaptic reflex test-Wall’s testwas delivered once every second. In the data presentation technique used in this paper, each response is made visible on a slowly moving film strip by beam intensification of the appropriate portion of a stimulus-synchronized oscilloscope sweep. Each response appears as a vertical bar on the horizontally moving film strip. The height of the bar above the dark base line is the amplitude of the response. Time interval between bars is always 1 sec. At the end of the experiment, the tip of the Wall’s test pipette was broken in place, using the dissecting microscope to visually control for movement. The cat was then perfused with 10% formaldehyde, and the segment of cord containing the pipette was removed for histological confirmation of the pipette position in the ventral horn. Kliiver-stained, 10 pm serial sections proved to be satisfactory for this purpose. RESULTS Close arterial injections of nicotine produced, in addition to the wellknown depression of the monosynaptic reflex (6)) an increase in excitability of presynaptic terminals. Ten close arterial injections of nicotine were made in doses ranging from 5 to 33 pg in seven separate preparations. Nine of these injections produced an increase in excitability of primary afferent terminals in the ventral horn. The only injection which did not produce the effect was one of the two 5 pg-injections. The increase in the amplitude of the Wall’s test response that measured these excitability changes ranged from 61% to 150% above control values. The maximum increase in excitability occurred between 2 and 4 set after start of the injection. The halfdecay time from this maximum ranged from 3 to 40 sec. Data collected during one such injection are presented in Fig. M-D. In this experiment the drug-induced increase in excitability of presynaptic terminals (Fig. 1A) was equivalent to a supramaximal conditioning shock to a nearby dorsal root, or to doubling the intensity of the 0.2 msec constant current pulse through the test pipette (from 4.5 to 9.0 r-A). The probable substratum of these presynaptic excitability changes, a depolarization of primary afferents, could be observed in d-c records of slow potential changes in the same dorsal root (Fig. 1C).
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’
AND
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’
before (A-D) and 3 FIG. 1. Effects of close arterial injections of 20 pg nicotine min after 2 mg/kg iv mecamylamine (E-G). rl and E, Wall’s test of the excitability of dorsal root S-l terminals in ventral horn of segment S-l. B and F, monosynaptic reflex recorded in the ipsilateral ventral root of segment L-7 as a result of a stimulus to dorsal root L-7. C and G, continuous d-c record from dorsal root S-l. An upward deflection represents relative negativity of the electrode closest to the cord. Records C and G were taken from the same dorsal root electrodes as A and E, however, C and G were continuous d-c records and .tl and E were a-c records of series of individual responses triggered by pipette stimuli (note difference in calibration). D, blood pressure. Base line equals 0 mmHg. H, diagram of the arrangement of stimulating and recording electrodes. This cat had both adrenal glands removed during surgical preparation for this experiment. The black dot over each record indicates duration of the nicotine injection.
In five of the ten close arterial injections of nicotine, a simultaneous record of the monosynaptic reflex was made. In each case, an increase in presynaptic terminal excitability was paralleled by monosynaptic reflex depression. The reflex depression ranged from 62% to 10070 of control values with the maximum depression occurring between 2 and 4 set after start of the injection. As illustrated in Fig. lB, however, the half-decay time for the nicotine-induced reflex depression was at least double that of the excitability increase in presynaptic terminals and ranged from 23 set to 10 min. This difference in decay times for the drug effects may mean that if presynaptic inhibition does contribute to the early reflex depression, it may not be able to account for all of the depression. Another possibility is that Wall’s technique is not sensitive enough to observe subtle but significant excitability changes in presynaptic terminals. Due probably to the known variability of spinal preparations, there was no clear correlation between the dose of nicotine and magnitude of the effect in different animals. A dose of 5 pg, for example, produced in one cat the same increase in presynaptic excitability (83%) as an injection of 33 ,~g produced in another identical high-spinal preparation. A parallel variability existed for the nicotine effect on the monosynaptic reflex among different animals.
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Changes in systemic blood pressure were not the cause of nicotine’s effect on presynaptic terminals. Artificial increases in blood pressure produced by rapid injections of Dextran were not accompanied by any changes in presynaptic terminal excitability. Conversely, nicotine produced a clear increase in presynaptic terminal excitability (Fig. 1A) in an adrenalectomized animal in which no changes in systemic blood pressure were observed (Fig. 10). This latter result also excluded the possibility that nicotine was acting through the release of catecholamines from the adrenal glands. Three different approaches were used to control for the possibility that the observed presynaptic effects could be due to an increased afferent barrage triggered by nicotine’s known actions on peripheral receptors. As stated in Methods, all nicotine injections were given after a preparatory injection of gallamine, a synthetic curare substitute. Gallamine is known to block nicotine’s effect on muscle spindles (ll), and because of its pharmacologic similarity to curare, should block nicotine’s action at cutaneous receptors (7, 19, 23). This pharmacologic blockade of peripheral receptors was confirmed by our second control, which consisted of recording from the distal stump of a dorsal rootlet in one of our preparations. No increase in the afferent barrage was detected in this animal following maximum injections of nicotine or eserine. The third and most important control for cholinergic effects on peripheral receptors was the use of three completely deafferented preparations in which all dorsal roots on both sides of the cord were cut berow segment L-l, at which point a low spinal section was made. Monosynaptic reflex depression, together with increased excitability of presynaptic terminals, were observed following injection of nicotine in two of these deafferented preparations (Fig. 2A and C) . In the third deafferented cat, intracellular techniques were used to observe a transient reduction in amplitude of the monosynaptic EPSP following injection of 10 pg nicotine. The amplitude was reduced by as much as 38% with return to within 9% of control values in 3 min. Although depression of EPSP is not a sufficient condition for the demonstration of presynaptic inhibition, it is a necessary condition, and as such it is significant that this depression was present in a deafferented preparation following a dose of nicotine within the 5 to 33 rg range already shown to produce increases in presynaptic terminal excitability. An injection of atropine (2 “g/kg) was routinely given to protect the cat from cardiac arrest during injections of nicotine or eserine. There was no effect on presynaptic terminal excitability or on the monosynaptic reflex as a result of atropine injection. The central effects of nicotine could be blocked, however, by mecamylamine (2 mg/kg iv), a secondary amine with established CNS nicotinic-blocking properties at this dose (20). Fig. lE-G shows the blocking action of this drug for the same dose of nicotine
792
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2 m rec.
AND
DECIMA
“RL,
FIG. 2. Effect of a close arterial injection of 25 pg nicotine on presynaptic and motorpool excitability. Location of the pipette combined with arrangement of the recording electrodes enabled simultaneous testing of the excitability of presynaptic terminals and pool of motorneurons. A, Wall’s test of excitability of dorsal root L-7 terminals in the ventral horn. B, motorpool excitability tested by the same stimuli testing presynaptic terminals. Response to the test stimulus was recorded in ventral root L-7 with 0.25 msec latency. C, left to right, samples of monosynaptic reflex recorded in ventral root L-7 before, 6 set after, and 25 min after nicotine injection. Note absence of monosynaptic reflex in the record taken 6 set after injection. D, diagram of arrangement of stimulating and recording electrodes. This cat was a completely deafferented preparation. Black dots over records A and B indicate the duration of nicotine injection.
illcstrated in Fig. lA-D. Mecamylamine injection pc~ SC did not produce changes in either presynaptic terminal excitability or the monosynaptic reflex. Intravenous injections of eserine (2 mg/kg) also increased the excitability of presynaptic terminals while depressing the monosynaptic reflex. Data recorded during one such injection are presented in Fig. 3. Four injections of eserine in four different cats produced a maximum increase in excitability of presynaptic terminals that ranged from 2670 to 71% above control values. This maximum was observed between 27 and 51 set after start of the injection. The half-decay time of the excitability change ranged from 23 to 92 sec. This eserine effect on primary afferent terminals was observed in a completely deafferented preparation. As in the case of nicotine, the drug-induced depression of the monosynaptic reflex lasted more than twice as long as the excitability increase of presynaptic terminals. Mecamylamine also blocked the effect of eserine on the monosynaptic reflex, whereas atropine and gallamine did not. Conduction velocities were measured for the gastrocnemius medialis afferent fibers of which the central terminals showed the eserine-induced excitability increase documented in Fig. 3A. The conduction velocities
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FIG. 3. Effects of eserine sulfate (2 mg/kg iv). A, Wall’s test of excitability of central terminals of gastrocnemius medialis afferent fibers. B, monosynaptic reflex recorded in contralateral ventral root L-7 after stimulus to dorsal root L-7. C, blood pressure. Base line equals 0 mmHg. Black bars over records A-C indicate duration of eserine injection. D, single response to Wall’s test of central gastrocnemius medialis afferent terminals taken from record A and recorded at a fast sweep. E, single monosynaptic reflex response taken from record B and recorded at same sweep speed as D. F, diagram of arrangement of stimulating and recording electrodes. The gastrocnemius medialis nerve was deefferented on the left side by acute section of ventral roots of L-6, L-7, and S-l.
observed (110 to 128 m/set) indicated that the presynaptic terminals affected belonged to the afferent fibers of the monosynaptic reflex arc (14). Depression of the monosynaptic reflex by nicotine at the doses used in our experiments has been attributed to Renshaw-cell inhibition of the motoneuron (6, 8). Two independent methods in three separate experiments indicated that contrary to the predictions of this hypothesis, the excitability of the motorpoo1 increased after injections of nicotine or eserine. In the deafferented cord experiment illustrated in Fig. 2, the current pulse delivered through the pipette not only stimulated primary afferent terminals in the ventral horn but also produced a discharge in the ventral roots. The latter had a latency of 0.25 msec which indicated that it was the result of a direct stimulation of motoneurons through the pipette as opposed to transynaptic stimulation ( 16). Figure 2B documents the increase in this ventral root response that followed the administration of nicotine. This fact lends additional support to the belief that we were observing a direct test of motoneuron excitability because the monosynaptic reflex was completely abolished for a considerable period by this same nicotine injection (Fig. ZC). The increased excitability of the motorpool followed a briefer time
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FIG. 4. Effect of close arterial injection of 16 pg nicotine on the antidromic field potential in the gastrocnemius medialis motorpool. ‘4, each bar represents the average of five antidromic field-potential amplitudes. Shaded bar indicates time of nicotine injection. B, left to right, samples of antidromic field potential before, during, and after the nicotine effect. Arrows in r2 indicate the bars from which the sample records were taken. C, diagram of stimulating and recording arrangement. Left dorsal roots L-6 through S-2 were cut.
course than the increased excitability of presynaptic terminals in Fig. 2kt, but at no time was there reduction of the excitability of the motorpool below control values. In two other experiments, a focal pipette recorded the field potential which resulted from the antidromic invasion of the gastrocnemius medialis motor-pool. The increase in amplitude of this field potential after the close arterial injection of 16 pg nicotine is depicted in Fig. 4A. This injection was repeated in the same experiment with the same result, and an eserine injection (2 mg/kg iv) in a separate experiment produced a similar increase in size of the antidromic field. The growth of the antidromic field potential could mean that a larger number of motoneurons was invaded or that the somaclendritic tree of each individual motoneuron was more completely invaded. In either case, the implication is that the excitability of the motorpool was increased, not diminished, by these drugs ( 17). Injections of either nicotine or eserine are known to stimulate muscle spindles (1, 12, 21)) and nicotine has also been reported to excite cutaneous receptors (4, 7, 23). However, impulses in cutaneous and muscle afferent:;
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can produce depolarization in the central terminals of Group Ia afferents, resulting in presynaptic inhibition of the monosynaptic reflex (2, 9, 13). One might expect, therefore, that an injection of nicotine or eserine would increase the afferent barrage to the cord and depress the monosynaptic reflex through presynaptic inhibition. The operation of just such a presynaptic mechanism has already been demonstrated for large doses of nicotine (> 80 pg/kg iv) (11). However, smaller doses reportedly inhibit the monosynaptic reflex by a central mechanism that is blocked by mecamylarnine, presumably via activation of Renshaw interneurons ( 11). These interneurons are known to increase their spontaneous activity after injections of nicotine or eserine (8) and have also been shown to have inhibitory synapses on alpha motoneurons (10). Our observations indicate that presynaptic inhibition always contributes to the nicotine-induced monosynaptic reflex depression in the dose range investigated (5 to 33 pg). We used essentially the same close arterial injection technique used by Curtis et al. in their classical work on the effect of drugs on the monosynaptic reflex (6). All our nicotine injections were within or below the 10 pg-to-50 pg range used by these same investigators. Our only injections outside this range were two 5-pg injections of nicotine, one of which produced a dramatic 83% increase in the excitability of presynaptic terminals with a half-decay time of 29 sec. This 5-pg dose compares favorably with the 0.2 pg-to-2 pg dose of nicotine reported by Eccles et al. to be necessary for the observation of an increase in Renshaw-cell spontaneous activity (8). We have also determined that the monosynaptic reflex depression produced by eserine (2 mg/kg iv) has a presynaptic inhibitory component. Whereas the reflex depression produced by either nicotine or eserine outlasts considerably the presynaptic excitability increase measured by Wall’s test, a presynaptic inhibitory mechanism is certainly in operation during the initial phase of the reflex depression because the excitability of the motorpool is increased over control levels (Figs, 2 and 4). The peripheral actions of nicotine and eserine mentioned above would be the least controversial explanation for our observations, but there are several reasons for believing that the presynaptic inhibition observed is the result of a central action of these drugs. First, all our preparations were paralyzed with the synthetic curare substitute, gallamine, just prior to each injection of nicotine or eserine to block peripheral cholinergic effects. We confirmed the effectiveness of this treatment when we were unable to record any increase in background discharge from the distal stump of a dorsal rootlet following maximal injections of nicotine or eserine. Most significantly, while gallamine did block the peripheral effects of these cholinergic drugs, it was not expected to have central effects in the doses used and in fact did not prevent the action of nicotine or eserine on the
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monosynaptic reflex or central Ia afferent terminals. These findings are important in the light of the recent demonstration that a large number of unmyelinated axons in cat ventral roots have their cell bodies in the dorsal root ganglia (5) and carry information from visceral receptors (personal communication). However, there are no reasons a priori to expect that these receptors are pharmacologically different from others studied so far (7, 19, 23) and it is thus very likely that gallamine had also blocked nicotinic effects in these visceral receptors. The second reason is that the nicotineinduced primary afferent depolarization was blocked by mecamylamine (Fig. 1)) a drug that does block CNS nicotinic receptors (20) but does not influence the spindle acceleratory effect of large doses of nicotine ( 11) . Finally, the strongest argument for the central action of these drugs rests on the observation that nicotine and eserine both increased the excitability of presynaptic terminals during reflex depression in classically deafferented low-spinal preparations (Fig. 2). It is therefore concluded that the initial monosynaptic reflex depression following injections of nicotine or eserine has a significant presynaptic inhibitory component as a consequence of a drug-induced depolarization of the central terminals of Group Ia afferent fibers. In addition, selective drug antagonism and experiments in classical deafferented preparations (spinal cord sections at L-l and complete dorsal rhizotomy of lumbar and sacral segments) indicate that the primary afferent depolarization observed is the result of a central action of these drugs. To state that this nicotinic effect occurs centrally does not necessarily presuppose knowledge of its mechanism. The two possible alternatives: (a) direct effect of the drug on the tested terminals and (b) effect mediated via some interneuronal pool, remain still to be investigated. REFERENCES 1. ALBUQUERQUE, E. X., and C. M. SMITH. 1964. Specificity of excitation of muscle spindle afferents by cholinergic substances. J. Phamzacol. E.rb. Ther. 146: 344353. 2. ANDBN, N. E., M. G. M. JUKES, A. LUNDBERG, and L. VYKLICKG. 1966. The effect of DOPA on the spinal cord-3. Depolarization evoked on the central terminals of ipsilateral Ia afferents by volleys in the flexor reflex afferents. Acta Physiol. Stand. 68: 322-336. 3. BARAKAN, T. H., C. B. B. DOWNMAN, and J. C. ECCLES. 1949. Electrical potentials generated by antidromic volleys in quadriceps and hamstring motoneurons. J. Newrophysiol. 12 : 393-424. 4. BROWN, G. L., and J. A. B. GRAY. 1948. Some effects of nicotine-like substances and their relation to sensory nerve endings. J. Physiol (London) 107: 306-317. 5. COGGESHALL, R. E., J. D. COULTER, and W. D. WILLIS. 1974. Unmyelinated axons in the ventral roots of the cat lymbosacral enlargement. J. Camp. Neztrol. 153: 39-58.
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D. R., J. C. ECCLES, and R. M. Ecn~. 1957. Pharmacological studies on spinal reflexes. 1. Physiol. (London) 136 : 420-434. DOUGLAS, W. W., and J. A. B. GRAY. 1953. The excitant action of acetylcholine and other substances on cutaneous sensory pathways and its prevention by hexamethonium and d-turbocurarine. J. Physiol. (London) 119: 118-128. ECCLES, J. C., R. M. ECCLES, and P. FATT. 1956. Pharmacological investigation of a central synapse operated by acetylcholine. J. PhysioZ. (London) 131: 154-169. ECCLES, J. C., R. M. EC~LES, and F. MAGNI. 1961. Central inhibitory action attributable to presynaptic depolarization produced by muscle afferent volleys. J. Physiol. (London) 159: 147-166. ECCLES, J. C., P. FATT, and K. KOKETSU. 1954. Cholinergic and inhibitory synapses in a pathway from motor-axon collaterals to motoneurons. J. Physiol. (London) 126 : 524-562. GINZEL, K. H., E. ELDRED, and Y. SASAKI. 1969. Comparative study of the actions of nicotine and succinylcholine on the monosynaptic reflex and spindle afferent activity. Znt. J. Neuropharmacol. 8: 515-533. HUNT, C. C. 1952. Drug effects on mammalian muscle spindles. Fed. Proc. 11: 75. JANKOWSKA, E., S. LUND, and A. LUNDBERG. 1966. The effect of DOPA on the spinal cord-4. Depolarization evoked in the central terminals of contralateral Ia afferent terminals by volleys in the flexor reflex afferents. Acta Physiol. Stand. 68: 337-341. LLOYD, D. P. C. 1943. Neuron patterns controlling the transmission of ipsilateral hind limb reflexes in the cat. J. Neurophysiol. 6: 293-315. M~SZAROS, J. 1971. The effect of some cholinomimetic drugs on presynaptic inhibition in the lateral geniculate nucleus. Neuropharmacology 10 : 67-76. RENSHAW, B. 1940. Activity in the simplest spinal reflex pathways. 1. Neuro-
6. CURTIS, 7.
8.
9. 10.
II.
12. 13.
14. 15. 16.
physiol. 17.
18. 19. 20.
21.
3 : 373-387.
B. 1942. Effects of presynaptic volleys on spread of impulses over the soma of the motorneuron. J. Neurophysiol 5 : 235-243. SCHWEITZER, A., and S. WRIGHT. 1938. Action of nicotine on the spinal cord. J. Physiol. (Londort) 94: 136-147. SKOUBY, A. P. 1953. The influence of acetylcholine, curarine and related substances on the threshold for chemical pain stimuli. Acta Physiol. Stand. 29: 340-352. UEKI, S., K. KOKETSU, and E. F. DOMINO. 1961. Effects of mecamylamine on the Golgi recurrent collateral-Renshaw synapse in the spinal cord. Exp. Neural. 3: 141-148. VERHEY, B. A., and P. E. VOORHOEVE. 1963. Activation of group Ia and group 11 muscle spindle afferents by succinylcholine and other cholinergic drugs. Acta RENSHAW,
Physiol. Pharmacol 22. WALL, P. D. 1958.
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Excitability changes in afferent fiber terminations and their relation to slow potentials. J. Physiol. (London) 142: 1-21. 23. WATSON, P. J. 1970. Drug receptor sites in the rabbit saphenous nerve. ~r,jt. J. Phamacol. 40 : 102-112.
Presynaptic
Inhibition
Injections
I(.
( 1976)
of the Monosynaptic
of Nicotine J.
Dr~ar.twrut
50, 736-707
KEUI:OI.OGU
ENGELHARDT
of Anatomy, School Uttiuersity of California, Rereined
or Eserine AND
E.
Reflex in the Spinal
E. DECI~~A
by
Cat
l
of Alcdicirlc, and Brain Rcscarch Los A~rgelcs, Califorrtia 90024 October
Produced
Institute,
6,19ij
In completely deafferented cats with spinal cord transected and paralyzed with gallamine, the close arterial injection of nicotine (5 to 33 fig) into the spinal cord circulation or the intravenous injection of eserine (2 mg/kg) produced a transient increase in excitability of the central terminals of primary afferent fibers. Continuous d-c records from dorsal roots during nicotine injections indicated that a depolarization of the terminals was probably responsible for the observed excitability increase. Mecamylamine prcvented both the depolarization and the increase in excitability of primary afferent central terminals whereas atropine and gallamine did not. The monosynaptic reflex was always depressed following injections of nicotine or eserine, but the excitability of the motorpool was found to he increased by these drugs. It was concluded that the monosynaptic reflex depression following injections of nicotine or eserine had a significant presynaptic inhibitory component as a consequence of a drug-induced depolarization of the central terminals of Group la afferent fibers.
IKTRODUCTTON Since the work of Schweitzer and Wright ( lS), nicotine has been known to inhibit the monosynaptic reflex by a central action. Eccles and coworkers have established that drugs active at nicotinic receptors have a dramatic effect on Renshaw interneurons, producing a prolonged increase in the 1 The work presented partial fulfillment of Physiology. This work GMOO448/8-12. It is a Bevan and C. Su while Grinnell in preparation Brink of Merch, Sharp of Miss S. Henriksson
represents portions of a dissertation submitted by J. K. E. in requirements for the degree of Doctor of Philosophy in was supported by USPHS Grants NB 07154 and S-TOlpleasure to acknowledge some helpful discussion with Drs. J. A. this work was in progress, and the valuable criticism of Dr. A. of the manuscript. We also wish to think Dr. Norman G. & Dohme for providing mecamylamine. The technical assistance is gratefully acknowledged.
the
786 Copyright All rights
0 1976 by Academic Press, Inc. of reproduction in any form resewed.
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PRESYNAPTIC
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spontaneous activity of these cells at dosages which inhibit the monosynaptic reflex (6, 8). These same investigators advanced the hypothesis that cholinergic drugs inhibit the monosynaptic reflex by first exciting Renshaw interneurons. The hyperactive Renshaw cells would then inhibit the reflex by means of their inhibitory synapses on motoneurons (10). A recent report by MPszjros (15) that cholinergic drugs influence the excitability of presynaptic terminals in the lateral geniculate nucleus of cats raises the possibility that these drugs might have a similar effect on presynaptic terminals in the cat spinal cord. If cholinergic drugs were found to depolarize Group Ia afferent terminals, then presynaptic inhibition would be a reasonable alternative mechanism for the effect of cholinergic drugs on the monosynaptic reflex. The research reported here was specifically designed to examine this possibility. The results indicated that nicotine and eserine produce primary afferent depolarization by a central mechanism, METHODS Observations were made on 16 adult cats weighing 2.2 to 4.3 kg. Under ether anesthesia a tracheotomy was performed. The spinal cord was then cut at the level of C-l and the brain destroyed by interruption of its blood supply. After this procedure, anesthesia was discontinued and the animal was put on artificial respiration. A cannula was placed in the carotid artery to monitor blood pressure, and a second cannula was placed in a femoral vein for intravenous injections. Twelve cats were prepared for close arterial injection of drugs into the spinal cord circulation. The abdomen was opened in these preparations, and an Intramedic polyethylene cannula (Clay Adams, Inc.; i.d., 0.086 cm; o.d., 0.127 cm) was placed in the abdominal aorta via one external iliac artery with the tip just below the level of the renal artery. All branches of the abdominal aorta below the renal arteries were ligated except the lumbar arteries. This is a modification of the close arterial injection technique described by Curtis et al. (6). In one cat, a bilateral adrenalectomy was performed at this time. After the abdominal aorta cannula was in place, the laparotomy was closed. A lumbar laminectomy was then performed in these animals (as well as in those used for iv injection). Following the laminectomy, four cats had an additional spinal sectioning at segment L-l. Three of these low-spinal cats were completely deafferented by severing all dorsal roots on both sides that entered the cbrd below segment L-l. All deafferented cats were prepared for close arterial injections. After the cord was exposed, cats were mounted in a rigid metal frame, the dura opened and the cord covered with warm mineral oil. The rectal and cord pool temperature were maintained near 38.5 C by a heating pad under the body and a second heating element in the mineral oil which covered the cord. Ventral and/or dorsal roots (L-7 or S-l) were cut and
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proximal or distal stumps were placed on bipolar silver hook electrodes. When d-c recordings were to be made, nonpolarizable Ag-AgCl electrodes were used. The electrical activity picked up by the recording electrodes was differentially amplified by high input impedance amplifiers and stored on magnetic tape (Sanborn 3917A tape recorder). After each experiment, the tape was replayed into a Tektronix 565 oscilloscope and responses were photographed with a Grass kymographic camera. Prior to injection of nicotine or eserine, the cat was prepared with intravenous injections of 2 mg/kg atropine to prevent cardiac arrest and 5 to 10 mg/kg gallamine to block cholinergic effects at the neuromuscular junction. Before a close arterial injection, a sufficient amount of the nicotine solution was slowly injected to prefill the cannula in the abdominal aorta. Within 5 min of the preparatory drugs, 1 ml test solution was injected by hand in approximately 1 sec. Injections consisted of the appropriate amount of nicotine dissolved in 0.9% NaCl buffered to pH 7.4 with 5 mM phosphate buffer. All eserine injections were intravenous and consisted of 2 mg/ml eserine sulfate in 0.9% NaCl. Control injections of the carrier solution (0.9% NaCl) given from time to time, had no effect. The following drugs were used : atropine sulfate (Ruger Chemical Company) ; gallamine triethiodide (Flaxedil, American Cyanamid Company) ; eserine sulfate (Nutritional Biochemical Corporation) ; nicotine bitartrate (K and K Laboratories) ; nicotine alkaloid (Nutritional Biochemical Corporation) ; and Mecamylamine hydrochloride (Merck). The doses of the drugs administered are expressed in terms of the weight of the salt used, except those of nicotine, which are all given in terms of the base. The parameters routinely monitored during a drug injection were the excitability of primary afferent presynaptic terminals, the monosynaptic reflex, and the blood pressure. The excitability of presynaptic terminals was monitored by Wall’s technique (22). Current pulses were delivered through a glass micropipette filled with 0.9% NaCl (tip diameter lo-30 pm, resistance 3-10 MO). The tip of the pipette was sited near a pool of motoneurons by using the criterion of the antidromic field-potential recorded by the pipette after stimulus of a ventral root (3). After the pipette was in place, it was switched from the recording to a stimulating circuit, and the response in the appropriate dorsal root was observed at various stimulus intensities to insure that the experiment would be conducted in the linear portion of the stimulus-response curve. As long as the response remained in the linear portion of the stimulus-response curve and the stimulus remained constant, a drug-induced increase in the response could be interpreted as a proportional increase in excitability of the presynaptic terminals under study (22).
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Although various recording and stimulation arrangements were used and will be illustrated when the results are presented, stimuli were always delivered with a standard temporal relation. The first stimulus in a cycle was given to a dorsal root to elicit the monosynaptic reflex in the corresponding ventral root. The pulse used to test the excitability of presynaptic terminals (Wall’s test) always followed the monosynaptic reflex test stimulus by 500 msec. This cycle of monosynaptic reflex test-Wall’s testwas delivered once every second. In the data presentation technique used in this paper, each response is made visible on a slowly moving film strip by beam intensification of the appropriate portion of a stimulus-synchronized oscilloscope sweep. Each response appears as a vertical bar on the horizontally moving film strip. The height of the bar above the dark base line is the amplitude of the response. Time interval between bars is always 1 sec. At the end of the experiment, the tip of the Wall’s test pipette was broken in place, using the dissecting microscope to visually control for movement. The cat was then perfused with 10% formaldehyde, and the segment of cord containing the pipette was removed for histological confirmation of the pipette position in the ventral horn. Kliiver-stained, 10 pm serial sections proved to be satisfactory for this purpose. RESULTS Close arterial injections of nicotine produced, in addition to the wellknown depression of the monosynaptic reflex (6)) an increase in excitability of presynaptic terminals. Ten close arterial injections of nicotine were made in doses ranging from 5 to 33 pg in seven separate preparations. Nine of these injections produced an increase in excitability of primary afferent terminals in the ventral horn. The only injection which did not produce the effect was one of the two 5 pg-injections. The increase in the amplitude of the Wall’s test response that measured these excitability changes ranged from 61% to 150% above control values. The maximum increase in excitability occurred between 2 and 4 set after start of the injection. The halfdecay time from this maximum ranged from 3 to 40 sec. Data collected during one such injection are presented in Fig. M-D. In this experiment the drug-induced increase in excitability of presynaptic terminals (Fig. 1A) was equivalent to a supramaximal conditioning shock to a nearby dorsal root, or to doubling the intensity of the 0.2 msec constant current pulse through the test pipette (from 4.5 to 9.0 r-A). The probable substratum of these presynaptic excitability changes, a depolarization of primary afferents, could be observed in d-c records of slow potential changes in the same dorsal root (Fig. 1C).
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before (A-D) and 3 FIG. 1. Effects of close arterial injections of 20 pg nicotine min after 2 mg/kg iv mecamylamine (E-G). rl and E, Wall’s test of the excitability of dorsal root S-l terminals in ventral horn of segment S-l. B and F, monosynaptic reflex recorded in the ipsilateral ventral root of segment L-7 as a result of a stimulus to dorsal root L-7. C and G, continuous d-c record from dorsal root S-l. An upward deflection represents relative negativity of the electrode closest to the cord. Records C and G were taken from the same dorsal root electrodes as A and E, however, C and G were continuous d-c records and .tl and E were a-c records of series of individual responses triggered by pipette stimuli (note difference in calibration). D, blood pressure. Base line equals 0 mmHg. H, diagram of the arrangement of stimulating and recording electrodes. This cat had both adrenal glands removed during surgical preparation for this experiment. The black dot over each record indicates duration of the nicotine injection.
In five of the ten close arterial injections of nicotine, a simultaneous record of the monosynaptic reflex was made. In each case, an increase in presynaptic terminal excitability was paralleled by monosynaptic reflex depression. The reflex depression ranged from 62% to 10070 of control values with the maximum depression occurring between 2 and 4 set after start of the injection. As illustrated in Fig. lB, however, the half-decay time for the nicotine-induced reflex depression was at least double that of the excitability increase in presynaptic terminals and ranged from 23 set to 10 min. This difference in decay times for the drug effects may mean that if presynaptic inhibition does contribute to the early reflex depression, it may not be able to account for all of the depression. Another possibility is that Wall’s technique is not sensitive enough to observe subtle but significant excitability changes in presynaptic terminals. Due probably to the known variability of spinal preparations, there was no clear correlation between the dose of nicotine and magnitude of the effect in different animals. A dose of 5 pg, for example, produced in one cat the same increase in presynaptic excitability (83%) as an injection of 33 ,~g produced in another identical high-spinal preparation. A parallel variability existed for the nicotine effect on the monosynaptic reflex among different animals.
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Changes in systemic blood pressure were not the cause of nicotine’s effect on presynaptic terminals. Artificial increases in blood pressure produced by rapid injections of Dextran were not accompanied by any changes in presynaptic terminal excitability. Conversely, nicotine produced a clear increase in presynaptic terminal excitability (Fig. 1A) in an adrenalectomized animal in which no changes in systemic blood pressure were observed (Fig. 10). This latter result also excluded the possibility that nicotine was acting through the release of catecholamines from the adrenal glands. Three different approaches were used to control for the possibility that the observed presynaptic effects could be due to an increased afferent barrage triggered by nicotine’s known actions on peripheral receptors. As stated in Methods, all nicotine injections were given after a preparatory injection of gallamine, a synthetic curare substitute. Gallamine is known to block nicotine’s effect on muscle spindles (ll), and because of its pharmacologic similarity to curare, should block nicotine’s action at cutaneous receptors (7, 19, 23). This pharmacologic blockade of peripheral receptors was confirmed by our second control, which consisted of recording from the distal stump of a dorsal rootlet in one of our preparations. No increase in the afferent barrage was detected in this animal following maximum injections of nicotine or eserine. The third and most important control for cholinergic effects on peripheral receptors was the use of three completely deafferented preparations in which all dorsal roots on both sides of the cord were cut berow segment L-l, at which point a low spinal section was made. Monosynaptic reflex depression, together with increased excitability of presynaptic terminals, were observed following injection of nicotine in two of these deafferented preparations (Fig. 2A and C) . In the third deafferented cat, intracellular techniques were used to observe a transient reduction in amplitude of the monosynaptic EPSP following injection of 10 pg nicotine. The amplitude was reduced by as much as 38% with return to within 9% of control values in 3 min. Although depression of EPSP is not a sufficient condition for the demonstration of presynaptic inhibition, it is a necessary condition, and as such it is significant that this depression was present in a deafferented preparation following a dose of nicotine within the 5 to 33 rg range already shown to produce increases in presynaptic terminal excitability. An injection of atropine (2 “g/kg) was routinely given to protect the cat from cardiac arrest during injections of nicotine or eserine. There was no effect on presynaptic terminal excitability or on the monosynaptic reflex as a result of atropine injection. The central effects of nicotine could be blocked, however, by mecamylamine (2 mg/kg iv), a secondary amine with established CNS nicotinic-blocking properties at this dose (20). Fig. lE-G shows the blocking action of this drug for the same dose of nicotine
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FIG. 2. Effect of a close arterial injection of 25 pg nicotine on presynaptic and motorpool excitability. Location of the pipette combined with arrangement of the recording electrodes enabled simultaneous testing of the excitability of presynaptic terminals and pool of motorneurons. A, Wall’s test of excitability of dorsal root L-7 terminals in the ventral horn. B, motorpool excitability tested by the same stimuli testing presynaptic terminals. Response to the test stimulus was recorded in ventral root L-7 with 0.25 msec latency. C, left to right, samples of monosynaptic reflex recorded in ventral root L-7 before, 6 set after, and 25 min after nicotine injection. Note absence of monosynaptic reflex in the record taken 6 set after injection. D, diagram of arrangement of stimulating and recording electrodes. This cat was a completely deafferented preparation. Black dots over records A and B indicate the duration of nicotine injection.
illcstrated in Fig. lA-D. Mecamylamine injection pc~ SC did not produce changes in either presynaptic terminal excitability or the monosynaptic reflex. Intravenous injections of eserine (2 mg/kg) also increased the excitability of presynaptic terminals while depressing the monosynaptic reflex. Data recorded during one such injection are presented in Fig. 3. Four injections of eserine in four different cats produced a maximum increase in excitability of presynaptic terminals that ranged from 2670 to 71% above control values. This maximum was observed between 27 and 51 set after start of the injection. The half-decay time of the excitability change ranged from 23 to 92 sec. This eserine effect on primary afferent terminals was observed in a completely deafferented preparation. As in the case of nicotine, the drug-induced depression of the monosynaptic reflex lasted more than twice as long as the excitability increase of presynaptic terminals. Mecamylamine also blocked the effect of eserine on the monosynaptic reflex, whereas atropine and gallamine did not. Conduction velocities were measured for the gastrocnemius medialis afferent fibers of which the central terminals showed the eserine-induced excitability increase documented in Fig. 3A. The conduction velocities
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FIG. 3. Effects of eserine sulfate (2 mg/kg iv). A, Wall’s test of excitability of central terminals of gastrocnemius medialis afferent fibers. B, monosynaptic reflex recorded in contralateral ventral root L-7 after stimulus to dorsal root L-7. C, blood pressure. Base line equals 0 mmHg. Black bars over records A-C indicate duration of eserine injection. D, single response to Wall’s test of central gastrocnemius medialis afferent terminals taken from record A and recorded at a fast sweep. E, single monosynaptic reflex response taken from record B and recorded at same sweep speed as D. F, diagram of arrangement of stimulating and recording electrodes. The gastrocnemius medialis nerve was deefferented on the left side by acute section of ventral roots of L-6, L-7, and S-l.
observed (110 to 128 m/set) indicated that the presynaptic terminals affected belonged to the afferent fibers of the monosynaptic reflex arc (14). Depression of the monosynaptic reflex by nicotine at the doses used in our experiments has been attributed to Renshaw-cell inhibition of the motoneuron (6, 8). Two independent methods in three separate experiments indicated that contrary to the predictions of this hypothesis, the excitability of the motorpoo1 increased after injections of nicotine or eserine. In the deafferented cord experiment illustrated in Fig. 2, the current pulse delivered through the pipette not only stimulated primary afferent terminals in the ventral horn but also produced a discharge in the ventral roots. The latter had a latency of 0.25 msec which indicated that it was the result of a direct stimulation of motoneurons through the pipette as opposed to transynaptic stimulation ( 16). Figure 2B documents the increase in this ventral root response that followed the administration of nicotine. This fact lends additional support to the belief that we were observing a direct test of motoneuron excitability because the monosynaptic reflex was completely abolished for a considerable period by this same nicotine injection (Fig. ZC). The increased excitability of the motorpool followed a briefer time
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FIG. 4. Effect of close arterial injection of 16 pg nicotine on the antidromic field potential in the gastrocnemius medialis motorpool. ‘4, each bar represents the average of five antidromic field-potential amplitudes. Shaded bar indicates time of nicotine injection. B, left to right, samples of antidromic field potential before, during, and after the nicotine effect. Arrows in r2 indicate the bars from which the sample records were taken. C, diagram of stimulating and recording arrangement. Left dorsal roots L-6 through S-2 were cut.
course than the increased excitability of presynaptic terminals in Fig. 2kt, but at no time was there reduction of the excitability of the motorpool below control values. In two other experiments, a focal pipette recorded the field potential which resulted from the antidromic invasion of the gastrocnemius medialis motor-pool. The increase in amplitude of this field potential after the close arterial injection of 16 pg nicotine is depicted in Fig. 4A. This injection was repeated in the same experiment with the same result, and an eserine injection (2 mg/kg iv) in a separate experiment produced a similar increase in size of the antidromic field. The growth of the antidromic field potential could mean that a larger number of motoneurons was invaded or that the somaclendritic tree of each individual motoneuron was more completely invaded. In either case, the implication is that the excitability of the motorpool was increased, not diminished, by these drugs ( 17). Injections of either nicotine or eserine are known to stimulate muscle spindles (1, 12, 21)) and nicotine has also been reported to excite cutaneous receptors (4, 7, 23). However, impulses in cutaneous and muscle afferent:;
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can produce depolarization in the central terminals of Group Ia afferents, resulting in presynaptic inhibition of the monosynaptic reflex (2, 9, 13). One might expect, therefore, that an injection of nicotine or eserine would increase the afferent barrage to the cord and depress the monosynaptic reflex through presynaptic inhibition. The operation of just such a presynaptic mechanism has already been demonstrated for large doses of nicotine (> 80 pg/kg iv) (11). However, smaller doses reportedly inhibit the monosynaptic reflex by a central mechanism that is blocked by mecamylarnine, presumably via activation of Renshaw interneurons ( 11). These interneurons are known to increase their spontaneous activity after injections of nicotine or eserine (8) and have also been shown to have inhibitory synapses on alpha motoneurons (10). Our observations indicate that presynaptic inhibition always contributes to the nicotine-induced monosynaptic reflex depression in the dose range investigated (5 to 33 pg). We used essentially the same close arterial injection technique used by Curtis et al. in their classical work on the effect of drugs on the monosynaptic reflex (6). All our nicotine injections were within or below the 10 pg-to-50 pg range used by these same investigators. Our only injections outside this range were two 5-pg injections of nicotine, one of which produced a dramatic 83% increase in the excitability of presynaptic terminals with a half-decay time of 29 sec. This 5-pg dose compares favorably with the 0.2 pg-to-2 pg dose of nicotine reported by Eccles et al. to be necessary for the observation of an increase in Renshaw-cell spontaneous activity (8). We have also determined that the monosynaptic reflex depression produced by eserine (2 mg/kg iv) has a presynaptic inhibitory component. Whereas the reflex depression produced by either nicotine or eserine outlasts considerably the presynaptic excitability increase measured by Wall’s test, a presynaptic inhibitory mechanism is certainly in operation during the initial phase of the reflex depression because the excitability of the motorpool is increased over control levels (Figs, 2 and 4). The peripheral actions of nicotine and eserine mentioned above would be the least controversial explanation for our observations, but there are several reasons for believing that the presynaptic inhibition observed is the result of a central action of these drugs. First, all our preparations were paralyzed with the synthetic curare substitute, gallamine, just prior to each injection of nicotine or eserine to block peripheral cholinergic effects. We confirmed the effectiveness of this treatment when we were unable to record any increase in background discharge from the distal stump of a dorsal rootlet following maximal injections of nicotine or eserine. Most significantly, while gallamine did block the peripheral effects of these cholinergic drugs, it was not expected to have central effects in the doses used and in fact did not prevent the action of nicotine or eserine on the
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monosynaptic reflex or central Ia afferent terminals. These findings are important in the light of the recent demonstration that a large number of unmyelinated axons in cat ventral roots have their cell bodies in the dorsal root ganglia (5) and carry information from visceral receptors (personal communication). However, there are no reasons a priori to expect that these receptors are pharmacologically different from others studied so far (7, 19, 23) and it is thus very likely that gallamine had also blocked nicotinic effects in these visceral receptors. The second reason is that the nicotineinduced primary afferent depolarization was blocked by mecamylamine (Fig. 1)) a drug that does block CNS nicotinic receptors (20) but does not influence the spindle acceleratory effect of large doses of nicotine ( 11) . Finally, the strongest argument for the central action of these drugs rests on the observation that nicotine and eserine both increased the excitability of presynaptic terminals during reflex depression in classically deafferented low-spinal preparations (Fig. 2). It is therefore concluded that the initial monosynaptic reflex depression following injections of nicotine or eserine has a significant presynaptic inhibitory component as a consequence of a drug-induced depolarization of the central terminals of Group Ia afferent fibers. In addition, selective drug antagonism and experiments in classical deafferented preparations (spinal cord sections at L-l and complete dorsal rhizotomy of lumbar and sacral segments) indicate that the primary afferent depolarization observed is the result of a central action of these drugs. To state that this nicotinic effect occurs centrally does not necessarily presuppose knowledge of its mechanism. The two possible alternatives: (a) direct effect of the drug on the tested terminals and (b) effect mediated via some interneuronal pool, remain still to be investigated. REFERENCES 1. ALBUQUERQUE, E. X., and C. M. SMITH. 1964. Specificity of excitation of muscle spindle afferents by cholinergic substances. J. Phamzacol. E.rb. Ther. 146: 344353. 2. ANDBN, N. E., M. G. M. JUKES, A. LUNDBERG, and L. VYKLICKG. 1966. The effect of DOPA on the spinal cord-3. Depolarization evoked on the central terminals of ipsilateral Ia afferents by volleys in the flexor reflex afferents. Acta Physiol. Stand. 68: 322-336. 3. BARAKAN, T. H., C. B. B. DOWNMAN, and J. C. ECCLES. 1949. Electrical potentials generated by antidromic volleys in quadriceps and hamstring motoneurons. J. Newrophysiol. 12 : 393-424. 4. BROWN, G. L., and J. A. B. GRAY. 1948. Some effects of nicotine-like substances and their relation to sensory nerve endings. J. Physiol (London) 107: 306-317. 5. COGGESHALL, R. E., J. D. COULTER, and W. D. WILLIS. 1974. Unmyelinated axons in the ventral roots of the cat lymbosacral enlargement. J. Camp. Neztrol. 153: 39-58.
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D. R., J. C. ECCLES, and R. M. Ecn~. 1957. Pharmacological studies on spinal reflexes. 1. Physiol. (London) 136 : 420-434. DOUGLAS, W. W., and J. A. B. GRAY. 1953. The excitant action of acetylcholine and other substances on cutaneous sensory pathways and its prevention by hexamethonium and d-turbocurarine. J. Physiol. (London) 119: 118-128. ECCLES, J. C., R. M. ECCLES, and P. FATT. 1956. Pharmacological investigation of a central synapse operated by acetylcholine. J. PhysioZ. (London) 131: 154-169. ECCLES, J. C., R. M. EC~LES, and F. MAGNI. 1961. Central inhibitory action attributable to presynaptic depolarization produced by muscle afferent volleys. J. Physiol. (London) 159: 147-166. ECCLES, J. C., P. FATT, and K. KOKETSU. 1954. Cholinergic and inhibitory synapses in a pathway from motor-axon collaterals to motoneurons. J. Physiol. (London) 126 : 524-562. GINZEL, K. H., E. ELDRED, and Y. SASAKI. 1969. Comparative study of the actions of nicotine and succinylcholine on the monosynaptic reflex and spindle afferent activity. Znt. J. Neuropharmacol. 8: 515-533. HUNT, C. C. 1952. Drug effects on mammalian muscle spindles. Fed. Proc. 11: 75. JANKOWSKA, E., S. LUND, and A. LUNDBERG. 1966. The effect of DOPA on the spinal cord-4. Depolarization evoked in the central terminals of contralateral Ia afferent terminals by volleys in the flexor reflex afferents. Acta Physiol. Stand. 68: 337-341. LLOYD, D. P. C. 1943. Neuron patterns controlling the transmission of ipsilateral hind limb reflexes in the cat. J. Neurophysiol. 6: 293-315. M~SZAROS, J. 1971. The effect of some cholinomimetic drugs on presynaptic inhibition in the lateral geniculate nucleus. Neuropharmacology 10 : 67-76. RENSHAW, B. 1940. Activity in the simplest spinal reflex pathways. 1. Neuro-
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B. 1942. Effects of presynaptic volleys on spread of impulses over the soma of the motorneuron. J. Neurophysiol 5 : 235-243. SCHWEITZER, A., and S. WRIGHT. 1938. Action of nicotine on the spinal cord. J. Physiol. (Londort) 94: 136-147. SKOUBY, A. P. 1953. The influence of acetylcholine, curarine and related substances on the threshold for chemical pain stimuli. Acta Physiol. Stand. 29: 340-352. UEKI, S., K. KOKETSU, and E. F. DOMINO. 1961. Effects of mecamylamine on the Golgi recurrent collateral-Renshaw synapse in the spinal cord. Exp. Neural. 3: 141-148. VERHEY, B. A., and P. E. VOORHOEVE. 1963. Activation of group Ia and group 11 muscle spindle afferents by succinylcholine and other cholinergic drugs. Acta RENSHAW,
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Excitability changes in afferent fiber terminations and their relation to slow potentials. J. Physiol. (London) 142: 1-21. 23. WATSON, P. J. 1970. Drug receptor sites in the rabbit saphenous nerve. ~r,jt. J. Phamacol. 40 : 102-112.