Cholinergic properties of spino-bulbo-spinal reflex inhibition

Neuropharmaco/ogy, 1970,9, 185-190

CHOLINERGIC

Pergamon Press.

Printed in Gt. Britain.

PROPERTIES OF SPINO-BULBO-SPINAL REFLEX INHIBITION*

C. D. BARNES? Department of Anatomy and Physiology, Indiana University, Bloomington, Indiana 47401, U.S.A. (Accepted 22 April 1969)

Summary-Experiments were performed to determine whether the inhibitory phase of the spinobulbo-spinal (SBS) reflex had a cholinergic component and, if so, whether it was at the spinal or at the buibar level. Cats were decerebrated under ether anesthesia and then two laminectomies were performed. One laminectomy was done over the lumbar cord exposing L, and L, for stimulating and recording; the other was done at T,, for cold-blocking the cord. By stimulating the dorsal root of L, and recording from the ventral root, the L, monosynaptic reflex (MSR) obtained was used to test the effects of eserine and atropine, both directly and on the SBS inhibition produced by stimulating the dorsal root of Lg. Eserine (0.1 mg/kg, i.v.) was found to have an immediate effect of increasing SBS inhibition. It also produced, at a longer latency, a deep inhibitory phase of both the conditioned and unconditioned MSR. Atropine (4 mg/kg, i.v.), while having no effect when given alone, not only blocked the effects of a preceding dose of eserine, but usually abolished the SBS reflex altogether. The major effects of both eserine and atropine were found to be above the level of T,,.

SEVERAL recent investigations have described properties of a spino-bulbo-spinal (SBS) reflex system (SHIMAMURAand AKERT, 1965 ; SHIMAMURAand LIVINGSTON, 1963 ; SHIMAMURA et al., 1967), a system which depends upon a relay through the bulbar portion of the brain stem and recurrent projection to spinal motoneurons. Stimulation of a spinal dorsal root in decerebrate cats results in the well-known segmental mono- and poly-synaptic reflex responses in the spinal ventral root at the same segment. After a period of little or no ventral root activity, these responses are followed by an additional delayed reflex response (SHIMAMURA and LIVINGSTON, 1963). More recently it has been reported that there is an inhibitory influence associated with the SBS reflex, as well as the facilitatory response (SHIMAMURA et al., 1967). Both the spinal cord and the brain stem reticular formation have been studied by various means in the hope of discovering cholinoceptive cells. CURTIS and ECCLES (1958 a, b) using microelectrophoretic injection, confirmed the participation of acetylcholine (ACh) in the recurrent excitation of Renshaw cells. Whether or not other interneurons in the spinal cord are cholinoceptive is still at issue (CURTIS et al., 1966; WEIGHT and SALMOIRAGHI,1966). The site of action of cholinergic drugs in producing desynchronization of the EEG, according to RINALDI and HIMWICH (1955), STEINER and HIMWICH (1962) and IL’YUCHENOK and MASHKOVSKII(1961), is at the mesodiencephalic level. Adding support to the theory that this area contains cholinoceptive cells is the work of SALMOIRAGHIand STEINER (1963), who, *This work was supported in part by a grant NB05949 from the United States Public Health Service. iRecipient of Career Development Award K3-NB 34986 from the National Institute of Neurological Diseases and Blindness. 185

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with microelectrophoretic injection, reported cells in the reticular formation which increased their discharge rate with ACh. The present study was designed to take advantage of the anatomically distinct systems involved in segmental reflexes and the SBS reflex. Both reflexes, while being produced from a single stimulus delivered to a dorsal root, can result in inhibition of the monosynaptic reflex at L,. The SBS reflex was investigated to determine if its inhibitory phase has a cholinergic component, and, if so, whether it is at the spinal or bulbar level.

METHODS

Experiments were performed on 36 cats initially anesthetized with ether. After a tracheotomy, the animal was decerebrated at the intercollicular level and ether was discontinued. The left carotid artery was cannulated for recording blood pressure; the right femoral vein was cannulated for injection of drugs. Body temperature was monitored with a rectal thermometer and maintained by means of a heating pad. The animals were immobilized by mounting them in a rigid framework which included a head holder and clamps on the spinous processes of T,, L, and L,. A laminectomy was performed over the lower lumbar segments of the spinal cord. Here the dura was opened and everted, and the cavity thus formed was filled with warm mineral oil. The right dorsal and ventral roots (DR and VR) or b, L, and S, were cut just where they exited through the dura. DRL,, DRL, and VRL, were each positioned on pairs of platinum electrodes. The DRL, was stimulated with Textronix 161-162 generators, through a stimulus isolation transformer. A similar stimulation system driven by a ramp generator as described by SMITHand BARNES(1968) was used for DRL,. Stimulating electrodes were never placed closer than 8 mm from the root entry zone, nor closer than 4 mm from any part of the cord. The stimulus was a single 0*05-0*1 msec rectangular pulse, usually less than 0.5 V (never more than 1.0 V), but enough to be supramaximal for group IA fibers. Recordings were taken from VRL, and fed through a Tektronix 122 preamplifier into an oscilloscope. Photographs were taken from the oscilloscope with a Polaroid camera. A second laminectomy was performed over the lower thoracic cord and the animal was prepared for reversible cold block as previously described by BARNESet al. (1962). BEAMAN and DAVIS(1931) have shown this technique for blocking the spinal cord to result in a condition corresponding closely to that developed after surgical transection. The cold block has no initial activity due to injury potentials and has the added advantage of reversibility. All animals were immobilized by repeated doses of gallamine triethiodide (flaxadil), as needed, and were maintained on artificial respiration. Drugs used were atropine, at a dose of 4 mg/kg per injection, and eserine (physostigmine), at a dose of 0.1 mg/kg per injection. These were given intravenously.

RESULTS

The first series of experiments were done to determine what differential effects the drugs had on the MSR alone and on the SBS-inhibited MSR. Before beginning, a conditioningtesting interval sequence of L, MSR amplitudes was run (Fig. 1). When the L, MSR was conditioned at different intervals by DRL,, the following sequence could be seen: a brief facilitation, segmental inhibition, SBS facilitation, SBS inhibition. The SBS inhibition may recede after 100 msec or more, returning the MSR to control level, or it may be followed by a

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FIG. 1. Interval-amplitude distribution of the L, MSR conditioned by DRL, with the cord intact. The conditioning stimulus to DRLG was delivered at 0 and the test started 20 msec before and moved forward in time one and a half msec each two seconds. The resulting time exposure consists of the L, MSR amplitudes for conditioning-testing intervals of -20 to 160 msec with 119 points between.

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FIG. 2. Conditioned and unconditioned MSR. The upper half are the direct records, the lower half the two outlines superimposed. The uppermost trace (dotted line below) is the L, MSR. Each(E) indicates theadministration ofeserine0.1 mg/kg i.v. ;(A) indicates atropine4mg/kgi.v.

Neuro. f.p. 186.

COLD

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FIG. 4. Interval-amplitude distributions of the L, MSR of an animal with both the cord intact (WARM) and cold-blocked at T,, (COLD) under: (A) control conditions before any drugs; (B) first administration of eserine 0.1 mg/kg ix. ; (C) atropine 4 mg/kg i.v.; (D) second eserine O-1 mg/kg.

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FIG. 5. Interval-amplitude distribution of the L, MSR during the deep inhibitory phase following eserine. Reading from the top: (WARM) 10 min after eserine, before cold-block; (COLD) 15 min after eserine, partial cold-block at T,,; (WARM) 20 min after eserine, coldblock at Ti,, reversed; (CORD CUT) 30 min after eserine, 5 min after the cord was transected at T,,.

Cholineryic properties of spin+bulbo-spinal

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second short period of SBS facilitation. For each animal a conditioning-testing interval sequence was run to determine what interval would give the maximum SBS inhibition. Figure 2 represents data taken from an animal at 2 set intervals over a 180 min period while eserine, a cholinesterase inhibitor, and atropine, an acetylcholine inhibitor, were being administered. The L, MSR was tested once every 2 set, with a conditioning stimulus being delivered to DRL, every other time (every 4 set). This was done at an interval preceding DRL, stimulation to produce maximum SBS inhibition. The signal from VRL, was fed into both channels of a dual-trace oscilloscope. The oscilloscope was operating in the alternating mode so that one trace displayed the MSR amplitude alone every 4 set and the other trace displayed the SBS-inhibited MSR every 4 set, the two being staggered 2 set apart. Thus, by moving the trace slowly through the x axis, the simultaneous-continuous record of Fig. 2 was achieved. This technique allowed for the unconditioned MSR to serve as a continuous control with which to compare the SBS-conditioned MSR. The first injection of eserine (0.1 mg/kg) produced an immediate increase in the SBS inhibition but no change in the MSR. Six minutes after the eserine there was a transient reversal of the SBS effect from inhibition, but no change in the MSR. There was also a transient reversal of the SBS effect from inhibition to facilitation, but this did not show up in all animals and lasted for less than a minute when it did. About 7 min after the eserine administration there was a profound increase in SBS inhibition. A minute after this increase in SBS inhibition, there was a similar drop in the amplitude of the MSR to 35 ‘Aof its original value. The MSR gradually increased to 70 % over the next 11 min and then suddenly returned to 100% of its control value. At the same time, the SBS inhibition showed a sudden transient reversal to facilitation and then returned to an enhanced level. A second dose of eserine (0.1 mg/kg), administered 1 hr after the first, resulted in a reoccurence of the first sequence. Before recovery from the deep inhibition, however, a dose of atropine (4 mg/kg) was given. Atropine caused the unconditioned MSR to immediately return to about 95 % of its pre-eserine value and the SBS inhibition was abolished. A third dose of eserine (0.1 mg/kg) would be seen to re-establish the SBS-inhibited MSR to about 60 % of the control MSR amplitude. A second dose of atropine (4 mg/kg) once again would suppress the SBS inhibition so that the inhibited MSR was more than 90% of the control MSR. At the termination of each experiment the spinal cord was cut at T,,. Then the two MSR amplitudes became identical, showing that the origin of the SBS inhibition is above that level. Figure 3 is the SBS-inhibited MSR, following the same general procedure as in the animal of Fig. 2, but representing the average of five animals. The relationships are generally as those of Fig. 2 except the lack of a deep inhibitory phase in the eserine curve. This is a result of averaging. All animals displayed the deep inhibition but at differing latenties after eserine; at no single point in time were all the cats in this phase. To examine the effects of eserine and atropine along the time course of the SBS reflex, a second series of animals was run, utilizing the reversible cold-block technique. Figure 4 illustrates the results of a typical animal in this series. The first administration of eserine (0.1 mg/kg) resulted in an increase in the SBS inhibitory phase and a decrease in the facilitatory phase (4B-WARM). When the cord was cold-blocked, the segmental inhibition appeared slightly decreased in length of action, but there was a marked enhancement of the long facilitation (4B-COLD). This latter phenomenon, however, was more likely the result of incomplete cold block since all animals did not exhibit it. It was also seen that the control MSR amplitude (that before interval 0) was not as depressed now as when the cord was

C. D. BARNES

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FIG. 3. Conditioned MSR expressed as a percentage of the unconditioned MSR, average of five animals. Time 0 represents the beginning of an experiment for CONTROL, the injection of drug for ATROPINE and ESERINE, and 15 min after actual transection for CORD CUT.

intact. The administration of atropine (4 mg/kg) resulted in a dramatic decrease in the SBS inhibition, as well as the segmental inhibition, and an increase in the control MSR amplitude with the cord intact (4GWARM). Some inhibition did exist since now the conditioned L, MSR never did regain its full preconditioned value. When the cord was blocked, however, the segmental inhibition reappeared relatively unchanged (4CXOLD). A second administration of eserine (0.1 mg/kg) brought a return of the SBS inhibition but this was nowhere near the original value existing before atropine (4D-WARM). The distribution resulting from cold-blocking the cord was unchanged from that of atropine (4D-COLD). To verify the completeness of the cold-block, the cord was cut at T,,. The distribution 10 min after the transection was identical to the last cold-block record. A final series of experiments was run to determine whether the origin of the deep inhibition phase following eserine was above or below T,, or whether it was just general depression. With these animals, the alternator technique was used as in Fig. 2 to monitor both conditioned and unconditioned L, MSR. Eserine was given at the usual dose of 0.1 mg/kg. When the deep inhibition phase started, an interval-amplitude distribution as in Fig. 1 was taken. The cord was then rewarmed and a third distribution taken. To determine the completeness of the cold-block, a final interval amplitude distribution was taken a few minutes after the cord was cut at T,,. The results of such a sequence are shown in Fig. 5. In the animal used in Fig. 5, only a partial cold-block was performed by using cold brine of a higher temperature than usual. It was found that by doing this, the inhibitory phase of the SBS reflex could be greatly reduced, revealing a much longer SBS facilitation. As can be seen when the SBS inhibition path was blocked (5COLD), the deep inhibition phase of eserine was removed. That no spontaneous recovery had occurred was evidenced by the re-occurence of the deep inhibition when the cold-block was removed (5-WARM). When the cold-block record (5-COLD) was compared to that after the cord had been transected at Tlo, it could be seen that only a partial block had been performed. DISCUSSION

The results of the present study indicate that there are cholinoceptive cells involved with the SBS reflex. This is shown by the fact that eserine, an anticholinesterase, increases the

Cholinergic properties of spino-bulbo-spinal

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inhibitory phase of the SBS reflex without a change of comparable amount in the MSR itself or any component of segmental reflexes. In those cases where eserine does appear to affect the MSR, such as the deep inhibition phase, it is shown to be the result of an effect above T1,,, since it is gone when the cord is cold-blocked. The deep inhibition phase of eserine seems to be due to the greatly increased discharge rate of some descending inhibitory system, possible the same one responsible for the SBS inhibition. The only evidence for their being the same system is that a partial cold-block which stops SBS inhibition also stops eserine deep inhibition. The evidence presented by MATSUZAKI(1967, 1968) on eserine-produced paradoxical sleep is strongly suggestive that these deep inhibitory bouts in the spinal cord have the same origin. That eserine does have a segmental effect is not denied, for it is seen to decrease the MSR amplitude and to shorten slightly the duration of segmental inhibition, The segmental effects, though consistent, are usually not dramatic, whereas the SBS effects are quite marked. The transient reversal of the SBS influences on the MSR from inhibition to facilitation, as seen in Fig. 2, is interpreted as being a result of fluctuations in the relative strength of the two competing systems in this portion of the curve. The large reversal signaling the beginning and end of the deep inhibition phase of eserine could be explained if the site of eserine action was distinct from the SBS inhibition and facilitation cells, but still acted on them. If this were the case, the path to the facilitatory cells need only be shorter or more susceptible than that to the inhibitory cells to produce the observed effects. A likely possibility for such a site wouId be the cerebellum, as it is known to have great influence on descending systems. In addition, evidence for ACh-sensitive cells in the cerebellum has been presented (MCCANCE and PHILLIS, 1964). CRAWFORDet al. (I966), however, present a strong argument against ACh acting as the transmitter of any afferents to Purkinje cells. Though ACh was not tested, on the grounds of microelectrophoretic injection experiments, OBATAet al. (1967) present a case of gamma-aminobutyric acid (GABA) being a natural transmitter of the Purkinje cells themsefves. The application of the anticholinergic drug, atropine, following eserine, not only abolished the increase in SBS inhibition induced by the eserine, but usually, in the dose used in this study, resulted in the complete abolition of the SBS reflex. Atropine given alone, however, not preceded by eserine, had no marked effect. It is surprising that atropine does not antagonize the normally liberated ACh without the presence of eserine. Similar findings were also reported by FRAZIERand BOYARSKY (1967). In view of the consistent and proportionately larger effects of eserine on SBS inhibition than on segmental inhibition, and the ability of atropine to reverse these effects, it is felt that there are cholinoceptive cells involved in the inhibitory phase of the SBS reflex. Since the drugs effects are abolished when the cord is cold-blocked or transected, these cells must be at the bulbar end of the reflex rather than in the lumbar cord itself.

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