- Email: [email protected]
Influence of serotonin antagonists on bulbospinal systems
Brain Research, 61 (1973) 331-341
331
© Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands
INFLUENCE OF SEROTONIN ANTAGONISTS ON BULBOSPINAL SYSTEMS
HERBERT K. P R O U D F I T AND E D M U N D G. A N D E R S O N
Department of Pharmacology, University of Illinois, College of Medicine, P.O. Box 6998, Chicago, Ill. 60680 (U.S.A.) (Accepted April 2nd, 1973)
SUMMARY
We attempted to elucidate the function of the serotonin-containing bulbospinal neurons by directly stimulating the raphe nucleus of unanesthetized decerebrate cats. Such stimuli evoked both a dorsal and a ventral root potential, and produced a complex time-dependent facilitation and inhibition of segmentally evoked monosynaptic reflexes (MSR). The entire time course of the curve representing the brain stem-evoked facilitation and inhibition of the MSR was displaced in a facilitatory direction following administration of the serotonin (5-HT) antagonists cinanserin or methysergide. The apparent block of descending inhibition probably resulted from increased descending facilitation. These results occurred without an increase in motoneuronal excitability, since the segmental MSR remained unchanged. The 5-HT antagonists increased the brain stem-evoked dorsal root potential (DRP), but did not alter the ventral root potential. In contrast, these agents depressed the segmentally evoked DRP. Measurements of the DRP length constants in afferent fibers revealed that brain stem stimulation evoked DRPs in fibers of smaller diameter than dorsal root stimulation. Apparently none of the potentials evoked by raphe stimulation were mediated by serotonergic fibers since these potentials were not reduced by 5-HT antagonists. It is suggested that the serotonergic system is tonically active in the decerebrate cat, and that the increased facilitatory action on the conditioned MSR and brain stem DRP following a 5-HT antagonist results from release of tonic serotonergic inhibition of descending facilitatory fibers.
I NTRODUCTION
Histochemical studies of Dahlstr6m and Fuxel0,11 have shown that serotonincontaining cell bodies localized in the caudal raphe nuclei have axons which descend
332
H . K . P R O U D F I T A N D E. G . A N D E R S O N
in the spinal cord and terminate in the dorsal, lateral and intermediate horns. The tunctional role of this serotonergic system remains uncertain. Administration of 5hydroxytryptophan (5-HTP) to elevate cord levels of serotonin (5-hydroxytryptamine, 5-HT) has provided evidence that 5-HT exerts a facilitatory action on motoneurons4, but other studies have suggested the 5-HT system may inhibit flexor reflexes2,12. The function of these descending serotonergic neurons could be clarified by stimulating their somata, located in the brain stem raphe nuclei, and recording their influences on segmental systems. In a previous study from this laboratory 8, conditioning volleys delivered to the nucleus raphe magnus as well as the lateral reticular formation resulted in an early facilitation followed by inhibition of the segmentally evoked monosynaptic reflex (MSR). Several 5-HT antagonists blocked the inhibition of segmental MSRs induced by brain stem stimulation. Since the conduction velocity of the inhibitory system was too fast for the small diameter serotonergic nerves descending from the raphe nuclei, it was suggested that the inhibition might be mediated by a serotonergic interneuron located in the spinal cord. However, evidence for such an interneuron is not good 9 (see review by Andersona). The present study was initiated to re-investigate the function of the descending 5-HT system, by examining the entire time course of the effects of brain stem stimulation on the segmentally evoked MSR and, in addition, to assess the possibility of descending serotonergic actions on prima ry afferent terminals. Some of these results have been presented in a preliminary report ~4. METHODS
Cats were made decerebrate, under temporary ether anesthesia, by removal of the forebrain rostral to the inferior colliculus. A laminectomy was performed and the seventh lumbar (L7) spinal roots were dissected free and mounted for stimulation and/or recording in an oil pool maintained at 36-37 °C. Platinum hook electrodes were routinely used for stimulating and recording. The animals were artificially respired and immobilized with gallamine triethiodide. The caudal brain stem raphe nuclei were stimulated using stereotaxically placed concentric bipolar stainless steel electrodes. Thirty msec trains of 1-3 V (0.08-0.25 mA), 0.5 msec square wave pulses were applied to the electrodes at 300 Hz. Dorsal and ventral root potentials evoked by these stimuli were recorded at the L7 segment. The effects of brain stem stimulation on motoneuron excitability were assessed by measuring the MSR evoked by a test stimulus (0.01-0.1 V; 0.05 msec) to the dorsal root at various time intervals following a conditioning volley applied to the brain stem. Control MSRs were elicited in the absence of brain stem volleys. Dorsal root potentials (DRP) and reflexes (DRR) were recorded from the most caudal L7 dorsal rootlet. Potentials were quantified by summing 10 evoked potentials using a CAT 1000 computer of average transients and measuring either peak amplitude (MSR and DRR) or area (DRP). Length constants were determined for the fibers in which the dorsal root potentials were generated. Two platinum hook electrodes held in a micromanipulator were used to record the potentials from a dorsal rootlet. One electrode was placed near the
333
SEROTONIN AND BULBOSPINAL SYSTEMS
dorsal root entry zone and the other was placed on a dorsal rootlet about 10 mm from the first. The electrodes were moved in 1 mm increments away from their original position; the electrode separation remained constant. The length constant is defined as the distance at which the potential declined to 1/e of its original value. All brain stem electrode placements were verified histologically. The Prussian Blue technique of Adrian and Moruzzi 1 was used to stain for iron deposits left by a brief DC current application to the electrode. The serotonin antagonists used were cinanserin HCI (4 mg/kg) and methysergide bimaleate (1 mg/kg). All drugs were dissolved in normal saline and administered intravenously through an indwelling catheter placed in the anticubital vein. In some experiments intra-arterial injections to the spinal cord were made via a catheter inserted into the caudal mesenteric artery and guided up the abdominal aorta to a point about 1 cm caudal to the renal arteries. The testicular (ovarian), deep circumflex iliac, external iliac, and common iliac arteries were ligated leaving only the spinal arteries intact.
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Fig. l. Position of stimulating and recording electrodes. Trains of stimuli were applied to the brain stem in the area of the nucleus raphe magnus using a bipolar stimulating electrode. Brain stem stimulation evoked a ventral root potential (VRP) and dorsal root potential (DRP-BS) (upper left and right panels, respectively). Stimulation of the L7 dorsal root evoked a monosynaptic reflex and a dorsal root potential (DRP-S) (lower left and right panels, respectively). The potentials in the left panels were recorded from the L7 ventral root while those on the right were recorded from the most caudal L7 dorsal rootlet. Abbreviations: R, recording electrode; Rd, nucleus raphe dorsalis; Rm, nucleus raphe magnus; Ro, nucleus raphe obscurus; Rp, nucleus raphe pontis; Rpa, nucleus raphe pallidus; S, stimulating electrodes; T, trapezoid body.
334
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Fig. 2. A representative experiment illustrating the effect of cinanserin (4 mg/kg) on the MSR evoked at various times after a brain stem conditioning volley. The MSR evoked by stimulation of the L7 dorsal root is plotted as a percent of the unconditioned MSR. Intervals between brain stem conditioning stimuli and the test stimulus are plotted in milliseconds. The solid line represents the control brain stem conditioning curve while the dotted line is that after cinanserin administration.
RESULTS
Attempts were made to activate the descending serotonin-containing cells in the nucleus raphe magnus of the caudal brain stem using stereotaxically placed bipolar stimulating electrodes. Evoked potentials elicited by brain stem stimulation were recorded from both the dorsal and ventral roots. The brain stem dorsal root potential (DRP-BS) was a negative going wave with an onset latency of about 30 msec and a duration of 75-100 msec (Fig. 1). No D R R was observed following brain stem stimulation. The ventral root potential (VRP) evoked from the brain stem began 5-10 msec after the first pulse of the stimulus volley and lasted 40-50 msec (Fig. 1). To further study the influence of brain stem stimulation on segmental systems, a test MSR was evoked at various intervals following the brain stem conditioning stimuli. The test MSR was either facilitated or inhibited depending on the interval between the conditioning brain stem volley and the test stimulus to the L7 dorsal root (Fig. 2, solid line). The MSR was facilitated at intervals between 30 and 35 msec and between 70 and 120 msec. Inhibition of the MSR was usually observed within the 40-65 msec range. Since the raphe is a thin laminar structure, cell bodies and axons in areas surrounding the raphe magnus were probably activated by current spread from the stimulating electrode. Therefore, we tried to identify those influences of the descending system which were mediated by serotonergic fibers by determining their susceptibility to 5-HT antagonists. Cinanserin HCI (4 mg/kg) and methysergide bimaleate (1 mg/kg) were used to antagonize the effects of 5-HT. The injection of cinanserin into 6 cats dramatically altered the influence of brain stem stimulation on the MSR. Within 2-3 min, the effects of the conditioning volley on the MSR were shifted in a facilitatory direction throughout the entire time course
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Fig. 3. The effect of cinanserin (4 mg/kg) in a preparation with only facilitation of the MSR following brain stem stimulation. MSRs evoked at various times after brain stem conditioning stimuli are plotted as in Fig. 2. Solid line represents the pre-drug control and the dotted line was obtained after
cinanserin administration.
of the conditioning volley influence (Fig. 2). This resulted in a marked increase in the facilitatory effects of brain stem stimulation and either a decrease of inhibition or conversion to facilitation. In 4 additional cats, no inhibition was observed at any interstimulus interval, but only pure facilitatory effects resulted from brain stem stimulation (Fig. 3, solid line). In these preparations, the injection of cinanserin also smoothly displaced the brain stem conditioning curve in a facilitatory direction (Fig. 3). Thus, the influence of cinanserin on the conditioning curve was the same whether or not inhibitory actions were present (compare Figs. 2 and 3). Histological examination of the electrode placements in the above experiments revealed that half the locations were outside the nucleus raphe magnus (Fig. 4). No inhibition could be detected from electrode placements in the medial longitudinal fasciculus or the medial lemniscus. However, the effects of cinanserin on the brain stem conditioning curve were the same regardless of the stimulating electrode positions (cf. ref. 8). The MSR, unconditioned by brain stem stimulation, was not facilitated by the injection of cinanserin and in some experiments, it was slightly depressed. In addition, the VRP evoked from the brain stem was unaffected by the 5-HT antagonists in 10 of 12 animals. However, for an unknown reason, cinanserin markedly increased the brain stem VRP in two animals. Thus, in the majority of the animals tested, the facilitatory effect of brain stem stimulation following the injection of cinanserin appeared to occur without a significant increase in motoneuronal excitability.
336
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A N D E. G . A N D E R S O N
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Fig. 4. Relationship between brain stem electrode placement and the effectsof conditioning stimuli on the MSR. Each point represents the position of the electrode tips as determined by the Prussian Blue test. The open circles represent areas where conditioning stimulation produced pure facilitation of the MSR and the closed circles represent areas where stimulation produced a combination of facilitation and inhibition. Abbreviations: G VII, genu of the seventh nerve; ML, medial lemniscus; MLF, medial longitudinal fasciculus; N VII, seventh nerve; Py, pyramids; RM, nucleus raphe magnus; ST V, spinal tract of the fifth nerve.
Recovery from cinanserin's effect on the efficacy of brain stem conditioning stimuli began within 40 min of its injection, and was usually complete within 70 min. Cinanserin had no significant effect on blood pressure, producing only a small transient (1-2 min) decrease in blood pressure in some animals. The administration of another 5-HT antagonist, methysergide bimaleate, produced effects similar to those of cinanserin on the brain stem conditioning curve, shifting it in a facilitatory direction. Methysergide, however, markedly decreased the segmental MSR, an action that has been previously documented and shown to be unrelated to 5-HT antagonism 5,7,8. The serotonin antagonists could have produced their effects by acting at either the brain stem or the spinal cord. To distinguish these two possibilities cinanserin and methysergide were injected into the arterial supply to the spinal cord, or into the general circulation via the antecubital vein. Low doses of cinanserin (1-1.6 mg/kg) and methysergide (200 #g/kg) administered by close arterial injection shifted the conditioning curve in a facilitatory direction; as did higher intravenous doses. The low doses of cinanserin and methysergide produced no significant effects when administered intravenously. Similar results were obtained in a previous study 8. The displacement of the entire brain stem conditioning curve in a facilitatory direction by the serotonin antagonists suggested a generalized increase in motoneuron excitability. This was not the case; however, since the unconditioned MSR did not increase. An alternative explanation of this displacement is that the descending 5-HT system produces presynaptic inhibition of transmission from la primary afferent terminals to motoneurons. Thus, experiments were designed to examine the possibility that raphe stimulation produces presynaptic inhibition. DRPs and DRRs, which are indicators of primary afferent depolarization, were recorded following dorsal root and brain stem stimulation. Dorsal root stimulation evoked both a D R R and a DRP
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Fig. 5. The effectof cinanserin (4 mg/kg) on DRP-BS, DRP-S and DRR. The DRP-BS was evoked by trains of pulses applied to the brain stem while the DRP-S and DRR were evoked by single pulses to the L7 dorsal root. All potentials were recorded from the same L7 dorsal rootlet. Each point in the DRP curves represents the average area 4- S.E. (12 experiments for DRP-BS and 6 experiments for DRP-S). The points in the DRR curve represent the average peak amplitude in 6 experiments. The DRP areas and DRR amplitudes for the points in each experiment are the sum of 10 sweeps obtained using a signal averaging computer. The DRP areas and the DRR amplitudes are plotted as a percent of pre-drug values v e r s u s the time in minutes after cinanserin.
(DRP-S) while brain stem stimulation evoked only a D R P (DRP-BS). Susceptibility to blockade by serotonin antagonists was used as a criterion for the involvement o f serotonin in the generation of these potentials. DRPs were elicited by stimidating almost anywhere in the medial brain stem. All of the electrode placements illustrated in Fig. 4 evoked a DRP-BS. Cinanserin administration significantly increased the DRP-BS within 5 min in all of the 12 cats tested (Fig. 5). The injection of methysergide produced the same effects as cinanserin. In contrast, the dorsal root potential evoked from the L7 dorsal root was not increased, but tended to decrease following cinanserin, and the segmentally evoked D R R was clearly depressed (Fig. 5). The time course of the segmental D R R depression was very similar to that of the increase in the DRP-BS (Fig. 5). Within 5 min after cinanserin, the D R R was significantly decreased below
338
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control levels and by 30 rain it showed significant recovery. In 3 experiments methysergide was found to have effects similar to those of cinanserin. The differential effect of cinanserin on the DRP-BS and DRP-S suggested that these two DRPs were generated in different afferent terminals. To test this hypothesis, length constants were calculated in control cats for the fibers in which the DRP-BS and DRP-S were generated to determine whether these DRPs originated in fibers of the same size. The amplitude of the DRPs was measured at 1 mm intervals while movir~g the recording electrode away from the dorsal root entry zone. The amplitude of the DRPs measured along the dorsal root declined exponentially as the distance between the point of measurement and the entry zone increased (Fig. 6). F r o m these data, length constants were calculated for the fibers in which the DRPs were generated by determining the distance at which each D R P declined to 1/e of its original value. The length constant for the DRP-BS fibers in Fig. 6 was 3.4 mm while that for the DRP-S fibers was 8.1 mm. In 3 experiments, the length constant was invariably larger for the fibers carrying the DRP-S. These measurements indicate that the DRP-S is generated in fibers of larger diameter than those carrying the DRP-BS.
SEROTONIN AND BULBOSPINAL SYSTEMS
339
DISCUSSION
The objective of these experiments was to activate the serotonin-containing cell bodies in the nucleus raphe magnus and determine their effect on segmental motor reflexes. In most experiments, conditioning brain stem stimulation produced a complex facilitatory-inhibitory-facilitatory action on segmentally evoked MSRs as the interval between the conditioning and test stimuli was increased (Fig. 2). In some animals, no inhibitory effects could be elicited by brain stem stimulation. In these situations, a pure facilitatory effect was observed throughout the time course of the brain stem conditioning influence (Fig. 3). From these data it would appear that inhibition, when it is observed, is imposed on a period of facilitation. The position of the electrode in the brain stem has some influence on the presence or absence of inhibition (Fig. 4). On the basis of previous workT,s it was anticipated that the 5-HT antagonists would block brain stem evoked inhibition of the MSR. However, it was consistently found that the 5-HT antagonists produced a smooth displacement of the brain stem conditioning curve in a facilitatory direction with no change in the shape of the curve (Figs. 2 and 3). It is unlikely these effects occurred as the result of a decrease in inhibition, but rather from augmented facilitatory effects of brain stem stimulation. This conclusion is supported by the fact that the slow conduction velocity of the unmyelinated serotonergic fibers of less than 2 #m in diameter is not compatible with inhibitory actions carried by these fibers from the brain stem with latencies much less than 100 msec. These increased descending facilitatory actions cannot be mediated by direct increases in motoneuron excitability or decreases in a tonic presynaptic inhibition since the MSR evoked in the absence of brain stem stimulation was not altered by the 5-HT antagonists. Another possible explanation of the increased facilitatory effects of brain stem stimulation following administration of 5-HT antagonists is that brain stem stimulation produces a phasic, 5-HT mediated, presynaptic inhibition of la terminals. Thus, the 5-HT antagonists would decrease the phasic presynaptic inhibition of 1a afferent impulses by the brain stem without affecting the unconditioned segmental MSR. This possibility, however, is ruled out by the observation that the brain stem evoked DRP was increased rather than decreased by the 5-HT antagonists. Furthermore, the length constant of the DRP-BS being smaller than that of the DRP-S indicates that brain stem stimulation evokes a primary afferent depolarization in fibers of a smaller diameter than la fibers. These measurements are consistent with the observations of Carpenter e t aL 6 and Lundberg and VyklickyTM showing that brain stem stimulation evokes depolarization in group I[ and Iii primary afferents. The increase in the DRP-BS following the 5-HT antagonists suggests that the serotonergic system has an important influence on presynaptic inhibition in the flexor reflex afferents. Since the 5-HT antagonists failed to block any of the effects of brain stem stimulation, but selectively increased some of its effects, the brain stem stimulation apparently failed to evoke any observable activity in serotonergic pathways. Instead, the dramatic effects of the 5-HT antagonists on the brain stem conditioning curves most
340
H . K. P R O U D F I T A N D E. G. A N D E R S O N
likely results from the removal of tonic activity in the descending serotonergic neurons in the decerebrate cat. The simplest model, which explains most presently available data, would require the 5-HT neurons to tonically inhibit the facilitatory pathways descending from the brain stem. This inhibition must occur at the spinal level since the 5-HT antagonists, when administered by close intra-arterial injection to the spinal cord, exert their actions at doses which are ineffective when administered intravenously. This tonic inhibition could occur either presynaptically on the terminals of the facilitatory fibers descending from the brain stem or postsynaptically on interneurons in the facilitatory pathway. Removal of this tonic inhibition by the 5-HT antagonists would result in increased facilitatory effects of brain stem stimulation without modifying descending inhibitory actions, or altering the segmental reflexes. In addition, the increase in the DRP-BS and decrease in the D R R and DRP-S suggests that the 5-HT neurons exert a tonic influence on the pathways generating primary afferent depolarization. The opposite effects of the 5-HT antagonists on the DRP-BS and the DRP-S is partly explained by the different fibers in which these potentials are generated. There are too many unknown entities (e.g. tonic state of afferent terminal polarization, whether 5-HT hyperpolarizes or depolarizes at its site of action) to propose a definitive model to explain these actions. It is of interest to note; however, that Engberg et al. 12 have shown that 5-HT antagonists will decrease tonic inhibition of transmission from flexor reflex afferents in the decerebrate cat. Furthermore, it is tempting to combine these results with our observation of the increased DRP-BS (which probably occurs in group II and III primary afferents) following a 5-HT antagonist, and suggest that in the decerebrate cat the serotonergic system mediates a tonic depolarization of group II and Ill afferents. Blockade of this system by a 5-HT antagonist would result in a decreased presynaptic inhibition of flexor reflexes and an increased DRP-BS. The increased DRP-BS would result from removing tonic depolarization of flexor reflex afferent terminals, thus allowing a greater depolarizing response to brain stem stimulation. ACKNOWLEDGEMENTS
This study was supported by N I H Grant NS 05611. H.K.P. was supported by N I M H Grant 8396.
REFERENCES 1 ADRIAN, E. D., AND MORUZZI, G., Impulses in the pyramidal tract, J. PhysioL (Lond.), 97 (1939) 153-199. 2 AND~N, N.-E., JUKES, M. G. M., AND LUNDBERG, A., Spinal reflexes and monoamine liberation, Nature (Lond.), 202 (1964) 1222-1223. 3 ANDERSON,E. G., Bulbospinal serotonin-containing neurons and motor control, Fed. Proc., 31 (1972) 107-112. 4 ANDERSON,E. G., AND SmaUVA, T., The effects of 5-hydroxytrytophan and L-tryptophan on spinal synaptic activity, J. Pharmacol. exp. Ther., 153 (1966) 352-360. 5 BANNA, N. E., AND ANDERSON,E. G., The effects of 5-hydroxytryptamine antagonists on spinal neuronal activity, J. Pharmacol. exp. Ther., 162 (1968) 319-325.
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6 CARPENTER,D., ENGBERG,1., FUNKENSTEIN,H., ANDLUNDBERG,A., Decerebrate control of reflexes
to primary afferents, Acta physiol, seaM., 59 (1963) 424-437. 7 CLINESCHMIDT,B. V., AND ANDERSON,E. G., Lysergic acid diethylamide: Antagonism of supraspinal inhibition of spinal reflexes, Brain Research, 16 (1969) 296-300. 8 CLINESCHMIDT,B. V., AND ANDERSON, E. G., The blockade of bulbospinal inhibition by 5hydroxytryptamine antagonists, Exp. Brain Res., 11 (1970) 175-186. 9 CLINESCHMIDT,B. V., PIERCE,J. E., AND LOVENBERG,W., Tryptophan hydroxylase and serotonin in spinal cord and brain stem before and after chronic transection, J. Neurochem., 18 (1971) 1593-1596. 10 DAHLSTROM,A., AND FOXE, K., Evidence for the existence of monoamine-containingneurons in the central nervous system. I. Demonstration of monoamines in the cell bodies of brain stem neurons, Acta physiol, scand., 62, Suppl. 232 (1964) 1-55. 11 DAHLSTR~M,A., AND FUXE, K., Evidence for the existence of monoamine neurons in the central nervous system. II. Experimentally induced changes in the intraneuronal amine levels of bulbospinal neuron systems, Acta physiol, seand., 64, Suppl. 247 (1965) 1-36. 12 ENGBERG,I., LUNDBERG,A., ANDRYALL,R. W., Is the tonic decerebrate inhibition of reflex paths mediated by monoaminergic pathways?, Acta physiol, seand., 72 (t968) 123-133. 13 LUNDBERG,A., AND VYKLICKY,L., Inhibition of transmission to primary afferents by electrical stimulation of the brain stem, Arch. ital. Biol., 104 (1966) 86-97. 14 PROUDF1T,H. K., AND ANDERSON,E. G., Alteration by serotonin antagonists of the effects of bulbospinal stimulation on spinal reflex pathways, Fed. Proe., 31 (1972) 318.
331
© Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands
INFLUENCE OF SEROTONIN ANTAGONISTS ON BULBOSPINAL SYSTEMS
HERBERT K. P R O U D F I T AND E D M U N D G. A N D E R S O N
Department of Pharmacology, University of Illinois, College of Medicine, P.O. Box 6998, Chicago, Ill. 60680 (U.S.A.) (Accepted April 2nd, 1973)
SUMMARY
We attempted to elucidate the function of the serotonin-containing bulbospinal neurons by directly stimulating the raphe nucleus of unanesthetized decerebrate cats. Such stimuli evoked both a dorsal and a ventral root potential, and produced a complex time-dependent facilitation and inhibition of segmentally evoked monosynaptic reflexes (MSR). The entire time course of the curve representing the brain stem-evoked facilitation and inhibition of the MSR was displaced in a facilitatory direction following administration of the serotonin (5-HT) antagonists cinanserin or methysergide. The apparent block of descending inhibition probably resulted from increased descending facilitation. These results occurred without an increase in motoneuronal excitability, since the segmental MSR remained unchanged. The 5-HT antagonists increased the brain stem-evoked dorsal root potential (DRP), but did not alter the ventral root potential. In contrast, these agents depressed the segmentally evoked DRP. Measurements of the DRP length constants in afferent fibers revealed that brain stem stimulation evoked DRPs in fibers of smaller diameter than dorsal root stimulation. Apparently none of the potentials evoked by raphe stimulation were mediated by serotonergic fibers since these potentials were not reduced by 5-HT antagonists. It is suggested that the serotonergic system is tonically active in the decerebrate cat, and that the increased facilitatory action on the conditioned MSR and brain stem DRP following a 5-HT antagonist results from release of tonic serotonergic inhibition of descending facilitatory fibers.
I NTRODUCTION
Histochemical studies of Dahlstr6m and Fuxel0,11 have shown that serotonincontaining cell bodies localized in the caudal raphe nuclei have axons which descend
332
H . K . P R O U D F I T A N D E. G . A N D E R S O N
in the spinal cord and terminate in the dorsal, lateral and intermediate horns. The tunctional role of this serotonergic system remains uncertain. Administration of 5hydroxytryptophan (5-HTP) to elevate cord levels of serotonin (5-hydroxytryptamine, 5-HT) has provided evidence that 5-HT exerts a facilitatory action on motoneurons4, but other studies have suggested the 5-HT system may inhibit flexor reflexes2,12. The function of these descending serotonergic neurons could be clarified by stimulating their somata, located in the brain stem raphe nuclei, and recording their influences on segmental systems. In a previous study from this laboratory 8, conditioning volleys delivered to the nucleus raphe magnus as well as the lateral reticular formation resulted in an early facilitation followed by inhibition of the segmentally evoked monosynaptic reflex (MSR). Several 5-HT antagonists blocked the inhibition of segmental MSRs induced by brain stem stimulation. Since the conduction velocity of the inhibitory system was too fast for the small diameter serotonergic nerves descending from the raphe nuclei, it was suggested that the inhibition might be mediated by a serotonergic interneuron located in the spinal cord. However, evidence for such an interneuron is not good 9 (see review by Andersona). The present study was initiated to re-investigate the function of the descending 5-HT system, by examining the entire time course of the effects of brain stem stimulation on the segmentally evoked MSR and, in addition, to assess the possibility of descending serotonergic actions on prima ry afferent terminals. Some of these results have been presented in a preliminary report ~4. METHODS
Cats were made decerebrate, under temporary ether anesthesia, by removal of the forebrain rostral to the inferior colliculus. A laminectomy was performed and the seventh lumbar (L7) spinal roots were dissected free and mounted for stimulation and/or recording in an oil pool maintained at 36-37 °C. Platinum hook electrodes were routinely used for stimulating and recording. The animals were artificially respired and immobilized with gallamine triethiodide. The caudal brain stem raphe nuclei were stimulated using stereotaxically placed concentric bipolar stainless steel electrodes. Thirty msec trains of 1-3 V (0.08-0.25 mA), 0.5 msec square wave pulses were applied to the electrodes at 300 Hz. Dorsal and ventral root potentials evoked by these stimuli were recorded at the L7 segment. The effects of brain stem stimulation on motoneuron excitability were assessed by measuring the MSR evoked by a test stimulus (0.01-0.1 V; 0.05 msec) to the dorsal root at various time intervals following a conditioning volley applied to the brain stem. Control MSRs were elicited in the absence of brain stem volleys. Dorsal root potentials (DRP) and reflexes (DRR) were recorded from the most caudal L7 dorsal rootlet. Potentials were quantified by summing 10 evoked potentials using a CAT 1000 computer of average transients and measuring either peak amplitude (MSR and DRR) or area (DRP). Length constants were determined for the fibers in which the dorsal root potentials were generated. Two platinum hook electrodes held in a micromanipulator were used to record the potentials from a dorsal rootlet. One electrode was placed near the
333
SEROTONIN AND BULBOSPINAL SYSTEMS
dorsal root entry zone and the other was placed on a dorsal rootlet about 10 mm from the first. The electrodes were moved in 1 mm increments away from their original position; the electrode separation remained constant. The length constant is defined as the distance at which the potential declined to 1/e of its original value. All brain stem electrode placements were verified histologically. The Prussian Blue technique of Adrian and Moruzzi 1 was used to stain for iron deposits left by a brief DC current application to the electrode. The serotonin antagonists used were cinanserin HCI (4 mg/kg) and methysergide bimaleate (1 mg/kg). All drugs were dissolved in normal saline and administered intravenously through an indwelling catheter placed in the anticubital vein. In some experiments intra-arterial injections to the spinal cord were made via a catheter inserted into the caudal mesenteric artery and guided up the abdominal aorta to a point about 1 cm caudal to the renal arteries. The testicular (ovarian), deep circumflex iliac, external iliac, and common iliac arteries were ligated leaving only the spinal arteries intact.
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Fig. l. Position of stimulating and recording electrodes. Trains of stimuli were applied to the brain stem in the area of the nucleus raphe magnus using a bipolar stimulating electrode. Brain stem stimulation evoked a ventral root potential (VRP) and dorsal root potential (DRP-BS) (upper left and right panels, respectively). Stimulation of the L7 dorsal root evoked a monosynaptic reflex and a dorsal root potential (DRP-S) (lower left and right panels, respectively). The potentials in the left panels were recorded from the L7 ventral root while those on the right were recorded from the most caudal L7 dorsal rootlet. Abbreviations: R, recording electrode; Rd, nucleus raphe dorsalis; Rm, nucleus raphe magnus; Ro, nucleus raphe obscurus; Rp, nucleus raphe pontis; Rpa, nucleus raphe pallidus; S, stimulating electrodes; T, trapezoid body.
334
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Fig. 2. A representative experiment illustrating the effect of cinanserin (4 mg/kg) on the MSR evoked at various times after a brain stem conditioning volley. The MSR evoked by stimulation of the L7 dorsal root is plotted as a percent of the unconditioned MSR. Intervals between brain stem conditioning stimuli and the test stimulus are plotted in milliseconds. The solid line represents the control brain stem conditioning curve while the dotted line is that after cinanserin administration.
RESULTS
Attempts were made to activate the descending serotonin-containing cells in the nucleus raphe magnus of the caudal brain stem using stereotaxically placed bipolar stimulating electrodes. Evoked potentials elicited by brain stem stimulation were recorded from both the dorsal and ventral roots. The brain stem dorsal root potential (DRP-BS) was a negative going wave with an onset latency of about 30 msec and a duration of 75-100 msec (Fig. 1). No D R R was observed following brain stem stimulation. The ventral root potential (VRP) evoked from the brain stem began 5-10 msec after the first pulse of the stimulus volley and lasted 40-50 msec (Fig. 1). To further study the influence of brain stem stimulation on segmental systems, a test MSR was evoked at various intervals following the brain stem conditioning stimuli. The test MSR was either facilitated or inhibited depending on the interval between the conditioning brain stem volley and the test stimulus to the L7 dorsal root (Fig. 2, solid line). The MSR was facilitated at intervals between 30 and 35 msec and between 70 and 120 msec. Inhibition of the MSR was usually observed within the 40-65 msec range. Since the raphe is a thin laminar structure, cell bodies and axons in areas surrounding the raphe magnus were probably activated by current spread from the stimulating electrode. Therefore, we tried to identify those influences of the descending system which were mediated by serotonergic fibers by determining their susceptibility to 5-HT antagonists. Cinanserin HCI (4 mg/kg) and methysergide bimaleate (1 mg/kg) were used to antagonize the effects of 5-HT. The injection of cinanserin into 6 cats dramatically altered the influence of brain stem stimulation on the MSR. Within 2-3 min, the effects of the conditioning volley on the MSR were shifted in a facilitatory direction throughout the entire time course
335
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Fig. 3. The effect of cinanserin (4 mg/kg) in a preparation with only facilitation of the MSR following brain stem stimulation. MSRs evoked at various times after brain stem conditioning stimuli are plotted as in Fig. 2. Solid line represents the pre-drug control and the dotted line was obtained after
cinanserin administration.
of the conditioning volley influence (Fig. 2). This resulted in a marked increase in the facilitatory effects of brain stem stimulation and either a decrease of inhibition or conversion to facilitation. In 4 additional cats, no inhibition was observed at any interstimulus interval, but only pure facilitatory effects resulted from brain stem stimulation (Fig. 3, solid line). In these preparations, the injection of cinanserin also smoothly displaced the brain stem conditioning curve in a facilitatory direction (Fig. 3). Thus, the influence of cinanserin on the conditioning curve was the same whether or not inhibitory actions were present (compare Figs. 2 and 3). Histological examination of the electrode placements in the above experiments revealed that half the locations were outside the nucleus raphe magnus (Fig. 4). No inhibition could be detected from electrode placements in the medial longitudinal fasciculus or the medial lemniscus. However, the effects of cinanserin on the brain stem conditioning curve were the same regardless of the stimulating electrode positions (cf. ref. 8). The MSR, unconditioned by brain stem stimulation, was not facilitated by the injection of cinanserin and in some experiments, it was slightly depressed. In addition, the VRP evoked from the brain stem was unaffected by the 5-HT antagonists in 10 of 12 animals. However, for an unknown reason, cinanserin markedly increased the brain stem VRP in two animals. Thus, in the majority of the animals tested, the facilitatory effect of brain stem stimulation following the injection of cinanserin appeared to occur without a significant increase in motoneuronal excitability.
336
H. K. PROUDFIT
A N D E. G . A N D E R S O N
* FACILITATION • FACI LITATI O N INHIBITION
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Fig. 4. Relationship between brain stem electrode placement and the effectsof conditioning stimuli on the MSR. Each point represents the position of the electrode tips as determined by the Prussian Blue test. The open circles represent areas where conditioning stimulation produced pure facilitation of the MSR and the closed circles represent areas where stimulation produced a combination of facilitation and inhibition. Abbreviations: G VII, genu of the seventh nerve; ML, medial lemniscus; MLF, medial longitudinal fasciculus; N VII, seventh nerve; Py, pyramids; RM, nucleus raphe magnus; ST V, spinal tract of the fifth nerve.
Recovery from cinanserin's effect on the efficacy of brain stem conditioning stimuli began within 40 min of its injection, and was usually complete within 70 min. Cinanserin had no significant effect on blood pressure, producing only a small transient (1-2 min) decrease in blood pressure in some animals. The administration of another 5-HT antagonist, methysergide bimaleate, produced effects similar to those of cinanserin on the brain stem conditioning curve, shifting it in a facilitatory direction. Methysergide, however, markedly decreased the segmental MSR, an action that has been previously documented and shown to be unrelated to 5-HT antagonism 5,7,8. The serotonin antagonists could have produced their effects by acting at either the brain stem or the spinal cord. To distinguish these two possibilities cinanserin and methysergide were injected into the arterial supply to the spinal cord, or into the general circulation via the antecubital vein. Low doses of cinanserin (1-1.6 mg/kg) and methysergide (200 #g/kg) administered by close arterial injection shifted the conditioning curve in a facilitatory direction; as did higher intravenous doses. The low doses of cinanserin and methysergide produced no significant effects when administered intravenously. Similar results were obtained in a previous study 8. The displacement of the entire brain stem conditioning curve in a facilitatory direction by the serotonin antagonists suggested a generalized increase in motoneuron excitability. This was not the case; however, since the unconditioned MSR did not increase. An alternative explanation of this displacement is that the descending 5-HT system produces presynaptic inhibition of transmission from la primary afferent terminals to motoneurons. Thus, experiments were designed to examine the possibility that raphe stimulation produces presynaptic inhibition. DRPs and DRRs, which are indicators of primary afferent depolarization, were recorded following dorsal root and brain stem stimulation. Dorsal root stimulation evoked both a D R R and a DRP
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Fig. 5. The effectof cinanserin (4 mg/kg) on DRP-BS, DRP-S and DRR. The DRP-BS was evoked by trains of pulses applied to the brain stem while the DRP-S and DRR were evoked by single pulses to the L7 dorsal root. All potentials were recorded from the same L7 dorsal rootlet. Each point in the DRP curves represents the average area 4- S.E. (12 experiments for DRP-BS and 6 experiments for DRP-S). The points in the DRR curve represent the average peak amplitude in 6 experiments. The DRP areas and DRR amplitudes for the points in each experiment are the sum of 10 sweeps obtained using a signal averaging computer. The DRP areas and the DRR amplitudes are plotted as a percent of pre-drug values v e r s u s the time in minutes after cinanserin.
(DRP-S) while brain stem stimulation evoked only a D R P (DRP-BS). Susceptibility to blockade by serotonin antagonists was used as a criterion for the involvement o f serotonin in the generation of these potentials. DRPs were elicited by stimidating almost anywhere in the medial brain stem. All of the electrode placements illustrated in Fig. 4 evoked a DRP-BS. Cinanserin administration significantly increased the DRP-BS within 5 min in all of the 12 cats tested (Fig. 5). The injection of methysergide produced the same effects as cinanserin. In contrast, the dorsal root potential evoked from the L7 dorsal root was not increased, but tended to decrease following cinanserin, and the segmentally evoked D R R was clearly depressed (Fig. 5). The time course of the segmental D R R depression was very similar to that of the increase in the DRP-BS (Fig. 5). Within 5 min after cinanserin, the D R R was significantly decreased below
338
I-1. K. PROUDFIT AND E. G. ANDERSON 2.0
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from the dorsal root entry zone. DRP amplitude is plotted as the logarithm and the straight lines were fitted to the points using the least squares method.
control levels and by 30 rain it showed significant recovery. In 3 experiments methysergide was found to have effects similar to those of cinanserin. The differential effect of cinanserin on the DRP-BS and DRP-S suggested that these two DRPs were generated in different afferent terminals. To test this hypothesis, length constants were calculated in control cats for the fibers in which the DRP-BS and DRP-S were generated to determine whether these DRPs originated in fibers of the same size. The amplitude of the DRPs was measured at 1 mm intervals while movir~g the recording electrode away from the dorsal root entry zone. The amplitude of the DRPs measured along the dorsal root declined exponentially as the distance between the point of measurement and the entry zone increased (Fig. 6). F r o m these data, length constants were calculated for the fibers in which the DRPs were generated by determining the distance at which each D R P declined to 1/e of its original value. The length constant for the DRP-BS fibers in Fig. 6 was 3.4 mm while that for the DRP-S fibers was 8.1 mm. In 3 experiments, the length constant was invariably larger for the fibers carrying the DRP-S. These measurements indicate that the DRP-S is generated in fibers of larger diameter than those carrying the DRP-BS.
SEROTONIN AND BULBOSPINAL SYSTEMS
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DISCUSSION
The objective of these experiments was to activate the serotonin-containing cell bodies in the nucleus raphe magnus and determine their effect on segmental motor reflexes. In most experiments, conditioning brain stem stimulation produced a complex facilitatory-inhibitory-facilitatory action on segmentally evoked MSRs as the interval between the conditioning and test stimuli was increased (Fig. 2). In some animals, no inhibitory effects could be elicited by brain stem stimulation. In these situations, a pure facilitatory effect was observed throughout the time course of the brain stem conditioning influence (Fig. 3). From these data it would appear that inhibition, when it is observed, is imposed on a period of facilitation. The position of the electrode in the brain stem has some influence on the presence or absence of inhibition (Fig. 4). On the basis of previous workT,s it was anticipated that the 5-HT antagonists would block brain stem evoked inhibition of the MSR. However, it was consistently found that the 5-HT antagonists produced a smooth displacement of the brain stem conditioning curve in a facilitatory direction with no change in the shape of the curve (Figs. 2 and 3). It is unlikely these effects occurred as the result of a decrease in inhibition, but rather from augmented facilitatory effects of brain stem stimulation. This conclusion is supported by the fact that the slow conduction velocity of the unmyelinated serotonergic fibers of less than 2 #m in diameter is not compatible with inhibitory actions carried by these fibers from the brain stem with latencies much less than 100 msec. These increased descending facilitatory actions cannot be mediated by direct increases in motoneuron excitability or decreases in a tonic presynaptic inhibition since the MSR evoked in the absence of brain stem stimulation was not altered by the 5-HT antagonists. Another possible explanation of the increased facilitatory effects of brain stem stimulation following administration of 5-HT antagonists is that brain stem stimulation produces a phasic, 5-HT mediated, presynaptic inhibition of la terminals. Thus, the 5-HT antagonists would decrease the phasic presynaptic inhibition of 1a afferent impulses by the brain stem without affecting the unconditioned segmental MSR. This possibility, however, is ruled out by the observation that the brain stem evoked DRP was increased rather than decreased by the 5-HT antagonists. Furthermore, the length constant of the DRP-BS being smaller than that of the DRP-S indicates that brain stem stimulation evokes a primary afferent depolarization in fibers of a smaller diameter than la fibers. These measurements are consistent with the observations of Carpenter e t aL 6 and Lundberg and VyklickyTM showing that brain stem stimulation evokes depolarization in group I[ and Iii primary afferents. The increase in the DRP-BS following the 5-HT antagonists suggests that the serotonergic system has an important influence on presynaptic inhibition in the flexor reflex afferents. Since the 5-HT antagonists failed to block any of the effects of brain stem stimulation, but selectively increased some of its effects, the brain stem stimulation apparently failed to evoke any observable activity in serotonergic pathways. Instead, the dramatic effects of the 5-HT antagonists on the brain stem conditioning curves most
340
H . K. P R O U D F I T A N D E. G. A N D E R S O N
likely results from the removal of tonic activity in the descending serotonergic neurons in the decerebrate cat. The simplest model, which explains most presently available data, would require the 5-HT neurons to tonically inhibit the facilitatory pathways descending from the brain stem. This inhibition must occur at the spinal level since the 5-HT antagonists, when administered by close intra-arterial injection to the spinal cord, exert their actions at doses which are ineffective when administered intravenously. This tonic inhibition could occur either presynaptically on the terminals of the facilitatory fibers descending from the brain stem or postsynaptically on interneurons in the facilitatory pathway. Removal of this tonic inhibition by the 5-HT antagonists would result in increased facilitatory effects of brain stem stimulation without modifying descending inhibitory actions, or altering the segmental reflexes. In addition, the increase in the DRP-BS and decrease in the D R R and DRP-S suggests that the 5-HT neurons exert a tonic influence on the pathways generating primary afferent depolarization. The opposite effects of the 5-HT antagonists on the DRP-BS and the DRP-S is partly explained by the different fibers in which these potentials are generated. There are too many unknown entities (e.g. tonic state of afferent terminal polarization, whether 5-HT hyperpolarizes or depolarizes at its site of action) to propose a definitive model to explain these actions. It is of interest to note; however, that Engberg et al. 12 have shown that 5-HT antagonists will decrease tonic inhibition of transmission from flexor reflex afferents in the decerebrate cat. Furthermore, it is tempting to combine these results with our observation of the increased DRP-BS (which probably occurs in group II and III primary afferents) following a 5-HT antagonist, and suggest that in the decerebrate cat the serotonergic system mediates a tonic depolarization of group II and Ill afferents. Blockade of this system by a 5-HT antagonist would result in a decreased presynaptic inhibition of flexor reflexes and an increased DRP-BS. The increased DRP-BS would result from removing tonic depolarization of flexor reflex afferent terminals, thus allowing a greater depolarizing response to brain stem stimulation. ACKNOWLEDGEMENTS
This study was supported by N I H Grant NS 05611. H.K.P. was supported by N I M H Grant 8396.
REFERENCES 1 ADRIAN, E. D., AND MORUZZI, G., Impulses in the pyramidal tract, J. PhysioL (Lond.), 97 (1939) 153-199. 2 AND~N, N.-E., JUKES, M. G. M., AND LUNDBERG, A., Spinal reflexes and monoamine liberation, Nature (Lond.), 202 (1964) 1222-1223. 3 ANDERSON,E. G., Bulbospinal serotonin-containing neurons and motor control, Fed. Proc., 31 (1972) 107-112. 4 ANDERSON,E. G., AND SmaUVA, T., The effects of 5-hydroxytrytophan and L-tryptophan on spinal synaptic activity, J. Pharmacol. exp. Ther., 153 (1966) 352-360. 5 BANNA, N. E., AND ANDERSON,E. G., The effects of 5-hydroxytryptamine antagonists on spinal neuronal activity, J. Pharmacol. exp. Ther., 162 (1968) 319-325.
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6 CARPENTER,D., ENGBERG,1., FUNKENSTEIN,H., ANDLUNDBERG,A., Decerebrate control of reflexes
to primary afferents, Acta physiol, seaM., 59 (1963) 424-437. 7 CLINESCHMIDT,B. V., AND ANDERSON,E. G., Lysergic acid diethylamide: Antagonism of supraspinal inhibition of spinal reflexes, Brain Research, 16 (1969) 296-300. 8 CLINESCHMIDT,B. V., AND ANDERSON, E. G., The blockade of bulbospinal inhibition by 5hydroxytryptamine antagonists, Exp. Brain Res., 11 (1970) 175-186. 9 CLINESCHMIDT,B. V., PIERCE,J. E., AND LOVENBERG,W., Tryptophan hydroxylase and serotonin in spinal cord and brain stem before and after chronic transection, J. Neurochem., 18 (1971) 1593-1596. 10 DAHLSTROM,A., AND FOXE, K., Evidence for the existence of monoamine-containingneurons in the central nervous system. I. Demonstration of monoamines in the cell bodies of brain stem neurons, Acta physiol, scand., 62, Suppl. 232 (1964) 1-55. 11 DAHLSTR~M,A., AND FUXE, K., Evidence for the existence of monoamine neurons in the central nervous system. II. Experimentally induced changes in the intraneuronal amine levels of bulbospinal neuron systems, Acta physiol, seand., 64, Suppl. 247 (1965) 1-36. 12 ENGBERG,I., LUNDBERG,A., ANDRYALL,R. W., Is the tonic decerebrate inhibition of reflex paths mediated by monoaminergic pathways?, Acta physiol, seand., 72 (t968) 123-133. 13 LUNDBERG,A., AND VYKLICKY,L., Inhibition of transmission to primary afferents by electrical stimulation of the brain stem, Arch. ital. Biol., 104 (1966) 86-97. 14 PROUDF1T,H. K., AND ANDERSON,E. G., Alteration by serotonin antagonists of the effects of bulbospinal stimulation on spinal reflex pathways, Fed. Proe., 31 (1972) 318.