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Lysergic acid diethylamide: antagonism of supraspinal inhibition of spinal reflexes
296 lysergic
SHORT
acid diehylamide:
antagonism
of supraspinal
C‘OMM~INICATIONS
inhibition
of spinal
reflexes
The axons from serotonin (5_hydroxytryptamine, 5-HT) containing neurons whose somata are loca!ized in the caudal raphe nuclei of the brain stem descend ria the ventral and lateral funiculi of the spinal cord to terminate in the dorsal, lateral and ventral horns6s7. There is evidence that some of the 5-HT containing termina!s make close contact with motoneurones7. Although 5-HT applied microiontophoretically has been reported to hyperpolarize motoneurones 9, 5-hydroxytryptophan (5-HTP) injected into spinal cats increases motoneuronal excitability”,lO. an action readily reversed by 5-HT antagonist+. The significance ofthese observations is uncertain. In the present study we have attempted to directly activate the serotonin containing neurons of the bulbospinal system through stimulating electrodes placed in the caudal raphC nuclei, while observing the effects of stimulation on the segmental reflexes. For comparison stimulating electrodes were also placed in the ventromedial reticular formation. Since electrical stimulation could activate other descending pathways, it was anticipated that actions mediated by serotonergic neurons could be distinguished by their susceptibility to blockade by serotonin antagonists. Preliminary results of this study are reported herein. Cats of either sex weighing I .7-3.0 kg were decerebrated between the coliicuii under ether anesthesia. After discontinuing ether, two concentric bipolar electrodes with internal tip diameter less than 0.2 mm and tip separation I mm were positioned in the brain stem raphe and ventromedial reticular formation by stereotaxic technique. Using the Horsley-Clarke planes, co-ordinates for placing electrodes in the raphe and reticular formation were respectively: P7.5 mm (frontal plane), H-6 to -8 (horizontal plane), LO (sagittal plane) and P11, H-8 to -9, L1.5. Activation of the stimulating electrode while moving it in the horizontal plane through the raphe and reticular formation revealed both facilitatory and inhibitory effects on segmental reflexes. The facilitatory influences of brain stem stimulation were not depressed by the 5-HT antagonists and will not be discussed in this paper. Areas of maximal inhibition were located by observing disappearance of decrebrate rigidity during stimulation. Histological examination of electrode placements showed them to be in the nucleus raphe magnus and nucleus reticularis gigantocellularis. The brain stem was stimulated at 300 cjsec for 30 msec with monophasic stimuli of 0.1 msec duration and 25 msec later a spinal cord monosynaptic reflex (MSR) was evoked by stimulating a peripheral nerve or dorsal root. The voltage applied to the brain stem was adjusted to produce approximately 50 7, inhibition of the MSR. This was usually less voltage than required to inhibit decerebrate rigidity (range 0.3-I V). The MSR was evoked by stimulating either the gastrocnemius-soleus nerve, the superior peroneal nerve or a whole dorsal root at a frequency of 1S/mm with voltage supramaximal for the MSR. Electrical activity of the appropriate L7 or Sr ventral root was amplified and displayed on an oscilloscope. The size of the conditioned and unconditioned MSR was quantified by measuring the potential obtained from 10 consecutive reflexes summed in a computer of average transients. Brain Research,
16 (1969) 296-300
297
SHORT COMMUNICATIONS LSD
CONTROL RF
+GS ..... GS"" G S .... RF ~GS
....
20
MR +GS
°'1
GS
rain
.....
.....
MR +GS
23
rain
30
rain
P
MR +P .... P
MR +P
P R F + P .....
RF .... +P
32
mln
Fig, 1, I_SD induced blockade of supraspinal inhibition. The column on the left shows control spinal reflexes evoked by stimulating gastrocnemius-soleus (GS) or superior peroneal (P) nerves with or without previous stimulation of the median raph6 (MR) or reticular formation (RF). Spinal reflexes in the right hand column were recorded after LSD (0.25 mg/kg) injection at the time indicated beneath each frame. The first and largest spike in each recording is the monosynaptic reflex (MSR). Three successive reflexes with conditioning are superimposed on 3 successive reflexes without conditioning. A square wave calibration pulse 0.5 mV in height and 0.5 msec in width precedes the MSR.
As shown in the left-side c o l u m n o f Fig. 1, stimulation o f either the reticular f o r m a t i o n or the m e d i a n raph6 inhibited the M S R subsequently evoked from either flexor ( s u p e r i o r peroneal) or extensor (gastrocnemius-soleus) m o t o n e u r o n s . As a consequence o f injecting LSD, c o n d i t i o n i n g by p r i o r stimulation o f the b r a i n stem no longer inhibited the M S R s , and in fact, facilitation o f b o t h flexor and extensor (extensor shown in right side o f Fig. 1) M S R s was evident d u r i n g the 25 rain p e r i o d i m m e d i a t e l y after a d m i n i s t e r i n g LSD. Thereafter and for the next 20-30 min stimulating the b r a i n stem exerted neither inhibitory nor facilitatory influence on spinal M S R s . A f t e r injecting L S D the u n c o n d i t i o n e d M S R o f flexor and extensor m o t o neurons was transiently depressed (extensor shown in Fig. 1) and then recovered to Brain Research, 16 (1969) 296-300
298
SHORT COMMUNICATIONS
20-
0
o~ 20.o
~0-
60-20
0
20 40 60 Time (rain)
80
I00
Fig. 2. Antagonism by methysergide(0,5 mg/kg injected at arrow) of descending bulbospinal inhibition of the monosynapticreflex(MSR). The MSR was evoked by stimulatingan L7 dorsal root and resorded from the correspondingventral root. For each point, the averageheightof 10 consecutive MSRs without and 10 consecutiveMSRs with previous stimulationof n. reticularis gigantocellularis was determinedand percent inhibition or facilitationcalculatedfrom the resultant values.
slightly above the pre-drug level (flexor shown in Fig. 1). In 5 of 5 experiments, LSD (0.25-0.5 mg/kg) induced antagonism of supraspinal inhibition lasting from 45 to 150 min. Increasing the strength of brain stem stimulation surmounted the LSD blockade of supraspinal inhibition in some experiments, whereas in others increasing stimulus strengh produced facilitation of the subsequently evoked MSR. LSD caused approximately a 25 mm fall in blood pressure (diastolic). In some experiments, pressure was immediately restored to the control level by injecting 6% dextran solution, doxapram or methamphetamine. No association was observed between the LSD induced drop in blood pressure and the block of bulbospinal inhibition. Other antiserotonin agents were tested in addition to LSD. Methysergide bimaleate (0.5-1 mg/kg) proved a potent antagonist of supraspinal inhibition in 10 of 10 experiments (e.g., Fig. 2). In addition to reducing reticular inhibition, methysergide also decreased the unconditioned MSR to about one-half its control value. These actions of methysergide were independent of each other, inasmuch as injection of the adrenergic blocker ethobutamoxane HC1 (0.25-1 mg/kg) comparably decreased the MSR without blocking supraspinal inhibition. After recovery of the MSR to the preethobutamoxane value, a subsequent injection of either ethobutamoxane or methysergide failed to depress the MSR (i.e., the MSR becomes tolerant to depression by either drug). However, methysergide still blocked bulbospinal inhibition even though it failed to depress the unconditioned MSR. Other serotonin antagonists were less effective than LSD and methysergide. Cinanserin HCI (SQ 10,643; 4-6 mg/kg) gave variable results ranging from complete disinhibition to no effect, BOL (2-bromo-LSD; 1-1.5 mg/kg) effected transient disinhibition, and cyproheptadine HC1 (5 mg/kg) was clearly ineffective. Marked hypotension precluded investigating higher doses of BOL. Serotonin antagonism is the outstanding pharmacological characteristic shared by methysergide and LSD. This suggests that the blocking action effected by LSD and
Brain Research, 16 (1969) 296-300
SHORT COMMUNICATIONS
299
methysergide on bulbospinal inhibition results from their common ability to antagonize 5-HT. Assuming serotonin released from the descending serotonergic neurons inhibits the motoneurons, this would be a likely site at which LSD and methysergide could block bulbospinal inhibition. This would be consistent with the microiontophoretic data showing 5-HT hyperpolarized motoneurons 9, but cannot be readily reconciled with the increased motoneuronal excitability following 5-HTP administration2,1°. Furthermore, we have observed the overall conduction velocity for the inhibitory system from brain stem to spinal cord to exceed l0 m/sec. This is rather fast for the thin and possibly unmyelinated 5-HT containing neurons. Another possible site of action of LSD and methysergide could be the brain stem, perhaps by removing a background excitatory input to inhibitory areas. This would be consistent with the observations of Aghajanian et al. that LSD produced cessation of spontaneous firing of neurons in the raph61. Further evidence for the brain stem as a possible site of action is the occurrence of 5-HT containing neuron terminals in the ventromedial pontine and medullary reticular formation 8. Studying a random sample, Bradley and Wolstencroft found some pontine and medullary neurons to be excited and others depressed by 5-HT 4. Although their site of action is uncertain, the finding that LSD and methysergide block supraspinal inhibition resulting either from stimulating reticular formation or the raph6 possibly suggests a common final pathway or a common facilitatory input involving a 5-HT link. From anatomical studies, Taber et al. postulated a participatory role for the raphd nuclei in brain stem functions currently attributed solely to the reticular formation ll. Interestingly, Couch and Salmoiraghi recently found that cells in the raphd which were excited by electrical stimulation of the lateral reticular formation were also excited by 5-HT 5. This study was supported in part by United States Public Health Service Grants R01-NB 05611 and T01-NB 5262. Department of Pharmacology, University of Illinois, College of Medicine, Chicago, Ill. 60612 (U.S.A.)
B. V. CLINESCHMIDT EDMUND G. ANDERSON
1 AGHAJAN1AN,G. K., FOOTF,W. E., AND SHEARD, M. H., Lysergic acid diethylamide: sensitive neuronal units in the midbrain raph6, Science, 161 (1968) 706-708. 2 ANDERSON, E. G., AND SHIBUYA, T., The effect of 5-hydroxytryptophan and i-tryptophan on spinal synaptic activity, J. Pharmacol. exp. Ther., 153 (1966) 352-360. 3 BANNA, N. R., AND ANDERSON, E. G., The effects of 5-hydroxytryptamine antagonists on spinal neuronal activity, J. Pharmacol. exp. Ther., 162 (1968) 319-325. 4 BRADLEY, P. B., AND WOLSTENCROFT,J. H., Actions of drugs on single neurones in the brainstem, Brit. med. Bull., 21 (1965) 15-18. 5 COUCH,J. R., AND SALMOIRAGHI, G. C., The responses of neurones in midline puns and lower midbrain to norepinephrine (NE) and serotonin (5-HT), Fed. Proc., 28 (1969) 443. 6 DAHLSTROM, A., AND FUXE, K., Evidence for the existence of monoamine-containing neurons in the central nervous system. I. Demonstration of monoamines in the cell bodies of brain stem neurons, Acta physiol, scand., 62 (1964) Suppl. 232. 7 DAHLSTR()M,A., AND FUXE, K., Evidence for the existence of monoamine neurons in the central Brain Research, 16 (1969) 296-300
300
8
9 10
11
SHORT COMMUNICATIONS
nervous system. I1. Experimentally induced changes in the intraneuronal amine levels of bulbospinal neuron systems, Acta physiol, scand., 64 (1965) Suppl. 247. FuxE, K., Evidence for the existence of monoamine neurons in the central nervous system. IV. Distribution of monoamine nerve terminals in the central nervous system, Acta physiol. stand., 64 (1965) Suppl 247. PHILLIS, J. W., TEBECIS, A. K., AND YORK, D. H., Depression of spinal motoneurones by noradrenaline, 5-hydroxytryptamine, and histamine, Earop. J. Pharmacok, 4 (1968) 471-475. SHIBUYA, T., AND ANDERSON, E. G., The influence of chronic cord transection on the effects of 5-hydroxytryptophan, l-tryptophan and pargyline on spinal neuronal activity, J, Pharmacol. exp. Ther., 164 (1968) 185-190. TABER, E., BRODAL, A., AND WALBERG, F., The raph6 nuclei of the brain stem m the cat. 1. Normal topography and cytoarchitecture and general discussioo, J. cornp. NeuroL, 114 (1960) 161-182.
(Accepted August 5th, 1969)
Brain Research, 16 (1969) 296-300
SHORT
acid diehylamide:
antagonism
of supraspinal
C‘OMM~INICATIONS
inhibition
of spinal
reflexes
The axons from serotonin (5_hydroxytryptamine, 5-HT) containing neurons whose somata are loca!ized in the caudal raphe nuclei of the brain stem descend ria the ventral and lateral funiculi of the spinal cord to terminate in the dorsal, lateral and ventral horns6s7. There is evidence that some of the 5-HT containing termina!s make close contact with motoneurones7. Although 5-HT applied microiontophoretically has been reported to hyperpolarize motoneurones 9, 5-hydroxytryptophan (5-HTP) injected into spinal cats increases motoneuronal excitability”,lO. an action readily reversed by 5-HT antagonist+. The significance ofthese observations is uncertain. In the present study we have attempted to directly activate the serotonin containing neurons of the bulbospinal system through stimulating electrodes placed in the caudal raphC nuclei, while observing the effects of stimulation on the segmental reflexes. For comparison stimulating electrodes were also placed in the ventromedial reticular formation. Since electrical stimulation could activate other descending pathways, it was anticipated that actions mediated by serotonergic neurons could be distinguished by their susceptibility to blockade by serotonin antagonists. Preliminary results of this study are reported herein. Cats of either sex weighing I .7-3.0 kg were decerebrated between the coliicuii under ether anesthesia. After discontinuing ether, two concentric bipolar electrodes with internal tip diameter less than 0.2 mm and tip separation I mm were positioned in the brain stem raphe and ventromedial reticular formation by stereotaxic technique. Using the Horsley-Clarke planes, co-ordinates for placing electrodes in the raphe and reticular formation were respectively: P7.5 mm (frontal plane), H-6 to -8 (horizontal plane), LO (sagittal plane) and P11, H-8 to -9, L1.5. Activation of the stimulating electrode while moving it in the horizontal plane through the raphe and reticular formation revealed both facilitatory and inhibitory effects on segmental reflexes. The facilitatory influences of brain stem stimulation were not depressed by the 5-HT antagonists and will not be discussed in this paper. Areas of maximal inhibition were located by observing disappearance of decrebrate rigidity during stimulation. Histological examination of electrode placements showed them to be in the nucleus raphe magnus and nucleus reticularis gigantocellularis. The brain stem was stimulated at 300 cjsec for 30 msec with monophasic stimuli of 0.1 msec duration and 25 msec later a spinal cord monosynaptic reflex (MSR) was evoked by stimulating a peripheral nerve or dorsal root. The voltage applied to the brain stem was adjusted to produce approximately 50 7, inhibition of the MSR. This was usually less voltage than required to inhibit decerebrate rigidity (range 0.3-I V). The MSR was evoked by stimulating either the gastrocnemius-soleus nerve, the superior peroneal nerve or a whole dorsal root at a frequency of 1S/mm with voltage supramaximal for the MSR. Electrical activity of the appropriate L7 or Sr ventral root was amplified and displayed on an oscilloscope. The size of the conditioned and unconditioned MSR was quantified by measuring the potential obtained from 10 consecutive reflexes summed in a computer of average transients. Brain Research,
16 (1969) 296-300
297
SHORT COMMUNICATIONS LSD
CONTROL RF
+GS ..... GS"" G S .... RF ~GS
....
20
MR +GS
°'1
GS
rain
.....
.....
MR +GS
23
rain
30
rain
P
MR +P .... P
MR +P
P R F + P .....
RF .... +P
32
mln
Fig, 1, I_SD induced blockade of supraspinal inhibition. The column on the left shows control spinal reflexes evoked by stimulating gastrocnemius-soleus (GS) or superior peroneal (P) nerves with or without previous stimulation of the median raph6 (MR) or reticular formation (RF). Spinal reflexes in the right hand column were recorded after LSD (0.25 mg/kg) injection at the time indicated beneath each frame. The first and largest spike in each recording is the monosynaptic reflex (MSR). Three successive reflexes with conditioning are superimposed on 3 successive reflexes without conditioning. A square wave calibration pulse 0.5 mV in height and 0.5 msec in width precedes the MSR.
As shown in the left-side c o l u m n o f Fig. 1, stimulation o f either the reticular f o r m a t i o n or the m e d i a n raph6 inhibited the M S R subsequently evoked from either flexor ( s u p e r i o r peroneal) or extensor (gastrocnemius-soleus) m o t o n e u r o n s . As a consequence o f injecting LSD, c o n d i t i o n i n g by p r i o r stimulation o f the b r a i n stem no longer inhibited the M S R s , and in fact, facilitation o f b o t h flexor and extensor (extensor shown in right side o f Fig. 1) M S R s was evident d u r i n g the 25 rain p e r i o d i m m e d i a t e l y after a d m i n i s t e r i n g LSD. Thereafter and for the next 20-30 min stimulating the b r a i n stem exerted neither inhibitory nor facilitatory influence on spinal M S R s . A f t e r injecting L S D the u n c o n d i t i o n e d M S R o f flexor and extensor m o t o neurons was transiently depressed (extensor shown in Fig. 1) and then recovered to Brain Research, 16 (1969) 296-300
298
SHORT COMMUNICATIONS
20-
0
o~ 20.o
~0-
60-20
0
20 40 60 Time (rain)
80
I00
Fig. 2. Antagonism by methysergide(0,5 mg/kg injected at arrow) of descending bulbospinal inhibition of the monosynapticreflex(MSR). The MSR was evoked by stimulatingan L7 dorsal root and resorded from the correspondingventral root. For each point, the averageheightof 10 consecutive MSRs without and 10 consecutiveMSRs with previous stimulationof n. reticularis gigantocellularis was determinedand percent inhibition or facilitationcalculatedfrom the resultant values.
slightly above the pre-drug level (flexor shown in Fig. 1). In 5 of 5 experiments, LSD (0.25-0.5 mg/kg) induced antagonism of supraspinal inhibition lasting from 45 to 150 min. Increasing the strength of brain stem stimulation surmounted the LSD blockade of supraspinal inhibition in some experiments, whereas in others increasing stimulus strengh produced facilitation of the subsequently evoked MSR. LSD caused approximately a 25 mm fall in blood pressure (diastolic). In some experiments, pressure was immediately restored to the control level by injecting 6% dextran solution, doxapram or methamphetamine. No association was observed between the LSD induced drop in blood pressure and the block of bulbospinal inhibition. Other antiserotonin agents were tested in addition to LSD. Methysergide bimaleate (0.5-1 mg/kg) proved a potent antagonist of supraspinal inhibition in 10 of 10 experiments (e.g., Fig. 2). In addition to reducing reticular inhibition, methysergide also decreased the unconditioned MSR to about one-half its control value. These actions of methysergide were independent of each other, inasmuch as injection of the adrenergic blocker ethobutamoxane HC1 (0.25-1 mg/kg) comparably decreased the MSR without blocking supraspinal inhibition. After recovery of the MSR to the preethobutamoxane value, a subsequent injection of either ethobutamoxane or methysergide failed to depress the MSR (i.e., the MSR becomes tolerant to depression by either drug). However, methysergide still blocked bulbospinal inhibition even though it failed to depress the unconditioned MSR. Other serotonin antagonists were less effective than LSD and methysergide. Cinanserin HCI (SQ 10,643; 4-6 mg/kg) gave variable results ranging from complete disinhibition to no effect, BOL (2-bromo-LSD; 1-1.5 mg/kg) effected transient disinhibition, and cyproheptadine HC1 (5 mg/kg) was clearly ineffective. Marked hypotension precluded investigating higher doses of BOL. Serotonin antagonism is the outstanding pharmacological characteristic shared by methysergide and LSD. This suggests that the blocking action effected by LSD and
Brain Research, 16 (1969) 296-300
SHORT COMMUNICATIONS
299
methysergide on bulbospinal inhibition results from their common ability to antagonize 5-HT. Assuming serotonin released from the descending serotonergic neurons inhibits the motoneurons, this would be a likely site at which LSD and methysergide could block bulbospinal inhibition. This would be consistent with the microiontophoretic data showing 5-HT hyperpolarized motoneurons 9, but cannot be readily reconciled with the increased motoneuronal excitability following 5-HTP administration2,1°. Furthermore, we have observed the overall conduction velocity for the inhibitory system from brain stem to spinal cord to exceed l0 m/sec. This is rather fast for the thin and possibly unmyelinated 5-HT containing neurons. Another possible site of action of LSD and methysergide could be the brain stem, perhaps by removing a background excitatory input to inhibitory areas. This would be consistent with the observations of Aghajanian et al. that LSD produced cessation of spontaneous firing of neurons in the raph61. Further evidence for the brain stem as a possible site of action is the occurrence of 5-HT containing neuron terminals in the ventromedial pontine and medullary reticular formation 8. Studying a random sample, Bradley and Wolstencroft found some pontine and medullary neurons to be excited and others depressed by 5-HT 4. Although their site of action is uncertain, the finding that LSD and methysergide block supraspinal inhibition resulting either from stimulating reticular formation or the raph6 possibly suggests a common final pathway or a common facilitatory input involving a 5-HT link. From anatomical studies, Taber et al. postulated a participatory role for the raphd nuclei in brain stem functions currently attributed solely to the reticular formation ll. Interestingly, Couch and Salmoiraghi recently found that cells in the raphd which were excited by electrical stimulation of the lateral reticular formation were also excited by 5-HT 5. This study was supported in part by United States Public Health Service Grants R01-NB 05611 and T01-NB 5262. Department of Pharmacology, University of Illinois, College of Medicine, Chicago, Ill. 60612 (U.S.A.)
B. V. CLINESCHMIDT EDMUND G. ANDERSON
1 AGHAJAN1AN,G. K., FOOTF,W. E., AND SHEARD, M. H., Lysergic acid diethylamide: sensitive neuronal units in the midbrain raph6, Science, 161 (1968) 706-708. 2 ANDERSON, E. G., AND SHIBUYA, T., The effect of 5-hydroxytryptophan and i-tryptophan on spinal synaptic activity, J. Pharmacol. exp. Ther., 153 (1966) 352-360. 3 BANNA, N. R., AND ANDERSON, E. G., The effects of 5-hydroxytryptamine antagonists on spinal neuronal activity, J. Pharmacol. exp. Ther., 162 (1968) 319-325. 4 BRADLEY, P. B., AND WOLSTENCROFT,J. H., Actions of drugs on single neurones in the brainstem, Brit. med. Bull., 21 (1965) 15-18. 5 COUCH,J. R., AND SALMOIRAGHI, G. C., The responses of neurones in midline puns and lower midbrain to norepinephrine (NE) and serotonin (5-HT), Fed. Proc., 28 (1969) 443. 6 DAHLSTROM, A., AND FUXE, K., Evidence for the existence of monoamine-containing neurons in the central nervous system. I. Demonstration of monoamines in the cell bodies of brain stem neurons, Acta physiol, scand., 62 (1964) Suppl. 232. 7 DAHLSTR()M,A., AND FUXE, K., Evidence for the existence of monoamine neurons in the central Brain Research, 16 (1969) 296-300
300
8
9 10
11
SHORT COMMUNICATIONS
nervous system. I1. Experimentally induced changes in the intraneuronal amine levels of bulbospinal neuron systems, Acta physiol, scand., 64 (1965) Suppl. 247. FuxE, K., Evidence for the existence of monoamine neurons in the central nervous system. IV. Distribution of monoamine nerve terminals in the central nervous system, Acta physiol. stand., 64 (1965) Suppl 247. PHILLIS, J. W., TEBECIS, A. K., AND YORK, D. H., Depression of spinal motoneurones by noradrenaline, 5-hydroxytryptamine, and histamine, Earop. J. Pharmacok, 4 (1968) 471-475. SHIBUYA, T., AND ANDERSON, E. G., The influence of chronic cord transection on the effects of 5-hydroxytryptophan, l-tryptophan and pargyline on spinal neuronal activity, J, Pharmacol. exp. Ther., 164 (1968) 185-190. TABER, E., BRODAL, A., AND WALBERG, F., The raph6 nuclei of the brain stem m the cat. 1. Normal topography and cytoarchitecture and general discussioo, J. cornp. NeuroL, 114 (1960) 161-182.
(Accepted August 5th, 1969)
Brain Research, 16 (1969) 296-300