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A brain stem cholinergic system activated by vestibular volleys
Neuropharmacology, 1970, 9, 391-394 Pergamon Press. Printed in Gt. Britain.
PRELIMINARY NOTE A BRAIN STEM CHOLINERGIC SYSTEM ACTIVATED BY VESTIBULAR VOLLEYS* C. D. BARNES~"and O. POMPEIANO Istituto di Fisiologia Umana, Cattedra II, UniversitY.di Pisa, Italia (Accepted 15 January 1970) Summary--In decerebrate cats, repetitive stimulation of the VIIIth cranial nerve elicits two effects in the lumbar cord which have the same time course. They are: facilitation of the ipsilateral extensor monosynaptic reflex and dorsal root potential. The dorsal root potential is due to primary afferent depolarization in the group I afferents from both extensor and flexor muscles of the ipsilateral hindlimb as well as in cutaneous afferents. The administration of physostigmine results in the VIIIth nerve-produced facilitation of the gastrocnemius-soleus monosynaptic reflex being markedly reduced or reversed to inhibition. Similarly, the dorsal root potential recorded from Ln is reduced in amplitude. Both of these effects of physostigmine are reversed by atropine. MATSUZAK! (MATSUZAKI et al., 1967; MATSUZAKI, 1968) has demonstrated that the administration of physostigmine in decerebrate cats produces some of the concomitants of paradoxical sleep. This phenomenon includes tonic suppression of the neck muscular activity and phasic events characterized by the electrical activity of the pontine reticular formation, and rapid eye movements. All of these reactions appear indistinguishable from spontaneously-occurring paradoxical sleep which may occur in decerebrate preparations (MATSUZAKI, 1968). The time course of this phenomenon and its reversibility by atropine is similar to that found in a spino-bulbo-spinal reflex inhibition after the administration of physostigmine (BARNES, 1970). While the abolition of the postural tonus associated with paradoxical sleep is due to activation of the suppressive areas of the brainstem leading to postsynaptic inhibition of the spinal motoneurones (GASSELL et al., 1965), the rapid eye movements are dependent on vestibular nuclei. This dependency has been demonstrated by the disappearance of these reactions after vestibular lesion (POMPEIANOand MORRISON, 1965 ; MORRISONand POMPEIANO, 1966) and by the relationship in time with high frequency bursts of activity in the medial and descending vestibular nuclei (BIZZl et al., 1964). The persistence of the rhythmic pontine activity after vestibular lesion indicates the existence of a pathway from the pons which initiates the vestibular discharge, thus leading to the R E M episodes. The vestibular lesion, however, reduces the percentage of time spent by the animal in paradoxical sleep (POMPEIANO and MORRISON, 1966). This indicates that some part of the efferent vestibular discharge is fed into the pontine center thus facilitating the appearance of the tonic events typical of this *This investigation was supported in part by Public Health Service Research Grants NB 07685 and NB 07834. tRecipient of Career Development Award NB 34986. Present address: Department of Anatomy and Physiology, Indiana University, Bloomington, Indiana, 47401, U.S.A. 391
C. D. BARNESand O. POMPEI'ANO
392
phase of sleep. Repetitive stimulation of the VIIIth cranial nerve results in enhancement of the extensor monosynaptic reflex (Cook et al., 1968). The present study was undertaken to determine if a cholinergic system affects the spinal cord response to VIIIth nerve stimulation. METHODS
Thirty unanesthetized cats, decerebrate at the precollicular levels, were used. The initial surgery was done under ether anesthesia. After decerebration the ether was discontinued. Recordings were initiated no sooner than two hours after the cessation of anesthesia. The left VIIIth nerve was stimulated intradurally with a bipolar concentric electrode with rectangular pulses 0.2 msec in duration, at a frequency of 500 c/s for 200 msec. A pair of recording electrodes with an interelectrode distance of about 0.5 m m was placed sterotaxically in the ascending medial longitudinal fasciculus. Stimulus intensities were expressed in multiples of the threshold voltage (T) required to evoke the monosynaptic discharge of the second order vestibular neurons. Both hindlimbs were denervated, with the exception of the left gastrocnemius-soleus muscles, and the ventral roots L6-S 2 were cut. Monosynaptic reflexes elicited from stimulation of the gastrocnemius-soleus nerve with rectangular pulses 0.05 msec in duration were recorded from the ventral root of L 7 or Sv The gastrocnemiussoleus monosynaptic reflex was tested at 25 msec intervals from the beginning of VIIIth nerve stimulation for the first 175 msec and at 100 msec intervals for the next 800 msec. Dorsal root potentials were recorded from the most caudal rootlet of L e with a d.c. preamplifier. Drugs were administered through a catheter in the femoral vein.
RESULTS
Stimulation of the VIIIth cranial nerve with a stimulus intensity of more than 1T results in a facilitation of the gastrocnemius-soleus monosynaptic reflex (Fig. 1). After the administration of physostigmine (0.1 mg/kg), an anticholinesterase, the facilitatory pattern was reduced markedly and in many cases transformed to inhibition. After VIIIth nerve % 150
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FIG. 1. Changes in amplitude of the gastrocnemius-soleus monosynaptic reflex evoked by a 200 msec tetanus of 500/sec applied to the ipsilateral VIIIth nerve at an intensity 3 times threshold for an ascending medial longitudinal fasciculus response. Each point represents the average from six cats with 5 or more measurements at each interval from each. Filled circles: Pre-drug control. Open circles: 10 min after 0.1 mg/kg physostigmine. Triangles: 10 rain after 1 mg/kg atropine in the previously physostigmine-treated cats.
A brain stem cholinergic system activated by vestibular volleys
393
stimulation the gastrocnemius-soleus monosynaptic reflex returned to control amplitude (Table 1). Physostigmine also reduced the amplitude of the dorsal root potential by VIIIth nerve stimulation. The increase in dorsal root potential amplitude with increasing stimulus strength is of the same form after physostigmine as before but the post-drug potentials have only half the amplitude of their control equivalents. Though the effects persisted in varying degrees for more than one hour, they could be reversed within seconds by a 1 mg/kg dose TABLE I.
M E A N AND STANDARD ERROR OF THE GASTROCNEMIUS--SOLEUS MONO-
SYNAPTIC REFLEX FOLLOWI NG THE ONSET OF
VIIITH
NERVE TETANUS IN THE SIX
ANIMALS IN F I G . 1
Time (msec)
Control
Physostigmine
Atropine sulfate
0 25 50 75 100 125 150 175 250 350 450 550 650 750 850 950
100 153,19 1 3 2 i 13 145.55 154±6 163±6 131 ± 14 1 1 3 i 15 84,3 73±4 802_ 1 83±3 89,6 90,6 89,7 92--4
100 117-5=19 76___29 89±29 72±28 77±31 70,28 75±31 8 7 ± 12 8 5 ± 15 824-14 84,6 8 3 ± I0 82,8 91 ± 9 94± I
100 142±8 1 2 3 i 14 154±41 161±42 158±38 143,47 123,46 115zc20 99zE7 97-- 11 93±12 91 ± 7 98,8 101 i 4 103 ± 7
of atropine sulfate, an anticholinergic drug. Atropine sulfate returned the physostigmine depressed reflex to control levels during the VIIIth nerve stimulation but usually to significantly above control values for several hundred msec. after the conditioning stimulus. Given by itself, however, atropine sulfate had no effect. To control against the effects being of peripheral origin three animals were run using neostigmine and atropine methylnitrate, agents which only poorly cross the blood-brain barrier. The effect was not produced by neostigmine (0.1 mg/kg i.v.) and the physostigmine produced inhibition was not reversed by atropine methylnitrate (1 mg/kg i.v.). DISCUSSION
In normal decerebrate cats, the monosynaptic reflex facilitation from VIIIth nerve stimulation is associated with primary afferent depolarization in group I muscle afferents (Cook et al., 1968). The finding indicates that the reduced postsynaptic efficacy of the orthodromic volleys due to primary afferent depolarization is overwhelmed by the large interneuronal discharge induced by vestibular volleys. The possibility that the physostigmine effect is due to potentiation of the presynaptic mechanism is disproved by the fact that the VIIIth nerve-produced dorsal root potential is actually depressed by the drug. One cannot dismiss the possibility that the depression of the monosynaptic reflex induced by the physostigmine is a result of an upset in the balance between VIIIth nerve-induced presynaptic inhibition and postsynaptic facilitation favoring the inhibition, however. An
394
C. D. BARNESand O. POMPEIANO
alternative e x p l a n a t i o n might be that physostigmine opens a pathway for the vestibular volleys to activate the descending pathway responsible for paradoxical sleep-induced postsynaptic inhibition.
REFERENCES BARNES, C. D. (1970). Cholinergic properties of spino-bulbo-spinalreflex inhibition. Neuropharmacology 9: 185-190. BlzzI, E., POMPEIANO,O. and SOMOGYI,I. (1964), Spontaneous activity of single vestibular neurons of unrestrained cats during sleep and wakeflflness. Archs itaL Biol. 102: 308-330. COOK,W. A., JR., CANO~ANO,A. and POMPEIANO,O. (1968). Vestibular influences on primary afferents in the spinal cord. Pflugers Arch. 299: 334-338. GASSEC, M. M., MARCH~AFAVA,P. L. and POMPEIANO,O. (1965). An analysis of the supraspinal influences acting on motoneurons during sleep in the unrestrained cat. Archs itaL Biol. 103: 25~[4. MATSUZAKf,M. (1968). Differential effects of Na-butyrate and physostigmine on brainstem activities of para-sleep. Brain Res. 11 : 251-255. MATSUZAKI,M., OKADA,Y. and SHUrO, S. (1967), Cholinergic actions related to paradoxical sleep induction in the mesencephalic cat. Experientia 23: 1029-1030. MORR~SON, A. R. and POMPEIANO,O. (1966). Vestibular influences during sleep II. Effects of vestibular lesions on the pyramidal discharge during desynchronized sleep. Archs itaL Biol. 104: 214-230. POMPEtANO, O. and MORRISON,A. R. (1965). Vestibular influences during sleep~I. Abolition of the rapid eye movements of desynchronized sleep following vestibular lesions. Archs itaL Biol. 103: 569-595. POMPEIANO,O. and MORRISON,A. R. (1966). Vestibular influences during sleep--IV. Functional relations between vestibular nuclei and lateral geniculate nucleus during desynchronized sleep. Archs ital. Biol. 104: 425~[58.
PRELIMINARY NOTE A BRAIN STEM CHOLINERGIC SYSTEM ACTIVATED BY VESTIBULAR VOLLEYS* C. D. BARNES~"and O. POMPEIANO Istituto di Fisiologia Umana, Cattedra II, UniversitY.di Pisa, Italia (Accepted 15 January 1970) Summary--In decerebrate cats, repetitive stimulation of the VIIIth cranial nerve elicits two effects in the lumbar cord which have the same time course. They are: facilitation of the ipsilateral extensor monosynaptic reflex and dorsal root potential. The dorsal root potential is due to primary afferent depolarization in the group I afferents from both extensor and flexor muscles of the ipsilateral hindlimb as well as in cutaneous afferents. The administration of physostigmine results in the VIIIth nerve-produced facilitation of the gastrocnemius-soleus monosynaptic reflex being markedly reduced or reversed to inhibition. Similarly, the dorsal root potential recorded from Ln is reduced in amplitude. Both of these effects of physostigmine are reversed by atropine. MATSUZAK! (MATSUZAKI et al., 1967; MATSUZAKI, 1968) has demonstrated that the administration of physostigmine in decerebrate cats produces some of the concomitants of paradoxical sleep. This phenomenon includes tonic suppression of the neck muscular activity and phasic events characterized by the electrical activity of the pontine reticular formation, and rapid eye movements. All of these reactions appear indistinguishable from spontaneously-occurring paradoxical sleep which may occur in decerebrate preparations (MATSUZAKI, 1968). The time course of this phenomenon and its reversibility by atropine is similar to that found in a spino-bulbo-spinal reflex inhibition after the administration of physostigmine (BARNES, 1970). While the abolition of the postural tonus associated with paradoxical sleep is due to activation of the suppressive areas of the brainstem leading to postsynaptic inhibition of the spinal motoneurones (GASSELL et al., 1965), the rapid eye movements are dependent on vestibular nuclei. This dependency has been demonstrated by the disappearance of these reactions after vestibular lesion (POMPEIANOand MORRISON, 1965 ; MORRISONand POMPEIANO, 1966) and by the relationship in time with high frequency bursts of activity in the medial and descending vestibular nuclei (BIZZl et al., 1964). The persistence of the rhythmic pontine activity after vestibular lesion indicates the existence of a pathway from the pons which initiates the vestibular discharge, thus leading to the R E M episodes. The vestibular lesion, however, reduces the percentage of time spent by the animal in paradoxical sleep (POMPEIANO and MORRISON, 1966). This indicates that some part of the efferent vestibular discharge is fed into the pontine center thus facilitating the appearance of the tonic events typical of this *This investigation was supported in part by Public Health Service Research Grants NB 07685 and NB 07834. tRecipient of Career Development Award NB 34986. Present address: Department of Anatomy and Physiology, Indiana University, Bloomington, Indiana, 47401, U.S.A. 391
C. D. BARNESand O. POMPEI'ANO
392
phase of sleep. Repetitive stimulation of the VIIIth cranial nerve results in enhancement of the extensor monosynaptic reflex (Cook et al., 1968). The present study was undertaken to determine if a cholinergic system affects the spinal cord response to VIIIth nerve stimulation. METHODS
Thirty unanesthetized cats, decerebrate at the precollicular levels, were used. The initial surgery was done under ether anesthesia. After decerebration the ether was discontinued. Recordings were initiated no sooner than two hours after the cessation of anesthesia. The left VIIIth nerve was stimulated intradurally with a bipolar concentric electrode with rectangular pulses 0.2 msec in duration, at a frequency of 500 c/s for 200 msec. A pair of recording electrodes with an interelectrode distance of about 0.5 m m was placed sterotaxically in the ascending medial longitudinal fasciculus. Stimulus intensities were expressed in multiples of the threshold voltage (T) required to evoke the monosynaptic discharge of the second order vestibular neurons. Both hindlimbs were denervated, with the exception of the left gastrocnemius-soleus muscles, and the ventral roots L6-S 2 were cut. Monosynaptic reflexes elicited from stimulation of the gastrocnemius-soleus nerve with rectangular pulses 0.05 msec in duration were recorded from the ventral root of L 7 or Sv The gastrocnemiussoleus monosynaptic reflex was tested at 25 msec intervals from the beginning of VIIIth nerve stimulation for the first 175 msec and at 100 msec intervals for the next 800 msec. Dorsal root potentials were recorded from the most caudal rootlet of L e with a d.c. preamplifier. Drugs were administered through a catheter in the femoral vein.
RESULTS
Stimulation of the VIIIth cranial nerve with a stimulus intensity of more than 1T results in a facilitation of the gastrocnemius-soleus monosynaptic reflex (Fig. 1). After the administration of physostigmine (0.1 mg/kg), an anticholinesterase, the facilitatory pattern was reduced markedly and in many cases transformed to inhibition. After VIIIth nerve % 150
A
IOO
.
......
o
o
~
5o 0
200
I
I
I
i
400
600
800
I000 i'~$e¢
FIG. 1. Changes in amplitude of the gastrocnemius-soleus monosynaptic reflex evoked by a 200 msec tetanus of 500/sec applied to the ipsilateral VIIIth nerve at an intensity 3 times threshold for an ascending medial longitudinal fasciculus response. Each point represents the average from six cats with 5 or more measurements at each interval from each. Filled circles: Pre-drug control. Open circles: 10 min after 0.1 mg/kg physostigmine. Triangles: 10 rain after 1 mg/kg atropine in the previously physostigmine-treated cats.
A brain stem cholinergic system activated by vestibular volleys
393
stimulation the gastrocnemius-soleus monosynaptic reflex returned to control amplitude (Table 1). Physostigmine also reduced the amplitude of the dorsal root potential by VIIIth nerve stimulation. The increase in dorsal root potential amplitude with increasing stimulus strength is of the same form after physostigmine as before but the post-drug potentials have only half the amplitude of their control equivalents. Though the effects persisted in varying degrees for more than one hour, they could be reversed within seconds by a 1 mg/kg dose TABLE I.
M E A N AND STANDARD ERROR OF THE GASTROCNEMIUS--SOLEUS MONO-
SYNAPTIC REFLEX FOLLOWI NG THE ONSET OF
VIIITH
NERVE TETANUS IN THE SIX
ANIMALS IN F I G . 1
Time (msec)
Control
Physostigmine
Atropine sulfate
0 25 50 75 100 125 150 175 250 350 450 550 650 750 850 950
100 153,19 1 3 2 i 13 145.55 154±6 163±6 131 ± 14 1 1 3 i 15 84,3 73±4 802_ 1 83±3 89,6 90,6 89,7 92--4
100 117-5=19 76___29 89±29 72±28 77±31 70,28 75±31 8 7 ± 12 8 5 ± 15 824-14 84,6 8 3 ± I0 82,8 91 ± 9 94± I
100 142±8 1 2 3 i 14 154±41 161±42 158±38 143,47 123,46 115zc20 99zE7 97-- 11 93±12 91 ± 7 98,8 101 i 4 103 ± 7
of atropine sulfate, an anticholinergic drug. Atropine sulfate returned the physostigmine depressed reflex to control levels during the VIIIth nerve stimulation but usually to significantly above control values for several hundred msec. after the conditioning stimulus. Given by itself, however, atropine sulfate had no effect. To control against the effects being of peripheral origin three animals were run using neostigmine and atropine methylnitrate, agents which only poorly cross the blood-brain barrier. The effect was not produced by neostigmine (0.1 mg/kg i.v.) and the physostigmine produced inhibition was not reversed by atropine methylnitrate (1 mg/kg i.v.). DISCUSSION
In normal decerebrate cats, the monosynaptic reflex facilitation from VIIIth nerve stimulation is associated with primary afferent depolarization in group I muscle afferents (Cook et al., 1968). The finding indicates that the reduced postsynaptic efficacy of the orthodromic volleys due to primary afferent depolarization is overwhelmed by the large interneuronal discharge induced by vestibular volleys. The possibility that the physostigmine effect is due to potentiation of the presynaptic mechanism is disproved by the fact that the VIIIth nerve-produced dorsal root potential is actually depressed by the drug. One cannot dismiss the possibility that the depression of the monosynaptic reflex induced by the physostigmine is a result of an upset in the balance between VIIIth nerve-induced presynaptic inhibition and postsynaptic facilitation favoring the inhibition, however. An
394
C. D. BARNESand O. POMPEIANO
alternative e x p l a n a t i o n might be that physostigmine opens a pathway for the vestibular volleys to activate the descending pathway responsible for paradoxical sleep-induced postsynaptic inhibition.
REFERENCES BARNES, C. D. (1970). Cholinergic properties of spino-bulbo-spinalreflex inhibition. Neuropharmacology 9: 185-190. BlzzI, E., POMPEIANO,O. and SOMOGYI,I. (1964), Spontaneous activity of single vestibular neurons of unrestrained cats during sleep and wakeflflness. Archs itaL Biol. 102: 308-330. COOK,W. A., JR., CANO~ANO,A. and POMPEIANO,O. (1968). Vestibular influences on primary afferents in the spinal cord. Pflugers Arch. 299: 334-338. GASSEC, M. M., MARCH~AFAVA,P. L. and POMPEIANO,O. (1965). An analysis of the supraspinal influences acting on motoneurons during sleep in the unrestrained cat. Archs itaL Biol. 103: 25~[4. MATSUZAKf,M. (1968). Differential effects of Na-butyrate and physostigmine on brainstem activities of para-sleep. Brain Res. 11 : 251-255. MATSUZAKI,M., OKADA,Y. and SHUrO, S. (1967), Cholinergic actions related to paradoxical sleep induction in the mesencephalic cat. Experientia 23: 1029-1030. MORR~SON, A. R. and POMPEIANO,O. (1966). Vestibular influences during sleep II. Effects of vestibular lesions on the pyramidal discharge during desynchronized sleep. Archs itaL Biol. 104: 214-230. POMPEtANO, O. and MORRISON,A. R. (1965). Vestibular influences during sleep~I. Abolition of the rapid eye movements of desynchronized sleep following vestibular lesions. Archs itaL Biol. 103: 569-595. POMPEIANO,O. and MORRISON,A. R. (1966). Vestibular influences during sleep--IV. Functional relations between vestibular nuclei and lateral geniculate nucleus during desynchronized sleep. Archs ital. Biol. 104: 425~[58.