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Brain stem neurons governing the discharges of sympathetic nerves
Journal of the Autonomic Nervous System, 5 (1982) 55-61
55
Elsevier Biomedical Press
Brain stem neurons governing the discharges of sympathetic nerves Gerard L. Gebber and Susan M. Barman Department of Pharmacology and Toxicology, Michigan State University, Eus't Lansing, MI 48824 (U.S.A.) (Received December 20th, 1980) (Accepted August 1st, 1981 )
Key words: brain stem oscillator--medullary neurons--spike-triggered averaging-sympathetic nerve rhythms
Abstract
Recent work from our laboratory revealed that the brain stem is inherently capable of generating a 2-6 cycles/s rhythm in sympathetic nerve discharge (SND) of the baroreceptor denervated cat. It is believed that this rhythm is representative of the fundamental organization of those brain stem networks responsible for the background discharges in sympathetic nerves. As a consequence, an attempt was made to identify brain stem neurons with activity patterns related to the 2-6 cycles/s rhythm in inferior cardiac SND of the baroreceptor denervated cat. Specifically, the relationships between the spontaneous discharges of single brain stem neurons and the inferior cardiac nerve were analyzed with the technique of spike-triggered averaging. Neurons whose discharges were synchronized either to the rising or falling phase of inferior cardiac SND were located in the classic pressor region of the lateral medullary reticular formation. These neurons may comprise or receive inputs from the oscillator responsible for the 2-6 cycles/s rhythm in SND. In addition, neurons which reset the 2-6 cycles/s rhythm were identified. These neurons may provide inputs to the 2-6 cycles/s oscillator in the brain stem.
Introduction
A basic topic in research on the neurophysiology of preganglionic sympathetic neurons concerns the manner in which the background discharges of these cells are generated. The problem was brought into focus when Bernard [6] demonstrated that transection of the cervical spinal cord led to a pronounced fall in blood pressure. This simple yet extremely important experiment demonstrated that sympathetic nerves which innervate the cardiovascular system are tonically active and that this 0165-1838/82/0000-0000/$02.75 © 1982 Elsevier Biomedical Press
56 activity arises primarily in neuronal circuits located in the brain. Subsequent w o r k performed by Dittmar [9] and Owsjannikow [15] in the 1870s implicated the lower brain st.em as the region responsible for the background discharges in sympathetic nerves. Our comprehension of the mechanisms and circuitry involved in generating sympathetic nerve discharge (SND), however, remains largely incomplete. The purpose of the current paper is to summarize the results of recent experiments on this topic which have been performed in our laboratory.
The 2-6 cycles/s rhythm in SND--an indicator of the fundamental organization of the brain stem sympathetic generator An important characteristic of the discharges of sympathetic nerve bundles is rhythmicity. That is, the central nervous system is capable of synchronizing the discharges of populations of sympathetic neurons in a periodic fashion. One of the prominent rhythms in SND is the cardiac-related periodicity. As first demonstrated by Adrian et al. [1] and Bronk et al. [7], the discharges of pre- and postganglionic nerves are synchronized into bursts (i.e. slow waves as we record them with a preamplifier bandpass of 1-1000 Hz) which are locked in a 1:1 relation to the cardiac cycle. A typical example of this rhythm in the discharges of the splanchnic sympathetic nerve of the cat is shown in panels IA and IB of Fig. 1. Until recently, the cardiac-related rhythm in SND was considered to result as a simple consequence of the baroreceptor reflexes [8,13]. That is, this rhythm was believed to arise from variations in the level of baroreceptor-induced inhibition of the tonic central drive to sympathetic nerves during each cardiac cycle. Results obtained in our laboratory since 1975 have led us to revise the traditional view for the generation of the cardiac-related rhythm in SND. We have proposed
A 150r
I Baro. Intact B .
SND 11 Baro. Den.
B
,,5 A /!
SND J
__J
Fig. I. Splanchnic SND before and after baroreceptor denervation. I: baroreceptor reflexes intact. A, oscilloscopic traces of blood pressure in mm Hg (top) and splanchnic SND (bottom). B, R-wave-triggered averages of blood pressure and SND (64 trials). Address bin was I ms. II: A and B, same, but after baroreceptor denervation. Horizontal calibration is 200 ms. Vertical calibration for SND is 100 p,V.
57 that this rhythm is intrinsic to the central nervous system and that the function of the baroreceptor reflexes is to entrain the rhythm to the cardiac cycle. The experiments on which this proposal is based appear in our original articles [10,12,16] and are summarized in a recent review [11]. One of our observations is shown in panel IIA of Fig. 1. We found that a rhythm with a frequency (2-6 cycles/s) close to that of the heart rate persisted in SND after bilateral section of the carotid sinus, aortic depressor and vagus nerves (i.e. baroreceptor denervation). This rhythm could not be attributed to cardiac-related activity in other visceral afferents because the phase relations between SND and the cardiac cycle were completely disrupted after bilateral section of the carotid sinus, aortic depressor and vagus nerves. In this regard, note that the R-wave-triggered average of splanchnic SND approached a straight line after baroreceptor denervation (panel liB of Fig. 1). More recently, 'we demonstrated that the 2-6 cycles/s rhythm in SND of the baroreceptor denervated cat is not generated in the spinal cord [14] or forebrain [4]. Thus, in the absence of evidence for 2-6 cycles/s activity in non-baroreceptor afferents, it would appear that the rhythm is representative of the fundamental organization of those brain stem networks responsible for the background discharges in sympathetic nerves. Two possibilities immediately come to mind concerning the neural circuitry responsible for the 2-6 cycles/s rhythm in SND. First, the rhythm might be generated by a group of brain stem neurons with inherent pacemaker activity. Second, the rhythm might be generated as the consequence of interactions within and between different neuronal networks. For instance, the rising phase of the 2-6 cycles/s slow wave of SND (see Fig. 1) might result from the synchronous discharges of one group of brain stem neurons while the falling phase of the slow wave might be due to active inhibition produced by the synchronous activation of a second group of brain stem neurons. In view of these possibilities, we became interested in identifying those neuronal types which constitute the brain stem sympathetic generator.
Medullary neurons with activity patterns temporally related to inferior cardiac SND Spike-triggered averaging was used to study the relationships between the naturally occurring discharges of single brain stem neurons and the inferior cardiac sympathetic nerve in the baroreceptor denervated cat. The details of this method have been presented in a previous report from our laboratory [5]. Briefly, the spontaneous discharges (extracellularly recorded) of the brain stem unit were used to trigger individual sweeps of a computer so as to construct an average of accompanying changes in inferior cardiac SND. The spike was used as a midsignal trigger so as to provide information on whether brain stem unit activity was temporally related to preceding as well as to subsequently occurring inferior cardiac SND. The relationships between brain stem unit discharges and inferior cardiac nerve activity were verified in two ways. First, the spike-triggered average of SND was compared to the average of SND derived from a random pulse series with approximately the same frequency as the unit spike series. Deflections in the spike-triggered average which
5~ 2
Uni~l SND A/~t/~"!':,l/~ )
"\ I \ I
-5OO
0 Interval, ms 1.0
153- 3
u
I
÷500
Un~l* SND
C
0
1Y
i
i
/
0 Interval, ms 750
Rondompulse* SND I
0
Interval, s 10
I
-750
I
I
I
0
I
I
+750
Interval, ms
Fig. 2. Relationship between the discharges of a brain stern neuron and the inferior cardiac sympathetic nerve in a baroreceptor dene~'ated cat. A: midsignal spike-triggered average of inferior cardiac SND (1200 trials). Spike trigger occurred at time0. Address bin was I ms. Vertical calibration is 5#V. B: autocorrelogram of unit discharges. Address bin was 12 ms. Analysis based on 2432 spikes. C: autocorrelogram of inferior cardiac SND. Analysis time was 40 s. Address bin was It) ms. Fig. 3. Synchronization of medulla~, unit discharges to a point near the beginning of the falling phase of the 2-6 cycles/s slow wave in inferior cardiac SND of the baroreceptor denervated cat. Top trace is midsignal spike-triggered average of inferior cardiac SND: spike trigger occurred at time 0. Bottom trace is average of SND constructed with triggers from a random pulse train. Number of trials for each average was 860. Address bin was 1.5 ms. Vertical calibration is 5 ~,V.
e x c e e d e d t h o s e in the ' d u m m y ' a v e r a g e by at least a f a c t o r of two w e r e c o n s i d e r e d to r e p r e s e n t c h a n g e s in i n f e r i o r c a r d i a c n e r v e d i s c h a r g e t e m p o r a l l y r e l a t e d to the s p o n t a n e o u s a c t i v i t y of the b r a i n s t e m n e u r o n . S e c o n d , the d a t a w e r e f r a c t i o n a t e d i n t o several c o n t i n u o u s s e g m e n t s c o n t a i n i n g e q u a l n u m b e r s o f unit spikes. T h e d i s c h a r g e s of the b r a i n s t e m n e u r o n a n d i n f e r i o r c a r d i a c n e r v e w e r e c o n s i d e r e d t e m p o r a l l y r e l a t e d if a v e r a g e s o f S N D c o n s t r u c t e d f r o m successive d a t a s e g m e n t s w e r e essentially i d e n t i c a l in c o n t o u r a n d a m p l i t u d e . A strip ( 1 . 5 - 3 . 5 m m lateral to the m i d l i n e ) of the r e t i c u l a r f o r m a t i o n f r o m the level of the o b e x to the p o n t o m e d u l l a r y b o r d e r was e x p l o r e d for n e u r o n s w i t h a c t i v i t y p a t t e r n s r e l a t e d to t h o s e in the i n f e r i o r c a r d i a c n e r v e of b a r o r e c e p t o r d e n e r v a t e d cats. T h i s strip falls w i t h i n the classic m e d u l l a r y p r e s s o r r e g i o n [2,3]. T h e d i s c h a r g e s o f a p p r o x i m a t e l y 30% of the n e u r o n s s a m p l e d w e r e t e m p o r a l l y r e l a t e d to i n f e r i o r c a r d i a c S N D . T h e m a j o r i t y o f these 65 n e u r o n s w e r e l o c a t e d in n u c l e u s reticularis p a r v o c e l l u l a r i s a n d n u c l e u s r e t i c u l a r i s ventralis. T h e m i d s i g n a l spiket r i g g e r e d a v e r a g e s o f i n f e r i o r c a r d i a c S N D for units w i t h s y m p a t h e t i c - r e l a t e d a c t i v i t y fell i n t o t w o categories. A n e x a m p l e o f the first c a t e g o r y is s h o w n in Fig. 2A. T i m e 0
59 in the average of inferior cardiac SND marks the occurrence of the spike of the brain stem neuron. Thus, the portion of the average to the left of time 0 is for SND which preceded unit activity while that to the right of time0 is for SND which followed unit activity. A distinguishing characteristic of the first category of averages of SND is the appearance of a rhythm in the 2-6 cycles/s range both in activity which preceded and followed the midsignal spike trigger. The period of this rhythm was the same as that which appeared in the autocorrelograms of the discharges of the brain stem unit (Fig. 2B) and inferior cardiac nerve (Fig. 2C). Identical periodicities in the autocorrelograms and midsignal spike-triggered average of SND (a form of crosscorrelation analysis) clearly indicate that the 2-6 cycles/s rhythm in unit and inferior cardiac nerve activities were locked to each other. Thus, it is likely that neurons from which such averages of inferior cardiac nerve activity (Fig. 2A) were derived either comprised or received inputs from the oscillator responsible for the 2-6 cycles/s slow wave in SND of the baroreceptor denervated cat. The data in Figs. 2 and 3 support the view that the 2-6 cycles/s slow wave in inferior cardiac SND results as the consequence of alternations in the discharges of two groups of brain stem neurons. The position of time 0 on the 2-6 cycles/s slow wave in the midsignal spike-triggered average of SND is indicative of the point of highest probability of occurrence of brain stem unit discharge. In this respect, we found brain stem neurons whose highest probability of discharge occurred near the beginning of either the rising (Fig. 2A) or falling (Fig. 3) phase of the 2-6 cycles/s slow wave in SND. These observations raise the tantalizing possibility that the 2-6 cycles/s rhythm in SND of the baroreceptor denervated cat is generated by a brain stem oscillator which contains pairs of mutually inhibitory neurons. This hypothesis should be tested in future studies with intracellular recordings a n d / o r unit-~ unit cross-correlation analysis. Electrical stimuli applied through the microelectrode at sites of sympathetic-related unit activity usually elicited changes in inferior cardiac SND similar to those observed in the corresponding spike-triggered average. Typical examples are shown in Fig. 4. Note should be taken of the similarities in contour and time-to-peak effect in corresponding spike-triggered and poststimulus averages of inferior cardiac nerve activity. These results further support the view that neurons whose discharges were used to construct averages of inferior cardiac nerve activity of the type shown in Figs. 2 and 3 were contained in brain stem networks which govern SND. An example of the second categor£ of midsignal spike-triggered averages of inferior cardiac SND is shown in Fig. 5. The portion of the average of SND which preceded the spike trigger (i.e. to the left of time 0) was flat. This observation indicates that this brain stem neuron was not influenced by preceding activity in the oscillator responsible for the 2-6 cycles/s rhythm in SND. That is, this neuron neither received inputs from nor was contained in the 2-6 cycles/s oscillator. Rather, the appearance of a prominent 2-6 cycles/s rhythm in the portion of the average of SND which followed unit discharge (i.e. to the right of time 0) indicates that this neuron provided inputs to the brain stem oscillator. Furthermore, replication of the rhythm in the discharges of the inferior cardiac nerve to the right but not the left of time 0 in the midsignal spike-triggered average of SND indicates that the
6O 4
Sp trig
Sp,r'g
/
i~A,, ~, Un~l ~ SND
r
V'
I
Random pulse ~ SND '\\ ,\
"~
_j
[ -1.5
I
I
0 Interval, s
I
I
I
+15
Fig. 4. Comparison of changes in inferior cardiac SND following spontaneous discharges of brain stem units with those produced by single shocks (5 V: 0.1 ms) applied once every 2 s through the recording microelectrode. Top traces in A and B show spike-triggered averages of SND (959 trials in A and 857 trials in B) for different medulla~ neurons. Bottom traces show corresponding poststimulus averages of SND (64 trials). Address bin was 0.4 ms. Spike and stimulus triggers occurred at beginning of traces. Horizontal calibration is 40 ms. Vertical calibration is 20 ffV for traces in A and bottom trace in I:L and 5 #V for top trace in B. Fig. 5. Resetting of 2-6 cycles/s rhythm in inferior cardiac SND by medullary unit discharges in the baroreceptor denervated cat. Top trace is midsignal spike-triggered average of SND. Spike trigger occurred at time 0. Bottom trace is average of SND constructed with triggers from a random pulse train. Number of trials for each average was 769. Address bin was 3 ms. Vertical calibration is 20 ~V.
d i s c h a r g e s o f such n e u r o n s reset the b r a i n s t e m o s c d l a t o r . A l t h o u g h we h a v e b e e n successful in i d e n t i f y i n g b r a i n s t e m units w i t h a c t i v i t y p a t t e r n s r e l a t e d to t h o s e in the i n f e r i o r c a r d i a c nerve, it is i m p o s s i b l e to state at this t i m e w h e t h e r all o f these n e u r o n s w e r e c o n t a i n e d w i t h i n n e t w o r k s w h i c h specifically g o v e r n S N D . I n this regard, the 2 - 6 c y c l e s / s r h y t h m m a y be c o m m o n to a n u m b e r o f f u n c t i o n a l l y d i s t i n c t b r a i n s t e m n e t w o r k s in the b a r o r e c e p t o r d e n e r v a t e d cat. T h u s , it c a n n o t b e c a t e g o r i c a l l y r u l e d o u t t h a t s o m e o f the n e u r o n s w h o s e d i s c h a r g e s w e r e t e m p o r a l l y r e l a t e d to S N D m a y h a v e b e e n c o m p o n e n t s o f ' n o n - s y m p a t h e t i c ' networks which govern systems whose outputs were not measured. Nevertheless, similarities in c o r r e s p o n d i n g s p i k e - t r i g g e r e d a n d p o s t s t i m u l u s a v e r a g e s of i n f e r i o r c a r d i a c n e r v e a c t i v i t y f a v o r the v i e w that, in the m a j o r i t y o f i n s t a n c e s , we w e r e d e a l i n g w i t h n e u r o n s w h i c h specifically g o v e r n e d S N D .
61
Acknowledgement This study was supported by Public Health Service Grant HL-13187 and by a Michigan Heart Association Grant-in-Aid.
References 1 Adrian, E.D., Bronk, D.W. and Phillips, G., Discharges in mammalian sympathetic nerves, J. Physiol. (Lond.), 74 (1932) 115-133. 2 Alexander, R.S., Tonic and reflex functions of medullary sympathetic cardiovascular centers, J. Neurophysiol., 9 (1946) 205-217. 3 Bard, P., Anatomical organization of the central nervous system in relation to control of the heart and blood vessels, Physiol. Rev., 40 Suppl. 4 (1960) 3-26. 4 Barman, S.M. and Gebber, G.L., Sympathetic nerve rhythm of brain stem origin, Amer. J. Physiol., 239 (Regulatory.Integrative Comp. Physiol. 8) (1980) R42-R47. 5 Barman, S.M. and Gebber, G.L., Problems associated with the identification of brain stem neurons responsible for sympathetic nerve discharge, J. auton. Nerv. Syst., 3 (1981) 369-377. 6 Bernard, C., Lecons sur la Physiologie et la Pathologie du Systrme Nerveux, Vol. 1, Bailliere, Paris, 1863. 7 Bronk, D.W., Ferguson, L.K., Margaria, R. and Solandt, D.Y., The activity of the cardiac sympathetic centers, Amer. J. Physiol., 117 (1936) 237-249. 8 Cohen, M.I. and Gootman, P.M., Periodicities in efferent discharges of splanchnic nerve of the cat, Amer. J. Physiol., 218 (1970) 1092-1101. 9 Dittmar, C., Uber die Lage des sogenannten Gefasscentrums der Medulla oblongata, Ber. Verh. Saechs. Wiss. Leipzig Math. Phys., KI., 25 (1873) 449-479. 10 Gebber, G.L., Basis for phase relations between baroreceptor and sympathetic nervous discharge, Amer. J. Physiol., 230 (1976) 263-270. I I Gebber, G.L., Central oscillators responsible for sympathetic nerve discharge, Amer. J. Physiol., 239 (Heart Circulat. Physiol., 8) (1980) H143-HI55. 12 Gebber, G.L. and Barman, S.M., Brain stem vasomotor circuits involved in the genesis and entrainment of sympathetic nervous rhythms. In W. DeJong, A.P. Provoost and A.P. Shapiro (Eds.), Hypertension and Brain Mechanisms, Progress in Brain Research, Vol. 47, Elsevier, Amsterdam, 1977, pp. 61-75. 13 Green, J.H.and Heffron, P.E., Studies upon the relationship between baroreceptor and sympathetic activity, Quart. J. exp. Physiol., 53 (1968) 23-32. 14 McCall, R.B. and Gebber, G.L., Brain stem and spinal synchronization of sympathetic nervous discharge, Brain Res., 89 (1975) 139-143. 15 Owsjannikow, P., Die tonischen und reflectorischen Centren der Gefassnerven, Ber. Verh. Saechs. Wiss. Leipzig Math. Phys., KI., 23 (1871) 135-147. 16 Taylor, D.G. and Gebber, G.L., Baroreceptor mechanisms controlling sympathetic nervous rhythms of central origin, Amer. J. Physiol., 228 (1975) 1002-1013.
55
Elsevier Biomedical Press
Brain stem neurons governing the discharges of sympathetic nerves Gerard L. Gebber and Susan M. Barman Department of Pharmacology and Toxicology, Michigan State University, Eus't Lansing, MI 48824 (U.S.A.) (Received December 20th, 1980) (Accepted August 1st, 1981 )
Key words: brain stem oscillator--medullary neurons--spike-triggered averaging-sympathetic nerve rhythms
Abstract
Recent work from our laboratory revealed that the brain stem is inherently capable of generating a 2-6 cycles/s rhythm in sympathetic nerve discharge (SND) of the baroreceptor denervated cat. It is believed that this rhythm is representative of the fundamental organization of those brain stem networks responsible for the background discharges in sympathetic nerves. As a consequence, an attempt was made to identify brain stem neurons with activity patterns related to the 2-6 cycles/s rhythm in inferior cardiac SND of the baroreceptor denervated cat. Specifically, the relationships between the spontaneous discharges of single brain stem neurons and the inferior cardiac nerve were analyzed with the technique of spike-triggered averaging. Neurons whose discharges were synchronized either to the rising or falling phase of inferior cardiac SND were located in the classic pressor region of the lateral medullary reticular formation. These neurons may comprise or receive inputs from the oscillator responsible for the 2-6 cycles/s rhythm in SND. In addition, neurons which reset the 2-6 cycles/s rhythm were identified. These neurons may provide inputs to the 2-6 cycles/s oscillator in the brain stem.
Introduction
A basic topic in research on the neurophysiology of preganglionic sympathetic neurons concerns the manner in which the background discharges of these cells are generated. The problem was brought into focus when Bernard [6] demonstrated that transection of the cervical spinal cord led to a pronounced fall in blood pressure. This simple yet extremely important experiment demonstrated that sympathetic nerves which innervate the cardiovascular system are tonically active and that this 0165-1838/82/0000-0000/$02.75 © 1982 Elsevier Biomedical Press
56 activity arises primarily in neuronal circuits located in the brain. Subsequent w o r k performed by Dittmar [9] and Owsjannikow [15] in the 1870s implicated the lower brain st.em as the region responsible for the background discharges in sympathetic nerves. Our comprehension of the mechanisms and circuitry involved in generating sympathetic nerve discharge (SND), however, remains largely incomplete. The purpose of the current paper is to summarize the results of recent experiments on this topic which have been performed in our laboratory.
The 2-6 cycles/s rhythm in SND--an indicator of the fundamental organization of the brain stem sympathetic generator An important characteristic of the discharges of sympathetic nerve bundles is rhythmicity. That is, the central nervous system is capable of synchronizing the discharges of populations of sympathetic neurons in a periodic fashion. One of the prominent rhythms in SND is the cardiac-related periodicity. As first demonstrated by Adrian et al. [1] and Bronk et al. [7], the discharges of pre- and postganglionic nerves are synchronized into bursts (i.e. slow waves as we record them with a preamplifier bandpass of 1-1000 Hz) which are locked in a 1:1 relation to the cardiac cycle. A typical example of this rhythm in the discharges of the splanchnic sympathetic nerve of the cat is shown in panels IA and IB of Fig. 1. Until recently, the cardiac-related rhythm in SND was considered to result as a simple consequence of the baroreceptor reflexes [8,13]. That is, this rhythm was believed to arise from variations in the level of baroreceptor-induced inhibition of the tonic central drive to sympathetic nerves during each cardiac cycle. Results obtained in our laboratory since 1975 have led us to revise the traditional view for the generation of the cardiac-related rhythm in SND. We have proposed
A 150r
I Baro. Intact B .
SND 11 Baro. Den.
B
,,5 A /!
SND J
__J
Fig. I. Splanchnic SND before and after baroreceptor denervation. I: baroreceptor reflexes intact. A, oscilloscopic traces of blood pressure in mm Hg (top) and splanchnic SND (bottom). B, R-wave-triggered averages of blood pressure and SND (64 trials). Address bin was I ms. II: A and B, same, but after baroreceptor denervation. Horizontal calibration is 200 ms. Vertical calibration for SND is 100 p,V.
57 that this rhythm is intrinsic to the central nervous system and that the function of the baroreceptor reflexes is to entrain the rhythm to the cardiac cycle. The experiments on which this proposal is based appear in our original articles [10,12,16] and are summarized in a recent review [11]. One of our observations is shown in panel IIA of Fig. 1. We found that a rhythm with a frequency (2-6 cycles/s) close to that of the heart rate persisted in SND after bilateral section of the carotid sinus, aortic depressor and vagus nerves (i.e. baroreceptor denervation). This rhythm could not be attributed to cardiac-related activity in other visceral afferents because the phase relations between SND and the cardiac cycle were completely disrupted after bilateral section of the carotid sinus, aortic depressor and vagus nerves. In this regard, note that the R-wave-triggered average of splanchnic SND approached a straight line after baroreceptor denervation (panel liB of Fig. 1). More recently, 'we demonstrated that the 2-6 cycles/s rhythm in SND of the baroreceptor denervated cat is not generated in the spinal cord [14] or forebrain [4]. Thus, in the absence of evidence for 2-6 cycles/s activity in non-baroreceptor afferents, it would appear that the rhythm is representative of the fundamental organization of those brain stem networks responsible for the background discharges in sympathetic nerves. Two possibilities immediately come to mind concerning the neural circuitry responsible for the 2-6 cycles/s rhythm in SND. First, the rhythm might be generated by a group of brain stem neurons with inherent pacemaker activity. Second, the rhythm might be generated as the consequence of interactions within and between different neuronal networks. For instance, the rising phase of the 2-6 cycles/s slow wave of SND (see Fig. 1) might result from the synchronous discharges of one group of brain stem neurons while the falling phase of the slow wave might be due to active inhibition produced by the synchronous activation of a second group of brain stem neurons. In view of these possibilities, we became interested in identifying those neuronal types which constitute the brain stem sympathetic generator.
Medullary neurons with activity patterns temporally related to inferior cardiac SND Spike-triggered averaging was used to study the relationships between the naturally occurring discharges of single brain stem neurons and the inferior cardiac sympathetic nerve in the baroreceptor denervated cat. The details of this method have been presented in a previous report from our laboratory [5]. Briefly, the spontaneous discharges (extracellularly recorded) of the brain stem unit were used to trigger individual sweeps of a computer so as to construct an average of accompanying changes in inferior cardiac SND. The spike was used as a midsignal trigger so as to provide information on whether brain stem unit activity was temporally related to preceding as well as to subsequently occurring inferior cardiac SND. The relationships between brain stem unit discharges and inferior cardiac nerve activity were verified in two ways. First, the spike-triggered average of SND was compared to the average of SND derived from a random pulse series with approximately the same frequency as the unit spike series. Deflections in the spike-triggered average which
5~ 2
Uni~l SND A/~t/~"!':,l/~ )
"\ I \ I
-5OO
0 Interval, ms 1.0
153- 3
u
I
÷500
Un~l* SND
C
0
1Y
i
i
/
0 Interval, ms 750
Rondompulse* SND I
0
Interval, s 10
I
-750
I
I
I
0
I
I
+750
Interval, ms
Fig. 2. Relationship between the discharges of a brain stern neuron and the inferior cardiac sympathetic nerve in a baroreceptor dene~'ated cat. A: midsignal spike-triggered average of inferior cardiac SND (1200 trials). Spike trigger occurred at time0. Address bin was I ms. Vertical calibration is 5#V. B: autocorrelogram of unit discharges. Address bin was 12 ms. Analysis based on 2432 spikes. C: autocorrelogram of inferior cardiac SND. Analysis time was 40 s. Address bin was It) ms. Fig. 3. Synchronization of medulla~, unit discharges to a point near the beginning of the falling phase of the 2-6 cycles/s slow wave in inferior cardiac SND of the baroreceptor denervated cat. Top trace is midsignal spike-triggered average of inferior cardiac SND: spike trigger occurred at time 0. Bottom trace is average of SND constructed with triggers from a random pulse train. Number of trials for each average was 860. Address bin was 1.5 ms. Vertical calibration is 5 ~,V.
e x c e e d e d t h o s e in the ' d u m m y ' a v e r a g e by at least a f a c t o r of two w e r e c o n s i d e r e d to r e p r e s e n t c h a n g e s in i n f e r i o r c a r d i a c n e r v e d i s c h a r g e t e m p o r a l l y r e l a t e d to the s p o n t a n e o u s a c t i v i t y of the b r a i n s t e m n e u r o n . S e c o n d , the d a t a w e r e f r a c t i o n a t e d i n t o several c o n t i n u o u s s e g m e n t s c o n t a i n i n g e q u a l n u m b e r s o f unit spikes. T h e d i s c h a r g e s of the b r a i n s t e m n e u r o n a n d i n f e r i o r c a r d i a c n e r v e w e r e c o n s i d e r e d t e m p o r a l l y r e l a t e d if a v e r a g e s o f S N D c o n s t r u c t e d f r o m successive d a t a s e g m e n t s w e r e essentially i d e n t i c a l in c o n t o u r a n d a m p l i t u d e . A strip ( 1 . 5 - 3 . 5 m m lateral to the m i d l i n e ) of the r e t i c u l a r f o r m a t i o n f r o m the level of the o b e x to the p o n t o m e d u l l a r y b o r d e r was e x p l o r e d for n e u r o n s w i t h a c t i v i t y p a t t e r n s r e l a t e d to t h o s e in the i n f e r i o r c a r d i a c n e r v e of b a r o r e c e p t o r d e n e r v a t e d cats. T h i s strip falls w i t h i n the classic m e d u l l a r y p r e s s o r r e g i o n [2,3]. T h e d i s c h a r g e s o f a p p r o x i m a t e l y 30% of the n e u r o n s s a m p l e d w e r e t e m p o r a l l y r e l a t e d to i n f e r i o r c a r d i a c S N D . T h e m a j o r i t y o f these 65 n e u r o n s w e r e l o c a t e d in n u c l e u s reticularis p a r v o c e l l u l a r i s a n d n u c l e u s r e t i c u l a r i s ventralis. T h e m i d s i g n a l spiket r i g g e r e d a v e r a g e s o f i n f e r i o r c a r d i a c S N D for units w i t h s y m p a t h e t i c - r e l a t e d a c t i v i t y fell i n t o t w o categories. A n e x a m p l e o f the first c a t e g o r y is s h o w n in Fig. 2A. T i m e 0
59 in the average of inferior cardiac SND marks the occurrence of the spike of the brain stem neuron. Thus, the portion of the average to the left of time 0 is for SND which preceded unit activity while that to the right of time0 is for SND which followed unit activity. A distinguishing characteristic of the first category of averages of SND is the appearance of a rhythm in the 2-6 cycles/s range both in activity which preceded and followed the midsignal spike trigger. The period of this rhythm was the same as that which appeared in the autocorrelograms of the discharges of the brain stem unit (Fig. 2B) and inferior cardiac nerve (Fig. 2C). Identical periodicities in the autocorrelograms and midsignal spike-triggered average of SND (a form of crosscorrelation analysis) clearly indicate that the 2-6 cycles/s rhythm in unit and inferior cardiac nerve activities were locked to each other. Thus, it is likely that neurons from which such averages of inferior cardiac nerve activity (Fig. 2A) were derived either comprised or received inputs from the oscillator responsible for the 2-6 cycles/s slow wave in SND of the baroreceptor denervated cat. The data in Figs. 2 and 3 support the view that the 2-6 cycles/s slow wave in inferior cardiac SND results as the consequence of alternations in the discharges of two groups of brain stem neurons. The position of time 0 on the 2-6 cycles/s slow wave in the midsignal spike-triggered average of SND is indicative of the point of highest probability of occurrence of brain stem unit discharge. In this respect, we found brain stem neurons whose highest probability of discharge occurred near the beginning of either the rising (Fig. 2A) or falling (Fig. 3) phase of the 2-6 cycles/s slow wave in SND. These observations raise the tantalizing possibility that the 2-6 cycles/s rhythm in SND of the baroreceptor denervated cat is generated by a brain stem oscillator which contains pairs of mutually inhibitory neurons. This hypothesis should be tested in future studies with intracellular recordings a n d / o r unit-~ unit cross-correlation analysis. Electrical stimuli applied through the microelectrode at sites of sympathetic-related unit activity usually elicited changes in inferior cardiac SND similar to those observed in the corresponding spike-triggered average. Typical examples are shown in Fig. 4. Note should be taken of the similarities in contour and time-to-peak effect in corresponding spike-triggered and poststimulus averages of inferior cardiac nerve activity. These results further support the view that neurons whose discharges were used to construct averages of inferior cardiac nerve activity of the type shown in Figs. 2 and 3 were contained in brain stem networks which govern SND. An example of the second categor£ of midsignal spike-triggered averages of inferior cardiac SND is shown in Fig. 5. The portion of the average of SND which preceded the spike trigger (i.e. to the left of time 0) was flat. This observation indicates that this brain stem neuron was not influenced by preceding activity in the oscillator responsible for the 2-6 cycles/s rhythm in SND. That is, this neuron neither received inputs from nor was contained in the 2-6 cycles/s oscillator. Rather, the appearance of a prominent 2-6 cycles/s rhythm in the portion of the average of SND which followed unit discharge (i.e. to the right of time 0) indicates that this neuron provided inputs to the brain stem oscillator. Furthermore, replication of the rhythm in the discharges of the inferior cardiac nerve to the right but not the left of time 0 in the midsignal spike-triggered average of SND indicates that the
6O 4
Sp trig
Sp,r'g
/
i~A,, ~, Un~l ~ SND
r
V'
I
Random pulse ~ SND '\\ ,\
"~
_j
[ -1.5
I
I
0 Interval, s
I
I
I
+15
Fig. 4. Comparison of changes in inferior cardiac SND following spontaneous discharges of brain stem units with those produced by single shocks (5 V: 0.1 ms) applied once every 2 s through the recording microelectrode. Top traces in A and B show spike-triggered averages of SND (959 trials in A and 857 trials in B) for different medulla~ neurons. Bottom traces show corresponding poststimulus averages of SND (64 trials). Address bin was 0.4 ms. Spike and stimulus triggers occurred at beginning of traces. Horizontal calibration is 40 ms. Vertical calibration is 20 ffV for traces in A and bottom trace in I:L and 5 #V for top trace in B. Fig. 5. Resetting of 2-6 cycles/s rhythm in inferior cardiac SND by medullary unit discharges in the baroreceptor denervated cat. Top trace is midsignal spike-triggered average of SND. Spike trigger occurred at time 0. Bottom trace is average of SND constructed with triggers from a random pulse train. Number of trials for each average was 769. Address bin was 3 ms. Vertical calibration is 20 ~V.
d i s c h a r g e s o f such n e u r o n s reset the b r a i n s t e m o s c d l a t o r . A l t h o u g h we h a v e b e e n successful in i d e n t i f y i n g b r a i n s t e m units w i t h a c t i v i t y p a t t e r n s r e l a t e d to t h o s e in the i n f e r i o r c a r d i a c nerve, it is i m p o s s i b l e to state at this t i m e w h e t h e r all o f these n e u r o n s w e r e c o n t a i n e d w i t h i n n e t w o r k s w h i c h specifically g o v e r n S N D . I n this regard, the 2 - 6 c y c l e s / s r h y t h m m a y be c o m m o n to a n u m b e r o f f u n c t i o n a l l y d i s t i n c t b r a i n s t e m n e t w o r k s in the b a r o r e c e p t o r d e n e r v a t e d cat. T h u s , it c a n n o t b e c a t e g o r i c a l l y r u l e d o u t t h a t s o m e o f the n e u r o n s w h o s e d i s c h a r g e s w e r e t e m p o r a l l y r e l a t e d to S N D m a y h a v e b e e n c o m p o n e n t s o f ' n o n - s y m p a t h e t i c ' networks which govern systems whose outputs were not measured. Nevertheless, similarities in c o r r e s p o n d i n g s p i k e - t r i g g e r e d a n d p o s t s t i m u l u s a v e r a g e s of i n f e r i o r c a r d i a c n e r v e a c t i v i t y f a v o r the v i e w that, in the m a j o r i t y o f i n s t a n c e s , we w e r e d e a l i n g w i t h n e u r o n s w h i c h specifically g o v e r n e d S N D .
61
Acknowledgement This study was supported by Public Health Service Grant HL-13187 and by a Michigan Heart Association Grant-in-Aid.
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