Brain Research, 555 t 1991) 7(I-74 © 1991 Elsevier Science Publishers B.V. All rights reserved. (XI06-8993/91/$03.50 A l) 0 NIS 0(X)689939116825U
70
BRES 16825
Monosynaptic connection from caudal to rostral ventrolateral medulla in the baroreceptor reflex pathway S.K. Agarwal and F.R. Calaresu Department of Physiology, University of Western Ontario, London, Ont. (Canada) (Accepted 26 February 1991) Key words: Caudal ventrolateral medulla; Nucleus tractus solitarii; Rostral ventrolateral medulla; Arterial pressure; Baroreceptor
Experiments were done to test the hypothesis that caudal ventrolateral medulla (CVLM) neurons excited by activation of arterial baroreceptors and by stimulation of depressor sites in the nucleus tractus solitarii (NTS) project monosynaptically to the rostral ventrolateral medulla (RVLM). In urethan anaesthetized and artificially ventilated rats we recorded extracellular activity from 46 spontaneously firing units in the CVLM. Twenty of these units were excited by baroreceptor activation (1-3 #g phenylephrine i.v.) and of these 6 were excited (mean latency of 9.8 _+ 2.3 ms) by single pulses (0.1 ms, 30 _+ 8.3 #A) delivered once per second to a depressor site in the ipsilateral NTS. These 6 units were also antidromically activated with a latency of 4.1 + 0.12 ms by stimulation of a pressor region in the ipsilateral RVLM. These results provide evidence for the existence of an excitatory projection from the NTS to the CVLM which, in turn, projects monosynaptically to sympathoexcitatory neurons in the RVLM. INTRODUCTION
8,11,12,20,24o Indeed, in a recent study in the rabbit it has
Activation of sympathoinhibitory neurons in the caudal ventrolateral medulla (CVLM) reduces arterial pressure (AP) while inhibition of these neurons increases A P 8'9'17'25. The CVLM area is not defined precisely, but
been suggested that sympathoinhibition is mediated by an N T S - C V L M - R V L M pathway z2. The present study was designed to test the hypothesis that CVLM n e u r o n s excited by activation of arterial baroreceptors and by stimulation of depressor sites in the
it appears to be centered around the external formation
NTS send monosynaptic connections to the RVLM. This
of the nucleus ambiguus4 and to contain the A I noradrenergic cell group 25. The current view regarding one of
was done by monitoring the change in firing frequency of CVLM units following stimulation of the NTS and by attempting to demonstrate that C V L M n e u r o n s excited by NTS stimulation were antidromically activated by
the functions of the CVLM is that it mediates the decrease in A P to activation of baroreceptor fibers via an inhibitory projection from CVLM to sympathoexcitatory n e u r o n s situated in the rostral ventrolateral medulla ( R V L M ) 1-3'5'6'8"1°'18'21, which, in turn, project to spinal sympathetic preganglionic vasomotor neurons. Additional evidence regarding the role of the CVLM in the baroreceptor reflex has been obtained by anatomical studies which have shown that baroreceptor afferent fibres synapse on second order neurons located within the nucleus tractus solitarii (NTS) 13 which in turn project to the R V L M and CVLM m. Some reports suggest that an inhibitory projection from the NTS to the R V L M mediates sympathoinhibitory baroreflex responses 9'18, but other reports have favoured the notion that these sympathoinhibitory responses are mediated through an excitatory pathway from the NTS to the CVLM 24 from which an inhibitory pathway to the R V L M originates ~'
stimulation of pressor sites in the RVLM. MATERIALS AND METHODS Experiments were done in 19 adult male Wistar rats (250-350 g, Charles River, Montreal), anaesthetized with urethan (Sigma, St. Louis, MO, 1.4 g/kg, i.p. initially and supplemental doses of 0.14 g/kg s.c. when necessary). Rectal temperature was maintained at 37.5 + 5 °C with a thermostatically controlled heating blanket. The animals were paralyzed (decamethonium bromide, Sigma, 3.3 mg/kg i.v. initially, with 0.35 mg supplements every 15-30 min) and artificially ventilated with room air using a small animal ventilator (Harvard Apparatus, model 683). The femoral artery and vein were cannulated. The arterial cannula was connected to a pressure transducer (Statham P23 Db) which was connected to a Grass polygraph (model 7) for continuous recording of AP. A Grass tachograph (7P4C), triggered by the arterial pressure pulse was used to monitor heart rate (HR). The venous cannula was used to inject l-3/~g of phenylephrine (PE, Sigma, St. Louis, 0.1-0.3 ml of a 10 ~g/ml solution) for baroreceptor activation. The animal was placed
Correspondence: S.K. Agarwal, Department of Physiology, University of Western Ontario, London, Ont., Canada, N6A 5C1. Fax: (1) (519) 661-3827.
71 current to antidromically activate terminals of CVLM neurons 14. The RVLM stimulating electrode was moved in 200/~m steps from the ventral surface of the medulla. At the end of the experiment the recording sites in the CVLM were marked with iontophoretic deposits of Pontamine sky blue. Iron was deposited by passing a current through the tip of the stimulating electrodes in the NTS and the RVLM 2. Stimulation and recording sites were mapped on diagrams of transverse sections of the rat brain from an atlas )6. The location of the tips of the stimulating electrodes in the NTS and RVLM in transverse sections of the medulla is shown in Fig. 1A, B. At the start of each experiment a stimulating electrode was placed in the right RVLM at a site where electrical stimulation at 50 Hz, 0.1 ms pulse duration and 5-100 # A elicited increases in MAP of 30-40 mmHg. After a pressor site in the RVLM was found, another electrode was positioned in the ipsilateral NTS at a site where a clear depressor response in MAP of 20-30 mmHg was obtained with a 5 s train of stimuli at 50 Hz, 0.1 ms pulse duration and 5-100/~A. After placing the stimulating electrodes, the ipsilateral CVLM was searched for spontaneously active units. After a stable recording from a single unit in the CVLM was obtained, the response of each unit to an increase in mean AP was tested by recording the change in discharge elicited by an i.v. bolus injection of 1-3/~g phenylephrine (PE). For further characterization, the units were tested for rhythmicity of spontaneous activity in relation to the cardiac cycle by constructing a post R-wave time histogram 2. Only units identified by these two criteria were studied. When a barosensitive CVLM unit was found its response to stimulation of a depressor site in the NTS was tested by constructing peri-stimulus time histograms of unit activity. Stimuli to the NTS consisted of single pulses of 0.1 ms duration delivered once per second at an intensity ranging from 5 to 100/~A. Finally each CVLM unit responsive to NTS stimulation was tested for antidromic activation by stimulation of the RVLM.
in a stereotaxic apparatus with the bite bar 20 mm below the interaural line. The medulla was exposed and activity from spontaneously firing units in the CVLM was recorded extracellularly with glass electrodes filled with 0.5 M sodium acetate and 2% Pontamine sky blue (4-10 MI2 impedance measured at 1 kHz). The electrode was inclined 20° with respect to the vertical in the sagittal plane with the tip pointing rostrally and was advanced through the dorsal medulla into the CVLM (stereotaxic coordinates from interaurai line: -4.3 to --5.1 mm caudal to the interaural line, 1.8-2.1 mm lateral and 2.3-2.9 mm below the dorsal surface of the medulla) by a hydraulic microdrive (Narishige, model M08). Electrical activity was amplified through a preamplifier (Dagan 2400; bandpass 0.3-10 kHz), displayed on an oscilloscope (Tektronix R5103N) and discriminated by a Neurolog NL200 spike trigger. The frequency of firing of single units was recorded on a polygraph along with AP and HR. Unit activity derived from the spike trigger was fed to an IBM-AT computer for spike train analysis. The NTS and the RVLM could be stimulated by stainless-steel unipolar electrodes (tip diameter 3-10/~m, impedance - 1 M.Q) connected via a Grass PSIU-6 stimulus isolation unit to a Grass $88 stimulator. Electrodes were lowered stereotaxically to sites either in the NTS or in the RVLM from which a train of stimuli (50 Hz, 0.1 ms pulses, 5-100/~A for 5 s) elicited a decrease or an increase in HR and AP, respectively. The stereotaxic coordinates used for NTS stimulation were 0.6 mm rostral to the obex, 0.6 mm lateral to the midline and 0.6 mm below the dorsal surface of the medulla. For the RVLM the electrode was inclined 20° with respect to the vertical in the sagittal plane with the tip pointing caudally; the stereotaxic coordinates from the interaural line were: -2.6 to -3.3 mm caudal to the interaural line, 1.6-2.1 mm lateral and 7.2-8.0 mm below the surface of the cerebellum. The criteria for identifying an antidromic evoked action potential were: constant latency at threshold; ability to follow stimulation at 100 Hz; collision between spontaneous action potentials and evoked action potentials 14. We constructed depth threshold curves to identify sites requiring low stimulus
,,
_-4.3 ..B
A
1 4 / ~ _ _ ~ N , [ , ~ --"
"-""~
~..~)"~'("-2"6
:C\,,,
c;,~/'
-"'~-~-
"
v!,, \
',
r',
A.P':'--"--X .'""" _ ,z~ _~'_-.'
,.
c
..
..-.''
r,
N.4t~?oOt, LM?.t~_4.3 "':'~"~' .~,-~,71
,-.. \
~,,,*,~"-'>.', ~.~.,,"1,
,.,'~-..:--~..~
,':_.:,,,r
', ..
,,
.,.-_\
,
:,-s.,
i
---.
~
" i,,,,
Fig. 1. Location of sites of recording and stimulation in transverse sections of the rat medulla. Numbers indicate distances caudal to interaural line in millimetres, modified from Paxinos and Watson 16. A: sites of stimulation in the NTS where depressor responses were obtained. B: sites of stimulation in the RVLM where pressor responses were obtained. C: o, location of units in the CVLM which were activated by baroreceptors; ~l, units excited by electrical stimulation of NTS and antidromically activated from the RVLM. AP, area postrema; CU, cuneate nucleus; CVLM, caudal ventrolateral medulla; Gr, gracile nucleus; IO, inferior olive; NA, nucleus ambiguus; NTS, nucleus tractus solitarii; Pyr, pyramidal tract; RP, raphe pallidus; RVLM, rostral ventrolateral medulla; 4V, 4th ventricle; 7, facial nucleus; 10, dorsal motor nucleus of vagus; 12, hypoglossal nucleus.
72 RESULTS A region of the C V L M surrounded dorsally by the ventral aspect of the loose formation of the nucleus ambiguus and ventrally by the lateral reticular nucleus between 1.8 and 2.2 mm lateral to the midline was explored for spontaneously firing units. It is likely that the activity of units in this region was recorded from cell bodies for the following reasons. First, separation of the depolarization of the initial segment and of the somatodendritic region could be identified in most recordings. Second, the action potentials of these units had a duration greater than 1 ms; and third, electrical activity could be recorded over distances of several tens of micrometers of electrode tip displacement. Of the 46 spontaneously firing units from which recordings were obtained, 32 were identified as barosensitive neurons because they responded with an increase (n = 20) or a decrease (n = 12) in their activity during the rapid rise of arterial pressure (and baroreceptor mediated fall in H R ) induced by an i.v. bolus injection of 1-3 #g of PE. The 12 units which were inhibited by PE were not studied further because they did not exhibit an excitatory response to activation of baroreceptors and were therefore unlikely to be sympathoinhibitory C V L M units involved in the baroreceptor reflex. The mean spontaneous firing rate + S.E.M. of the 20 units which were excited was 12.8 + 1.7 spikes/s. The location of these neurons is shown in transverse sections of the medulla (Fig. 1C) and typical excitatory responses
of a unit to bolus injections of PE are shown in Fig. 2. Stimulation of depressor sites in the NTS increased the frequency of discharge of 6 of the 20 units excited by activation of baroreceptors, with a mean latency +_ S.E.M. of 9.8 + 2.3 ms (range 3-15 ms). This excitation lasted for 14.5 + 3.5 ms; during this period the firing rate was increased by 454 + 63.4% in comparison to control level in the 100 ms preceding stimulation. The average threshold intensity to produce excitation with single pulses was 30 + 8.3 ,uA. An example of excitatory responses of a C V L M neuron at different intensities of stimulation of the NTS is shown in Fig. 3. The remaining 14 units excited by activation of baroreceptors did not respond to NTS stimulation. Electrical stimulation of the R V L M evoked antidromic action potentials in the 6 neurons in the C V L M which were (i) excited by activation of baroreceptors, (ii)
A 20
=-
to
,a,4a,,, a,;.d,..L,a,,., , 0
t00
•
,
,
200
300
400
,
,
,
500
, 600
2 0 2 Sweeps
ms
B 20
A
B =
t0
200 t MAP
,,,,,=,;,,11
200 I
o
(mmHg) 100[
,,., , . l . .
,,..,., 1= =.,,,,.,,,,,, ,oo me
¢
j,,,,
BP (mmHg) 100
20
0= w =
10 1[
, ,.,.,,,I,. 2do
t 0 1 Sweeps
0 200
Unit activity (spikes/s)
Ido
10
.~
,
too
0.1 Fig. 2. Responses of a unit in the CVLM to baroreceptor activation induced by i.v. bolus injections of 1 (A) and 3 (B) pg of PE delivered at arrowheads. Note the increase in firing frequency and gradual return to control frequency as AP returns to basal levels.
102 Sweeps
aoo
300
,,,
400
,
soo
soo ms
Fig. 3. Peristimulus time histograms of activity of a CVLM neuron during electrical stimulation of the NTS at 0.1 ms, 1 pulse per s (A) 20/~A; (B) 30/~A; (C) 40/~A. Bin width 1 ms; stimulus artifact at 100 ms.
73
A
C
B ~00
u
•
b
A .2m 04.0
i
0.5
i
t.0 0ep~
/
i
1.5
m
! 2.1"
--
i 1ram
J
Fig. 4. Collision test and depth threshold curve to identify terminals of a unit in the CVLM responding to RVLM stimulation. A: records are 5 superimposed oscilloscope sweeps demonstrating a direct connection from a CVLM neuron to RVLM neurons by the collision test. Dots represent spontaneous spikes, the asterisk indicates an antidromically evoked action potential only after a critical delay of 2.2 ms (top record); in the bottom record a delay of 2 ms from a spontaneously occurring spike prevents antidromic potential. Arrowheads indicate stimulus (0.1 ms duration, 20/~A intensity) artifacts. Horizontal calibration, 2 ms; vertical calibration, 50 gV. B: depth threshold curve for antidromic activation of a CVLM neuron with two different onset latencies as indicated (0, 2.2 ms; ©, 4.0 ms) showing the presence of a discrete jump in latency. C: (a) represents the location of the CVLM neuron (located at 0.5 mm rostral to the obex, 1.9 mm right and 2.7 mm from the dorsal surface of the medulla); (b) solid vertical line shows the track of the stimulating electrode in the RVLM for the same neuron. The rostrocaudal plane of this section is 2.5 mm rostral to the obex.
excited by stimulation of the NTS, and (iii) showed cardiac cycle related rhythmicity. A n t i d r o m i c spike latencies were in the range of 1-10 ms (4.1 + 0.12 ms) c o r r e s p o n d i n g to an estimated m e a n conduction velocity of 0.62 + 0.08 m/s. T h e threshold intensity for producing antidromic spikes in neurons ranged b e t w e e n 5 and 100 /~A. T h e response of a C V L M neuron to the collision test and the d e p t h threshold curve for activation of this n e u r o n are shown in Fig. 4. DISCUSSION These e x p e r i m e n t s have shown that stimulation of b a r o r e c e p t o r s excites a population of C V L M neurons which are o r t h o d r o m i c a l l y excited by NTS stimulation and antidromically activated by R V L M stimulation. D e p t h threshold curves d e m o n s t r a t e that these neurons have extensive arborizations in the R V L M . The existence of this N T S - C V L M - R V L M p a t h w a y is s u p p o r t e d by the anatomical d e m o n s t r a t i o n of a pathway between NTS and C V L M 19, by microinjection experiments showing that NTS neurons project to the C V L M by a glutamatergic p a t h w a y which mediates vasodepressor responses 2, 23,24, and by the d e m o n s t r a t i o n that cardiovascular
neurons in the R V L M are inhibited by activation of cell bodies in the C V L M 1. T h e present study provides direct evidence that C V L M neurons are monosynaptically connected to the R V L M and that the same neurons are also excited o r t h o d r o m i c a l l y by NTS stimulation. Topographically these neurons are located in the same area where microinjections of L - g l u t a m a t e have been shown to elicit decreases in A P and inhibition of cardiovascular neurons in the R V L M 1. These results, t a k e n t o g e t h e r with our previous findings 1, d e m o n s t r a t e that an excitatory projection exists between the NTS and the C V L M which, in turn, provides a m o n o s y n a p t i c p r o j e c t i o n to the R V L M . Similar results have b e e n o b t a i n e d recently in the rabbit 22. Finally, we found that 12 s p o n t a n e o u s l y firing C V L M units were inhibited by bolus injections of P E and a n o t h e r group of 14 units did not respond. It is likely that these units are not involved in b a r o r e c e p t o r reflex function. Alternatively, it is possible that these neurons may be involved in controlling the activity of vasopressin secreting cells in the supraoptic nuclei TM. M o r e experiments are n e e d e d to investigate this possibility. In s u m m a r y , these results provide evidence for the existence of an excitatory p r o j e c t i o n from the NTS to the
74 CVLM
which,
in turn,
projects
monosynaptically
to
s y m p a t h o e x c i t a t o r y n e u r o n s in the R V L M .
REFERENCES 1 Agarwal, S.K., Gelsema, A.J. and Calaresu, ER., Neurons in rostral VLM are inhibited by chemical stimulation of caudal VLM in rats, Am. J. Physiol., 257 (1989) R265-R270. 2 Agarwal, S.K., Gelsema, A.J. and Calaresu, ER., Inhibition of rostral VLM by baroreceptor activation is relayed through caudal VLM, Am. J. Physiol., 258 (1990) R1271-R1278. 3 Barman, S.M. and Gebber, G.L., Axonal projection patterns of ventrolaterai meduUospinal sympathoexcitatory neurons, J. Neurophysiol., 53 (1985) 1551-1566. 4 Bieger, D. and Hopkins, D.A., Viscerotopic representation of the upper alimentary tract in the medulla oblongata in the rat: the nucleus ambiguus, J. Comp. Neurol., 262 (1987) 546-562. 5 Brown, D.L. and Guyenet, P.G., Electrophysiological study of cardiovascular neurons in the rostral ventrolateral medulla in rats, Circ. Res., 56 (1985) 359-369. 6 Dampney, R.A.L., Goodchild, A.K., Robertson, L.G. and Montgomery, W., Role of ventrolateral medulla in vasomotor regulation: a correlative anatomical and physiological study, Brain Res., 249 (1982) 223-235. 7 Day, T.A. and Sibbald, J.R., A 1 cell group mediates solitary nucleus excitation of supraoptic vasopressin cells, Am. J. Physiol., 257 (1989) R1020-R1026. 8 Gordon, EJ., Aortic baroreceptor reflexes are mediated by NMDA receptors in the caudal ventrolateral medulla, Am. J. Physiol., 252 (1987) R628-R633. 9 Granata, A.R., Kumada, M. and Reis, D.J., Sympathoinhibition by A~ noradrenergic neurons in the C~ area of the rostral medulla, J. Auton. Nerv. Syst., 14 (1985) 387-395. 10 Granata, A.R., Numao, Y., Kumada, M. and Reis, D.J., A1 noradrenergic neurons tonically inhibit sympathoexcitatory neurons of C1 area in rat brainstem, Brain Research, 377 (1986) 127-146. 11 Guyenet, P.G., Sun, M. and Brown, D.L., Role of GABA and excitatory amino acids in the medullary baroreflex pathways. In J. Ciriello, F.R. Calaresu, L. Renaud and C. Polosa (Eds.), Organization of the Autonomic Nervous System: Central and Peripheral Mechanisms, Liss, New York, 1987, pp. 215-225. 12 Guyenet, P.G., Filtz, T.M. and Donaldson, S.R., Role of excitatory amino acids in the rat vagal and sympathetic baroreflexes, Brain Research, 407 (1987) 272-284. 13 Kalia, M.P., Localization of aortic and carotid baroreceptor and chemoreceptor primary afferents in the brainstem. In J.P. Buckley and C.M. Ferrario (Eds.), Central Nervous System Mechanisms in Hypertension, Raven, New York, 1981, pp. 9-23.
Acknowledgements. This work was supported by a grant from the Medical Research Council of Canada. S.K.A. is a Fellow of the Canadian Heart and Stroke Foundation. D.J. McKitrick provided useful critical comments.
14 Lipski, J. Antidromic activation of neurons as an analytical tool in the study of the central nervous system, J. Neurosci. Meth., 4 (1981) 1-32. 15 McAllen, R.M. and Blessing, W.W., Neurons (presumably A~ cells) projecting from the caudal ventrolateral medulla to the region of the supraoptic nucleus respond to baroreceptor inputs in the rabbit, Neurosci. Lett., 73 (1987) 247-252. 16 Paxinos, G. and Watson, C., The Rat Brain in Stereotaxic Coordinates, Academic Press, Sydney, 1986. 17 Pilowsky, P.M., Morris, M.J., Minson, J.B., West, M.J., Chalmers, J.P., Willoughby, J.O. and Blessing, W.W., Inhibition of vasodepressor neurons in the caudal ventrolateral medulla of the rabbit increases both arterial pressure and the release of neuropeptide Y-like immunoreactivity from the spinal cord, Brain Research, 420 (1987) 380-384. 18 Ross, C.A. Ruggiero, D.A., Park, D.H., Job, J.H., Sved, A.E, Fernandez-Pardal, J., Saavedra, J.M. and Reis, D.J., Tonic vasomotor control by the rostral ventrolateral medulla: effect of electrical and chemical stimulation of CI adrenaline containing neurons on arterial pressure, heart rate, and plasma catecholamines and vasopressin, J. Neurosci., 4 (1984) 474-494. 19 Ross, C.A., Ruggiero, D.A. and Reis, D.J., Projections from the nucleus tractus solitarii to the rostral ventrolateral medulla, J. Comp. Neurol., 242 (1985) 511-534. 20 Somogyi, P., Minson, J.B., Morilak, D., Smith, 1.L., McIlhinney, J.R.A. and Chalmers, J., Evidence for an excitatory amino acid pathway in the brainstem and for its involvement in cardiovascular control, Brain Research, 496 (1989) 401-407. 21 Sun, M.K. and Guyenet, P.G., GABA mediated baroreceptor inhibition of reticulospinal neurons, Am. J. Physiol., 249 (1985) R672-R680. 22 Terui, N., Masuda, N., Saeki, Y. and Kumada, M., Activity of barosensitive neurons in the caudal ventrolateral medulla that send axonal projections to the rostral ventrolateral medulla in rabbits, Neurosci. Lett., 118 (1990) 211-214. 23 Urbanski, R.W. and Sapru, H.N., Evidence for a sympathoexcitatory pathway from the nucleus tractus solitarii to the ventrolateral medullary pressor area, J. Auton. Nerv. Syst., 23 (1988) 161-174. 24 Urbanski, R.W. and Sapru, H.N., Putative neurotransmitters in medullary cardiovascular regulation, J. Auton. Nerv. Syst., 25 (1988) 181-193. 25 Willette, R.N., Punnen, S., Krieger, A.J. and Sapru, H.N., Interdependence of rostral and caudal ventrolateral medullary areas in the control of blood pressure, Brain Research, 321 (1984) 169-174.
70
BRES 16825
Monosynaptic connection from caudal to rostral ventrolateral medulla in the baroreceptor reflex pathway S.K. Agarwal and F.R. Calaresu Department of Physiology, University of Western Ontario, London, Ont. (Canada) (Accepted 26 February 1991) Key words: Caudal ventrolateral medulla; Nucleus tractus solitarii; Rostral ventrolateral medulla; Arterial pressure; Baroreceptor
Experiments were done to test the hypothesis that caudal ventrolateral medulla (CVLM) neurons excited by activation of arterial baroreceptors and by stimulation of depressor sites in the nucleus tractus solitarii (NTS) project monosynaptically to the rostral ventrolateral medulla (RVLM). In urethan anaesthetized and artificially ventilated rats we recorded extracellular activity from 46 spontaneously firing units in the CVLM. Twenty of these units were excited by baroreceptor activation (1-3 #g phenylephrine i.v.) and of these 6 were excited (mean latency of 9.8 _+ 2.3 ms) by single pulses (0.1 ms, 30 _+ 8.3 #A) delivered once per second to a depressor site in the ipsilateral NTS. These 6 units were also antidromically activated with a latency of 4.1 + 0.12 ms by stimulation of a pressor region in the ipsilateral RVLM. These results provide evidence for the existence of an excitatory projection from the NTS to the CVLM which, in turn, projects monosynaptically to sympathoexcitatory neurons in the RVLM. INTRODUCTION
8,11,12,20,24o Indeed, in a recent study in the rabbit it has
Activation of sympathoinhibitory neurons in the caudal ventrolateral medulla (CVLM) reduces arterial pressure (AP) while inhibition of these neurons increases A P 8'9'17'25. The CVLM area is not defined precisely, but
been suggested that sympathoinhibition is mediated by an N T S - C V L M - R V L M pathway z2. The present study was designed to test the hypothesis that CVLM n e u r o n s excited by activation of arterial baroreceptors and by stimulation of depressor sites in the
it appears to be centered around the external formation
NTS send monosynaptic connections to the RVLM. This
of the nucleus ambiguus4 and to contain the A I noradrenergic cell group 25. The current view regarding one of
was done by monitoring the change in firing frequency of CVLM units following stimulation of the NTS and by attempting to demonstrate that C V L M n e u r o n s excited by NTS stimulation were antidromically activated by
the functions of the CVLM is that it mediates the decrease in A P to activation of baroreceptor fibers via an inhibitory projection from CVLM to sympathoexcitatory n e u r o n s situated in the rostral ventrolateral medulla ( R V L M ) 1-3'5'6'8"1°'18'21, which, in turn, project to spinal sympathetic preganglionic vasomotor neurons. Additional evidence regarding the role of the CVLM in the baroreceptor reflex has been obtained by anatomical studies which have shown that baroreceptor afferent fibres synapse on second order neurons located within the nucleus tractus solitarii (NTS) 13 which in turn project to the R V L M and CVLM m. Some reports suggest that an inhibitory projection from the NTS to the R V L M mediates sympathoinhibitory baroreflex responses 9'18, but other reports have favoured the notion that these sympathoinhibitory responses are mediated through an excitatory pathway from the NTS to the CVLM 24 from which an inhibitory pathway to the R V L M originates ~'
stimulation of pressor sites in the RVLM. MATERIALS AND METHODS Experiments were done in 19 adult male Wistar rats (250-350 g, Charles River, Montreal), anaesthetized with urethan (Sigma, St. Louis, MO, 1.4 g/kg, i.p. initially and supplemental doses of 0.14 g/kg s.c. when necessary). Rectal temperature was maintained at 37.5 + 5 °C with a thermostatically controlled heating blanket. The animals were paralyzed (decamethonium bromide, Sigma, 3.3 mg/kg i.v. initially, with 0.35 mg supplements every 15-30 min) and artificially ventilated with room air using a small animal ventilator (Harvard Apparatus, model 683). The femoral artery and vein were cannulated. The arterial cannula was connected to a pressure transducer (Statham P23 Db) which was connected to a Grass polygraph (model 7) for continuous recording of AP. A Grass tachograph (7P4C), triggered by the arterial pressure pulse was used to monitor heart rate (HR). The venous cannula was used to inject l-3/~g of phenylephrine (PE, Sigma, St. Louis, 0.1-0.3 ml of a 10 ~g/ml solution) for baroreceptor activation. The animal was placed
Correspondence: S.K. Agarwal, Department of Physiology, University of Western Ontario, London, Ont., Canada, N6A 5C1. Fax: (1) (519) 661-3827.
71 current to antidromically activate terminals of CVLM neurons 14. The RVLM stimulating electrode was moved in 200/~m steps from the ventral surface of the medulla. At the end of the experiment the recording sites in the CVLM were marked with iontophoretic deposits of Pontamine sky blue. Iron was deposited by passing a current through the tip of the stimulating electrodes in the NTS and the RVLM 2. Stimulation and recording sites were mapped on diagrams of transverse sections of the rat brain from an atlas )6. The location of the tips of the stimulating electrodes in the NTS and RVLM in transverse sections of the medulla is shown in Fig. 1A, B. At the start of each experiment a stimulating electrode was placed in the right RVLM at a site where electrical stimulation at 50 Hz, 0.1 ms pulse duration and 5-100 # A elicited increases in MAP of 30-40 mmHg. After a pressor site in the RVLM was found, another electrode was positioned in the ipsilateral NTS at a site where a clear depressor response in MAP of 20-30 mmHg was obtained with a 5 s train of stimuli at 50 Hz, 0.1 ms pulse duration and 5-100/~A. After placing the stimulating electrodes, the ipsilateral CVLM was searched for spontaneously active units. After a stable recording from a single unit in the CVLM was obtained, the response of each unit to an increase in mean AP was tested by recording the change in discharge elicited by an i.v. bolus injection of 1-3/~g phenylephrine (PE). For further characterization, the units were tested for rhythmicity of spontaneous activity in relation to the cardiac cycle by constructing a post R-wave time histogram 2. Only units identified by these two criteria were studied. When a barosensitive CVLM unit was found its response to stimulation of a depressor site in the NTS was tested by constructing peri-stimulus time histograms of unit activity. Stimuli to the NTS consisted of single pulses of 0.1 ms duration delivered once per second at an intensity ranging from 5 to 100/~A. Finally each CVLM unit responsive to NTS stimulation was tested for antidromic activation by stimulation of the RVLM.
in a stereotaxic apparatus with the bite bar 20 mm below the interaural line. The medulla was exposed and activity from spontaneously firing units in the CVLM was recorded extracellularly with glass electrodes filled with 0.5 M sodium acetate and 2% Pontamine sky blue (4-10 MI2 impedance measured at 1 kHz). The electrode was inclined 20° with respect to the vertical in the sagittal plane with the tip pointing rostrally and was advanced through the dorsal medulla into the CVLM (stereotaxic coordinates from interaurai line: -4.3 to --5.1 mm caudal to the interaural line, 1.8-2.1 mm lateral and 2.3-2.9 mm below the dorsal surface of the medulla) by a hydraulic microdrive (Narishige, model M08). Electrical activity was amplified through a preamplifier (Dagan 2400; bandpass 0.3-10 kHz), displayed on an oscilloscope (Tektronix R5103N) and discriminated by a Neurolog NL200 spike trigger. The frequency of firing of single units was recorded on a polygraph along with AP and HR. Unit activity derived from the spike trigger was fed to an IBM-AT computer for spike train analysis. The NTS and the RVLM could be stimulated by stainless-steel unipolar electrodes (tip diameter 3-10/~m, impedance - 1 M.Q) connected via a Grass PSIU-6 stimulus isolation unit to a Grass $88 stimulator. Electrodes were lowered stereotaxically to sites either in the NTS or in the RVLM from which a train of stimuli (50 Hz, 0.1 ms pulses, 5-100/~A for 5 s) elicited a decrease or an increase in HR and AP, respectively. The stereotaxic coordinates used for NTS stimulation were 0.6 mm rostral to the obex, 0.6 mm lateral to the midline and 0.6 mm below the dorsal surface of the medulla. For the RVLM the electrode was inclined 20° with respect to the vertical in the sagittal plane with the tip pointing caudally; the stereotaxic coordinates from the interaural line were: -2.6 to -3.3 mm caudal to the interaural line, 1.6-2.1 mm lateral and 7.2-8.0 mm below the surface of the cerebellum. The criteria for identifying an antidromic evoked action potential were: constant latency at threshold; ability to follow stimulation at 100 Hz; collision between spontaneous action potentials and evoked action potentials 14. We constructed depth threshold curves to identify sites requiring low stimulus
,,
_-4.3 ..B
A
1 4 / ~ _ _ ~ N , [ , ~ --"
"-""~
~..~)"~'("-2"6
:C\,,,
c;,~/'
-"'~-~-
"
v!,, \
',
r',
A.P':'--"--X .'""" _ ,z~ _~'_-.'
,.
c
..
..-.''
r,
N.4t~?oOt, LM?.t~_4.3 "':'~"~' .~,-~,71
,-.. \
~,,,*,~"-'>.', ~.~.,,"1,
,.,'~-..:--~..~
,':_.:,,,r
', ..
,,
.,.-_\
,
:,-s.,
i
---.
~
" i,,,,
Fig. 1. Location of sites of recording and stimulation in transverse sections of the rat medulla. Numbers indicate distances caudal to interaural line in millimetres, modified from Paxinos and Watson 16. A: sites of stimulation in the NTS where depressor responses were obtained. B: sites of stimulation in the RVLM where pressor responses were obtained. C: o, location of units in the CVLM which were activated by baroreceptors; ~l, units excited by electrical stimulation of NTS and antidromically activated from the RVLM. AP, area postrema; CU, cuneate nucleus; CVLM, caudal ventrolateral medulla; Gr, gracile nucleus; IO, inferior olive; NA, nucleus ambiguus; NTS, nucleus tractus solitarii; Pyr, pyramidal tract; RP, raphe pallidus; RVLM, rostral ventrolateral medulla; 4V, 4th ventricle; 7, facial nucleus; 10, dorsal motor nucleus of vagus; 12, hypoglossal nucleus.
72 RESULTS A region of the C V L M surrounded dorsally by the ventral aspect of the loose formation of the nucleus ambiguus and ventrally by the lateral reticular nucleus between 1.8 and 2.2 mm lateral to the midline was explored for spontaneously firing units. It is likely that the activity of units in this region was recorded from cell bodies for the following reasons. First, separation of the depolarization of the initial segment and of the somatodendritic region could be identified in most recordings. Second, the action potentials of these units had a duration greater than 1 ms; and third, electrical activity could be recorded over distances of several tens of micrometers of electrode tip displacement. Of the 46 spontaneously firing units from which recordings were obtained, 32 were identified as barosensitive neurons because they responded with an increase (n = 20) or a decrease (n = 12) in their activity during the rapid rise of arterial pressure (and baroreceptor mediated fall in H R ) induced by an i.v. bolus injection of 1-3 #g of PE. The 12 units which were inhibited by PE were not studied further because they did not exhibit an excitatory response to activation of baroreceptors and were therefore unlikely to be sympathoinhibitory C V L M units involved in the baroreceptor reflex. The mean spontaneous firing rate + S.E.M. of the 20 units which were excited was 12.8 + 1.7 spikes/s. The location of these neurons is shown in transverse sections of the medulla (Fig. 1C) and typical excitatory responses
of a unit to bolus injections of PE are shown in Fig. 2. Stimulation of depressor sites in the NTS increased the frequency of discharge of 6 of the 20 units excited by activation of baroreceptors, with a mean latency +_ S.E.M. of 9.8 + 2.3 ms (range 3-15 ms). This excitation lasted for 14.5 + 3.5 ms; during this period the firing rate was increased by 454 + 63.4% in comparison to control level in the 100 ms preceding stimulation. The average threshold intensity to produce excitation with single pulses was 30 + 8.3 ,uA. An example of excitatory responses of a C V L M neuron at different intensities of stimulation of the NTS is shown in Fig. 3. The remaining 14 units excited by activation of baroreceptors did not respond to NTS stimulation. Electrical stimulation of the R V L M evoked antidromic action potentials in the 6 neurons in the C V L M which were (i) excited by activation of baroreceptors, (ii)
A 20
=-
to
,a,4a,,, a,;.d,..L,a,,., , 0
t00
•
,
,
200
300
400
,
,
,
500
, 600
2 0 2 Sweeps
ms
B 20
A
B =
t0
200 t MAP
,,,,,=,;,,11
200 I
o
(mmHg) 100[
,,., , . l . .
,,..,., 1= =.,,,,.,,,,,, ,oo me
¢
j,,,,
BP (mmHg) 100
20
0= w =
10 1[
, ,.,.,,,I,. 2do
t 0 1 Sweeps
0 200
Unit activity (spikes/s)
Ido
10
.~
,
too
0.1 Fig. 2. Responses of a unit in the CVLM to baroreceptor activation induced by i.v. bolus injections of 1 (A) and 3 (B) pg of PE delivered at arrowheads. Note the increase in firing frequency and gradual return to control frequency as AP returns to basal levels.
102 Sweeps
aoo
300
,,,
400
,
soo
soo ms
Fig. 3. Peristimulus time histograms of activity of a CVLM neuron during electrical stimulation of the NTS at 0.1 ms, 1 pulse per s (A) 20/~A; (B) 30/~A; (C) 40/~A. Bin width 1 ms; stimulus artifact at 100 ms.
73
A
C
B ~00
u
•
b
A .2m 04.0
i
0.5
i
t.0 0ep~
/
i
1.5
m
! 2.1"
--
i 1ram
J
Fig. 4. Collision test and depth threshold curve to identify terminals of a unit in the CVLM responding to RVLM stimulation. A: records are 5 superimposed oscilloscope sweeps demonstrating a direct connection from a CVLM neuron to RVLM neurons by the collision test. Dots represent spontaneous spikes, the asterisk indicates an antidromically evoked action potential only after a critical delay of 2.2 ms (top record); in the bottom record a delay of 2 ms from a spontaneously occurring spike prevents antidromic potential. Arrowheads indicate stimulus (0.1 ms duration, 20/~A intensity) artifacts. Horizontal calibration, 2 ms; vertical calibration, 50 gV. B: depth threshold curve for antidromic activation of a CVLM neuron with two different onset latencies as indicated (0, 2.2 ms; ©, 4.0 ms) showing the presence of a discrete jump in latency. C: (a) represents the location of the CVLM neuron (located at 0.5 mm rostral to the obex, 1.9 mm right and 2.7 mm from the dorsal surface of the medulla); (b) solid vertical line shows the track of the stimulating electrode in the RVLM for the same neuron. The rostrocaudal plane of this section is 2.5 mm rostral to the obex.
excited by stimulation of the NTS, and (iii) showed cardiac cycle related rhythmicity. A n t i d r o m i c spike latencies were in the range of 1-10 ms (4.1 + 0.12 ms) c o r r e s p o n d i n g to an estimated m e a n conduction velocity of 0.62 + 0.08 m/s. T h e threshold intensity for producing antidromic spikes in neurons ranged b e t w e e n 5 and 100 /~A. T h e response of a C V L M neuron to the collision test and the d e p t h threshold curve for activation of this n e u r o n are shown in Fig. 4. DISCUSSION These e x p e r i m e n t s have shown that stimulation of b a r o r e c e p t o r s excites a population of C V L M neurons which are o r t h o d r o m i c a l l y excited by NTS stimulation and antidromically activated by R V L M stimulation. D e p t h threshold curves d e m o n s t r a t e that these neurons have extensive arborizations in the R V L M . The existence of this N T S - C V L M - R V L M p a t h w a y is s u p p o r t e d by the anatomical d e m o n s t r a t i o n of a pathway between NTS and C V L M 19, by microinjection experiments showing that NTS neurons project to the C V L M by a glutamatergic p a t h w a y which mediates vasodepressor responses 2, 23,24, and by the d e m o n s t r a t i o n that cardiovascular
neurons in the R V L M are inhibited by activation of cell bodies in the C V L M 1. T h e present study provides direct evidence that C V L M neurons are monosynaptically connected to the R V L M and that the same neurons are also excited o r t h o d r o m i c a l l y by NTS stimulation. Topographically these neurons are located in the same area where microinjections of L - g l u t a m a t e have been shown to elicit decreases in A P and inhibition of cardiovascular neurons in the R V L M 1. These results, t a k e n t o g e t h e r with our previous findings 1, d e m o n s t r a t e that an excitatory projection exists between the NTS and the C V L M which, in turn, provides a m o n o s y n a p t i c p r o j e c t i o n to the R V L M . Similar results have b e e n o b t a i n e d recently in the rabbit 22. Finally, we found that 12 s p o n t a n e o u s l y firing C V L M units were inhibited by bolus injections of P E and a n o t h e r group of 14 units did not respond. It is likely that these units are not involved in b a r o r e c e p t o r reflex function. Alternatively, it is possible that these neurons may be involved in controlling the activity of vasopressin secreting cells in the supraoptic nuclei TM. M o r e experiments are n e e d e d to investigate this possibility. In s u m m a r y , these results provide evidence for the existence of an excitatory p r o j e c t i o n from the NTS to the
74 CVLM
which,
in turn,
projects
monosynaptically
to
s y m p a t h o e x c i t a t o r y n e u r o n s in the R V L M .
REFERENCES 1 Agarwal, S.K., Gelsema, A.J. and Calaresu, ER., Neurons in rostral VLM are inhibited by chemical stimulation of caudal VLM in rats, Am. J. Physiol., 257 (1989) R265-R270. 2 Agarwal, S.K., Gelsema, A.J. and Calaresu, ER., Inhibition of rostral VLM by baroreceptor activation is relayed through caudal VLM, Am. J. Physiol., 258 (1990) R1271-R1278. 3 Barman, S.M. and Gebber, G.L., Axonal projection patterns of ventrolaterai meduUospinal sympathoexcitatory neurons, J. Neurophysiol., 53 (1985) 1551-1566. 4 Bieger, D. and Hopkins, D.A., Viscerotopic representation of the upper alimentary tract in the medulla oblongata in the rat: the nucleus ambiguus, J. Comp. Neurol., 262 (1987) 546-562. 5 Brown, D.L. and Guyenet, P.G., Electrophysiological study of cardiovascular neurons in the rostral ventrolateral medulla in rats, Circ. Res., 56 (1985) 359-369. 6 Dampney, R.A.L., Goodchild, A.K., Robertson, L.G. and Montgomery, W., Role of ventrolateral medulla in vasomotor regulation: a correlative anatomical and physiological study, Brain Res., 249 (1982) 223-235. 7 Day, T.A. and Sibbald, J.R., A 1 cell group mediates solitary nucleus excitation of supraoptic vasopressin cells, Am. J. Physiol., 257 (1989) R1020-R1026. 8 Gordon, EJ., Aortic baroreceptor reflexes are mediated by NMDA receptors in the caudal ventrolateral medulla, Am. J. Physiol., 252 (1987) R628-R633. 9 Granata, A.R., Kumada, M. and Reis, D.J., Sympathoinhibition by A~ noradrenergic neurons in the C~ area of the rostral medulla, J. Auton. Nerv. Syst., 14 (1985) 387-395. 10 Granata, A.R., Numao, Y., Kumada, M. and Reis, D.J., A1 noradrenergic neurons tonically inhibit sympathoexcitatory neurons of C1 area in rat brainstem, Brain Research, 377 (1986) 127-146. 11 Guyenet, P.G., Sun, M. and Brown, D.L., Role of GABA and excitatory amino acids in the medullary baroreflex pathways. In J. Ciriello, F.R. Calaresu, L. Renaud and C. Polosa (Eds.), Organization of the Autonomic Nervous System: Central and Peripheral Mechanisms, Liss, New York, 1987, pp. 215-225. 12 Guyenet, P.G., Filtz, T.M. and Donaldson, S.R., Role of excitatory amino acids in the rat vagal and sympathetic baroreflexes, Brain Research, 407 (1987) 272-284. 13 Kalia, M.P., Localization of aortic and carotid baroreceptor and chemoreceptor primary afferents in the brainstem. In J.P. Buckley and C.M. Ferrario (Eds.), Central Nervous System Mechanisms in Hypertension, Raven, New York, 1981, pp. 9-23.
Acknowledgements. This work was supported by a grant from the Medical Research Council of Canada. S.K.A. is a Fellow of the Canadian Heart and Stroke Foundation. D.J. McKitrick provided useful critical comments.
14 Lipski, J. Antidromic activation of neurons as an analytical tool in the study of the central nervous system, J. Neurosci. Meth., 4 (1981) 1-32. 15 McAllen, R.M. and Blessing, W.W., Neurons (presumably A~ cells) projecting from the caudal ventrolateral medulla to the region of the supraoptic nucleus respond to baroreceptor inputs in the rabbit, Neurosci. Lett., 73 (1987) 247-252. 16 Paxinos, G. and Watson, C., The Rat Brain in Stereotaxic Coordinates, Academic Press, Sydney, 1986. 17 Pilowsky, P.M., Morris, M.J., Minson, J.B., West, M.J., Chalmers, J.P., Willoughby, J.O. and Blessing, W.W., Inhibition of vasodepressor neurons in the caudal ventrolateral medulla of the rabbit increases both arterial pressure and the release of neuropeptide Y-like immunoreactivity from the spinal cord, Brain Research, 420 (1987) 380-384. 18 Ross, C.A. Ruggiero, D.A., Park, D.H., Job, J.H., Sved, A.E, Fernandez-Pardal, J., Saavedra, J.M. and Reis, D.J., Tonic vasomotor control by the rostral ventrolateral medulla: effect of electrical and chemical stimulation of CI adrenaline containing neurons on arterial pressure, heart rate, and plasma catecholamines and vasopressin, J. Neurosci., 4 (1984) 474-494. 19 Ross, C.A., Ruggiero, D.A. and Reis, D.J., Projections from the nucleus tractus solitarii to the rostral ventrolateral medulla, J. Comp. Neurol., 242 (1985) 511-534. 20 Somogyi, P., Minson, J.B., Morilak, D., Smith, 1.L., McIlhinney, J.R.A. and Chalmers, J., Evidence for an excitatory amino acid pathway in the brainstem and for its involvement in cardiovascular control, Brain Research, 496 (1989) 401-407. 21 Sun, M.K. and Guyenet, P.G., GABA mediated baroreceptor inhibition of reticulospinal neurons, Am. J. Physiol., 249 (1985) R672-R680. 22 Terui, N., Masuda, N., Saeki, Y. and Kumada, M., Activity of barosensitive neurons in the caudal ventrolateral medulla that send axonal projections to the rostral ventrolateral medulla in rabbits, Neurosci. Lett., 118 (1990) 211-214. 23 Urbanski, R.W. and Sapru, H.N., Evidence for a sympathoexcitatory pathway from the nucleus tractus solitarii to the ventrolateral medullary pressor area, J. Auton. Nerv. Syst., 23 (1988) 161-174. 24 Urbanski, R.W. and Sapru, H.N., Putative neurotransmitters in medullary cardiovascular regulation, J. Auton. Nerv. Syst., 25 (1988) 181-193. 25 Willette, R.N., Punnen, S., Krieger, A.J. and Sapru, H.N., Interdependence of rostral and caudal ventrolateral medullary areas in the control of blood pressure, Brain Research, 321 (1984) 169-174.
