1. health and fitness
2. disease

Brain Research, 574 (1992) 320-324
© 1992 Elsevier Science Publishers B.V. All rights reserved. 0006-8993/92/$05.00
320
BRES 25080
Electrical stimulation of nucleus tractus solitarius excites vagal
preganglionic cardiomotor neurons of the nucleus ambiguus in rats
S.K. Agarwal and F.R. Calaresu
Department of Physiology, University of Western Ontario London, Ont., N6A 5C1 (Canada)
(Accepted 26 November 1991)
Key words: Nucleus ambiguus; Nucleus tractus solitarius; Arterial pressure; Heart rate; Cardiovascular regulation; Vagal preganglionic
cardiomotor neuron
Recent evidence indicates that the cell bodies of vagal cardioinhibitory neurons are located principally in the external formation of the
nucleus ambiguus (NA). As activation of baroreceptor afferent fibers projecting to the nucleus tractus solitarius (NTS) elicits a decrease in
heart rate it is likely that there is a connection between the NTS and NA. To test the hypothesis that stimulation of the NTS can excite vagal
preganglionic cardiomotor neurons (VPCN) in the NA, activity from 78 neurons in the NA was recorded extracellularly before and during
stimulation of a depressor site in the NTS (1 Hz, 0.1 ms) in urethan anesthetized and artificially ventilated male Wistar rats. Sixteen neurons
were characterized as vagal preganglionic cardiomotor neurons (VPCN) because they were excited by baroreceptor activation (1-3 ~g phenylephrine i.v.) and showed rhythmicity of their spontaneous activity in synchrony with the cardiac cycle. Stimulation of the NTS increased
the firing rate of all these VPCN. The remaining 62 neurons could not be considered as VPCN because they either had respiratory rhythmicity or were not sensitive to baroreceptor activation, or they were sensitive to baroreceptor activation but did not display cardiac cycle
related rhythmicity. These results provide evidence for the existence of an excitatory pathway from NTS to vagal preganglionic cardiomotor
neurons in the NA.
The site of origin of vagal preganglionic c a r d i o m o t o r
neurons (VPCN) has b e e n a m a t t e r of controversy for a
long time 3. The results of electrophysiological and horseradish peroxidase ( H R P ) tracing studies have indicated
that the nucleus ambiguus ( N A ) is the major brainstem
site of efferent V P C N , although cardioinhibitory fibers
also rise from the dorsal m o t o r nucleus of the vagus in
some species (cat, rabbit) 4'5'8'9'14. The N A consists of
three major divisions (a) a rostral compact area which
contains oesophageal m o t o n e u r o n s ; (b) a semicompact
area which contains neurons innervating the pharynx and
the larynx, and (c) the loose (external) formation which
contains preganglionic p a r a s y m p a t h e t i c o r V P C N projecting to the heart and o t h e r thoracic organs 2. R e c e n t
anatomical and electrophysiological evidence suggests
that the ventrolateral division of the loose formation of
the N A is the main site of origin of the V P C N in most
mammals (see reviews, refs. 9,14).
Physiological investigations have d e m o n s t r a t e d that
some neurons in the N A r e s p o n d to stimulation of the
carotid sinus nerve as well as to increased sinus pressure ~A3 and can be antidromically activated by electrical
stimulation of the cardiac branches of t h e vagus 5A6'17.
F u r t h e r m o r e , it has b e e n found that most of these neu-
rons have a relatively low level of ongoing activity in
anesthetized cats 4'13, rabbits 1° and rats 18'19 and fire rhythmically in time with the cardiac cycle 1°. Finally, stimulation of the cell bodies of these neurons has b e e n shown
to produce cardiac slowing 1'6'7 and lesions of this area
p r o m o t e the d e v e l o p m e n t of hypertension in sinoaortic
d e n e r v a t e d rats 15.
A d d i t i o n a l information about the functional connections of b a r o r e c e p t o r reflex pathways has come from anatomical studies showing that b a r o r e c e p t o r afferent fibers synapse on second o r d e r neurons located within the
nucleus tractus solitarius x2 (NTS). The NTS in turn sends
projections to N A mainly from the medial NTS in cats TM
14, and there are also reciprocal connections between
N A to the NTS. Injections of Phaseolus vulgaris leucoagglutinin ( P H A - L ) into the commissural NTS result in
light labeling in the N A 21. Second o r d e r b a r o r e c e p t o r
neurons are generally assumed to project to V P C N in
and around the N A near the obex 14.
The present study was designed to investigate the
functional characteristics of the pathway from the NTS
to the N A by recording extracellular activity from spontaneously firing neurons in the N A during stimulation of
the NTS. O n l y N A neurons which were excited by
Correspondence and present address: S.K. Agarwal, Playfair Neuroscience Unit, The Toronto Hospital, Mc 11-409, 399 Bathurst Street,
Toronto, Ont., Canada M5T 2S8. Fax: (1) (416) 369-5397.
321
baroreceptor activation and displayed a clear cardiac
rhythm were classified as vagal preganglionic cardiomotor neurons.
Experiments were done in 14 adult male Wistar rats
(250-325 g, Charles River, Montreal), anesthetized with
urethan (Sigma, St.Louis, MO, 1.4 g/kg, i.p.). 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). Supplemental doses of urethan were administered
when necessary. 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 electrocardiogram (ECG, lead II) was recorded with subcutaneous electrodes. The venous cannula was used to inject 1-3 /~g of phenylephrine (PE,
Sigma, St. Louis, 0.1-0.3 ml of a 10/zg/ml solution) for
baroreceptor activation. The animal was placed in a stereotaxic apparatus, with the bite bar 20 mm below the
interaural line. The medulla was exposed by retracting
the dorsal neck muscles, incising the atlanto-occipital
membrane, and removing part of the occipital bone and
the dura. Rectal temperature was maintained at 37.5 +
0.5°C with a thermostatically controlled heating blanket.
Activity from spontaneously firing units in the nucleus
ambiguus (NA) was recorded extracellularly with glass
micropipettes filled with 0.5 M sodium acetate and 2%
pontamine sky blue (4-10 Mf~ 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 NA (stereotaxic coordinates: 0.7-0.3 m m rostral to the obex, 1.8-2.0 mm lateral and 2.0-2.3 mm below the dorsal surface of the brain) 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. Digitized
unit activity (derived from the spike trigger) along with
stimulation markers, the AP wave and E C G were also
fed to an IBM-AT computer using a Data Translation
board (DT 2801A). Data were analyzed by the use of a
custom-written program for spike train analysis and of
Macmillan's ASYSTANT-plus software for averaging
E C G and AP signals, together with neural activity.
Electrical stimuli to the NTS were applied through a
stainless steel unipolar electrode (tip diameter 3-10/~m,
impedance - 1 Mfl, shaft diameter approximately 175
#m insulated with Insl-X, Insl-X Co, Yonkers, N.Y., except for the tip) connected via a Grass PSIU-6 stimulus
isolation unit to a Grass $88 stimulator. The tip of the
electrode was lowered stereotaxicaUy to a site in the
NTS from which a train of pulses (50 Hz, 0.1 ms, 10-75
HA for 5 s) elicited a decrease in H R and AP. The stereotaxic coordinates used for the NTS electrode 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.
After a responsive site in the NTS was found, the stimulating electrode remained in that site throughout the
experiment. Values given in the text are means +
S.E.M., unless indicated otherwise.
For histological verification recording sites in the NA
were marked with iontophoretic deposits of pontamine
/12 ,)
"''
OL
,,o
PI
' ':::' ...-':-_1/
i
~" :~.~::'.-.
-4.2
.
.
.
/
.
~
I
-4.8
4:,,,')
I!
i
II
..... -%.-"
•
.*
-4.3
.,
/"
~-5.1
E\ "% /
I ,//
..4.7
I
I
lmm
Fig. 1. Diagrammatic transverse sections of medulla showing location of stimulation sites in the tight NTS (filled circles) and recording sites in the right NA (open circles). Numbers indicate distance
caudal to interaural line in millimeters. Sections modified from Paxinos and Watson2°. 10, dorsal motor nucleus of the vagus; 12, hypoglossal nucleus; CU, cuneate nucleus; Gr, gracile nucleus; IO,
inferior olive; LRN, lateral reticular nucleus; NA, nucleus ambiguus; NTS, nucleus tractus solitarii; py, pyramid; RO, raphe obscurus; RP, raphe pallidus; 4V, fourth ventricle.
322
B
A
HR 600[
50
(bpm) 400 I
200 L
MAP 200 I
(mmHg) 100[
.,=
40,
CL
30
0
O
L~
B P 200 f
(mmHg) 100
g
x
EO
x
x
U,.
0
,L.J-Jd=l=J
.I . i . l . . l
f.
.
.
.
.
j
"0
g
C
g
10
Unit
act i vity
( spikezls )
t),
-150
-100
Change In
-50
HR
0
(bpm)
Fig. 2. A: responses of a unit to baroreceptor activation induced by i.v. bolus injections of 1 and 3 ag PE (at arrowheads). Note an increase
in firing frequency to the same level with 1 and 3/~g PE. Time scale in seconds. B: relationship of firing rate and HR. Points representing
number of spikes and values of HR in 14 sample periods taken at different levels of HR. Dashed line represents line of best fit (r = 13.9483;
P < 0.001),
sky blue (15 /~A of cathodal current for 8 min). Iron
from the tip of the stimulating electrode in the NTS was
deposited by passing an anodal current of 20 #A for 30
s and was later revealed by the Prussian Blue reaction.
At the end of the experiments the animals were perfused
with 50 ml of phosphate buffer solution (PBS) followed
by 50 ml of a 10% formalin solution in PBS. For detecting iron deposits, 0.5 g of potassium ferrocyanide was
added to the 50 ml of 10% formalin. The brains were
removed and stored in formalin for 3-4 days. Frozen
transverse sections (50 am) were cut and stained with
thionine. Stimulation and recording sites were mapped
on diagrams of transverse sections of the rat brain from
an atlas 2°.
After placing the stimulating electrode in the NTS,
the ipsilateral NA was searched for spontaneously active
units. After a stable recording from a single unit in the
NA was obtained, each unit was tested for its barosensitivity by recording the change in discharge rate in response to an increase in mean AP elicited by an i.v. bolus injection of 1-3/~g phenylephrine (PE), and for the
presence of rhythmicity of discharge in relation to the
cardiac cycle. A Schmit trigger circuit was used to derive standardized pulses coincident with a point halfway
up the rising slope of the R wave of the ECG. These
pulses were used to construct peri R wave histograms of
NA unit activity. Units with an abruptly increased firing
frequency by baroreceptor activation and which displayed cardiac cycle synchronous rhythmicity were regarded as 'vagal preganglionic cardiomotor neurons'
(VPCN).
The external formation of the NA from 700 pm rostral to 300 am caudal to the obex was searched for
VPCN. Spontaneous activity was recorded from 78 neurons in and around the nucleus ambiguus (NA) and ventral to neurons with large spikes and a respiration-related discharge pattern. The electrical activity of the NA
units was probably recorded from cell bodies because
separation of the depolarization of the initial segment
and the somatodendritic region could be identified in
most recordings, the action potentials of these units had
a duration of >1 ms, and electrical activity could be recorded over distances of several tens of #m of electrode
tip displacement. Of these 78 units, only 16 were identified as VPCN and these neurons were located in the
ventrolateral portion of the NA (Fig. 1) because they
responded with an immediate increase in activity during
the rapid rise of blood pressure and decrease in heart
rate induced by an intravenous injection of 1-3 ag of
phenylephrine (PE), and they displayed a clear cardiac
323
was established. Fig. 2B shows this relationship for a
VPCN. W h e n the change in H R was more than 100 bpm
the change in firing frequency remained constant.
In peri R wave histograms of the discharge of each of
these 16 units, rhythmicity of their activity in synchrony
with the cardiac cycle was apparent at control A P values
(Fig. 3). The mean firing of these 16 V P C N at control
A P was 2.8 + 0.57 spikes/s. The location of these neurons is shown in Fig. 1. The remaining 62 units were either respiratory (65%, 40/62) or were not sensitive to
baroreceptor activation (16%, 10/62) or they were sensitive to baroreceptor activation but did not display cardiac cycle related rhythmicity (19%, 12/2). These units
were not considered VPCN and were not studied further.
Stimulation of the depressor site in the NTS increased
the discharge activity of all the 16 V P C N after an average latency of 7.4 + 1.2 ms (range 4-14 ms). This excitation lasted for 20.3 + 2.4 ms; during this period the
firing rate was increased by 576.5 + 146.7% in comparison to control level in the 100 ms preceding stimulation.
The average threshold intensity to produce excitation
with single pulses was 30.0 + 7.2/~A. A n example of an
excitatory response of V P C N at different intensities of
stimulation of the NTS is shown in Fig. 4. The histolog-
"I-
EE
80
c
t0
0
0
!00
3742 Sweeps
200
300
400
500
SO0
me
Fig. 3. Peri R wave histogram oE unit activity taken from control
period of recording from a vagal preganglionic cardiomotor neuron
in NA. Top trace: electrocardiogram; middle trace: AP; bottom
trace: unit activity. Bin width 1 ms. Triggering R wave for 3742
sweeps shown at time = 100 ms.
cycle related rhythmicity. Typical responses of a spontaneously firing V P C N to bolus injections of P E are shown
in Fig. 2A. In addition, these units increased their firing
frequency to a maximum beyond which firing frequency
was not increased (Fig. 2A) even though the H R response increased. A negative linear relationship (r =
0.9483) between the firing rate and the change in H R
A
C
20
2o
m to
:
10
g
1
|Ill
o
t54
I LIII'LL' ,,,...Ih
IIlillllk
,~o
Sweeps
2do
Ii.I i L'- i,IlJ-lh iilll, .ia. ,,,lI.,
a~o
4~o
s~o
ilili, li II
I II I
600
o
ms
t52
B
Sweeps
i
II lIlII
|l I||
II
h i ii
i i
I
ik i illl i
i I
2~o
3~o
4~o
5~o
6oo
200
300
400
500
600
ma
D
20
ca
I
~o
20
t0
I,
0
i52
t00
Sweeps
200
300
400
500
600
ms
0
15t
i00
Sweeps
me
Fig. 4. Peristimulus time histogram of activity of a vagal preganglionic cardiomotor neuron of NA during electrical stimulation of the NTS at
different current intensities. Shock artifact at 100 ms. A: 10/~A B: 20/~A C: 25/~A D: 30/~A.
324
ically identified stimulation sites in the NTS are shown
in Fig. 1.
O u r experiments showing that electrical stimulation of
NTS excites VPCN in the N A demonstrate the existence
of an excitatory pathway from the NTS to VPCN of NA.
To our knowledge this is the first electrophysiological
demonstration of this pathway. We recorded spontaneous neuronal activity of 78 units in the external formation of the NA, a location known to contain VPCN 14,
and we found 16 units that were excited by baroreceptor
activation, showed modulation of their activity in synchrony with the cardiac cycle at control levels of mean
arterial pressure, and had a low level of discharge rate.
neurons to baroreceptor activation, i.e. increase of discharge rate and rhythmicity of their activity in synchrony
with cardiac cycle, have been demonstrated previously in
neurons in the N A that could be antidromically activated
by stimulation of the cardiac branches of the vagus ~6'~7.
Third, these units were excited by NTS stimulation with
a mean latency of 7.4 _+ 1.2 ms (range 4-14 ms). This
latency, with an estimated distance from NTS to N A of
2 mm, and assuming no intervening synapses, corresponds to a m e a n conduction velocity of 0.27 m/s which
is in the range of size of small unmyelinated fibers. In
summary, considering all the evidence available it is rea-
The low level of activity is in agreement with earlier
studies in cats 4A3, rabbits 1° and rats 18'19. Units showing
sonable to conclude that the 16 neurons described here
indeed represent vagal preganglionic cardiomotor neurons which are excited by an excitatory pathway from
these characteristics are likely VPCN for the following
nucleus tractus solitarius to VPCN in the NA.
reasons. First, the ventrolateral portion of the N A from
which we recorded VPCN activity (see Fig. 1) is established as the location of cell bodies of cardiomotor fibers 9A4. Second, the characteristic responses of these
This study was supported by a grant from the Medical Research
Council of Canada to ER.C. and a Canadian Heart and Stroke
Foundation Fellowship to S.K.A.
1 Agarwal, S.K. and Calaresu, ER., Enkephalins, substance-P
and acetylcholine microinjected into the nucleus ambiguus elicit
vagal bradycardia in rats, Brain Res., 563 (1991) 203-208.
2 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.
3 Calaresu, ER., Faiers, A.A. and Mogenson, G.J., Central neural regulation of heart and blood vessels in mammals, Prog.
Neurobiol., 5 (1975) 1-35.
4 Ciriello, J. and Calaresu, ER., Medullary origin of vagal
preganglionic axons to the heart of the cat, J. Auton. Nerv.
Syst., 5 (1982) 9-22.
5 Ciriello, J. and Calaresu, ER., Distribution of vagal cardioinhibitory neurons in the medulla of the cat, Am. J. Physiol., 238
(1980) R57-R64.
6 Ermirio, R., Ruggeri, P., Cogo, C.E., Molinari, C. and Calaresu, ER., Neuronal and cardiovascular responses to ANF
microinjected into the nucleus ambiguus, Am. J.Physiol., 260
(1991) R1089-R1094.
7 Geis, G.S. and Wurster, R.D., Cardiac responses during stimulation of the dorsal motor nucleus and nucleus ambiguus in the
cat, Circ. Res., 46 (1980) 606-611.
8 Geis, G.S., Kozelka, J.W. and Wurster, R.D., Organization
and reflex control of vagal cardiomotor neurons, J. Auton.
Nerv. Syst., 3 (1981) 436-450.
9 Hopkins, D.A., The dorsal motor nucleus of the vagus nerve
and nucleus ambiguus: structure and connections. In R.
Hainsworth, P.N. McWilliams and D.A.S.G. Mary (Eds.),
Cardiogenic Reflexes, University Press, Oxford, 1987, pp.
185-203.
10 Jordan, D., Khalid, M.E.M., Schneiderman, N. and Spyer,
K.M., The location and properties of preganglionic vagal cardiomotor neurons in the rabbit, Pfltigers Arch., 395 (1982)
244-250.
11 Kalia, M., Feldman, J.L. and Cohen, M.I., Afferent projections to the inspiratory neuronal regions of the ventrolateral
nucleus of the tractus solitarius in the cat, Brain Res.,
171 (1979) 135-141.
12 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.
13 Lipski, J., McAllen, R.M. and Trzebski, A., Carotid baroreceptor and chemoreceptor inputs onto single medullary neurons, Brain Res., 107 (1976) 132-136.
14 Loewy, A.D. and Spyer, K.M., Vagal preganglionic neurons.
In A.D. Loewy and K.M. Spyer (Eds.), Central Regulation of
Autonomic Function, Oxford University Press, Oxford, 1990,
pp. 68-87.
15 Machado, B.H. and Brody, M.J., Effect of nucleus ambiguus
lesion on the development of neurogenic hypertension, Hypertension, 11 (1988) 1-135-I-138.
16 Maqbool, A., Batten, T.EC. and McWilliam, P.N., Ultrastrucrural relationship between GABAergic terminals and cardiac
vagal preganglionic motoneurons and vagal afferents in the cat:
a combined HRP tracing and immunogold labelling study, Eur.
J. Neurosci., 3 (1991) 501-513.
17 McAllen, R.M. and Spyer, K.M., The location of cardiac vagal
preganglionic motoneurons in the medulla of the cat, J. Physiol., 258 (1976) 187-204.
18 McAllen, R.M. and Spyer, K.M., The baroreceptor input to
cardiac vagal motoneurons, J. Physiol., 282 (1978) 365-374.
19 Nosaka, S., Yamamoto, T. and Yasunaga, K., Localization of
vagal cardioinhibitory preganglionic neurons within rat brain
stem, J. Comp. Neurol., 186 (1979) 79-92.
20 Nosaka, S., Yasunaga, K.and Tamai, S., Vagal cardiac preganglionic neurons: distribution, cell types and reflex discharges,
Am. J. Physiol., 243 (1982) R92-R98.
21 Paxinos, G. and Watson, C., The Rat Brain in Stereotaxic Coordinates, Academic Press, Sydney, 1986.
22 Sawchenko, EE., Cunningham, Jr., E.T. and Levin, M.C., Anatomical and biochemical specificity in central autonomic pathways. In J. Ciriello, ER. Calaresu, L.P. Renaud and C. Polosa
(Eds.), Organization of the Autonomic Nervous System: Central
and Peripheral Mechanisms, Liss, New York, 1987, pp.
267-281.