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
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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
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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-
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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
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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.
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
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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
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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-
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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
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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.
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