Cardiovascular effects of neurotensin microinjections into the nucleus of the solitary tract

ELSEVIER

Research report

Cardiovascular effects of neurotensin microinjections into the nucleus of the solitary tract

Abstract Neurotensin (NT)

immunorcactivity

nucleus of the solitary tract (NTS). analogues NT 1-8 and [D-Trp”]NT Microinjection

Hg).

hita

havtz bctm

dtmo~~a-;l~~d

to t-w

e\tcnhivcl>

distributed throughout the caudal

of NT ( 10 pmol) elicited decreases in arterial pressure (AP) ( - 34 + 3 mm Hg) and heart rate (HR) ( - 28 f 2 beats/min).

whereas microinjection mm

and bindin;

In this study. the cardiovascular effects of microinjectin g the tridecapeptide neurotensin (NT) or its into NTS were investigated in the chloralose-anesthetized. paralyzed and artificially ventilated rat.

of equimolar amounts of the NT fragment NT 1-8 elicited a significantly

smaller depressor response ( - II k 3

but the bradycardic (-

22 + -I beats/min) response was similar in magnitude to that elicited by NT. On the other hand. did not-elicit cardiovascular responses from sites in NTS. In addition. the prior injection of [D-T$']NT

microinjection of [D-Trp”]NT

into cardiovascular responsive sites in the NTS did not signiticantly reduce the AP or HR response to NT. The depressor response elicited by NT was not affected by bilateral vagotomy but was abolished by either CI-CZ nicotinic receptor blocker hexamethonium beats/mink

spinal cord transection or the i.v. administration of the

bromide. The cardiac slowing was partially attenuated by either bilateral vagotomy ( - 19 k 2

i.v. administration

of atropine methyl bromide ( - 17 f 3 beats/min). i.v. administration of hexamethonium bromide or by spinal cord transection ( - I 2 _+ _3 bcuts/min). and completely abolished after total autonomic blockade or by combined bilateral vagotomy and spinal cord transection. These data have demonstrated that within a restricted region of the caudal NTS ( - I I + 4 beats/min)

NT activates neurons that contribute to vasodepressor responses ah a result of sympatho-inhibition of vagi’l excitation

and sympatho-inhibition.

central cardiovascular rellcx

Furthcrmorc. these data suggest that NT may act as ;I ntlun~tr~‘nsmitIt’r or mclkk’tor

in

piitllWil~S.

1. Introduction The nucleus of the solitary tract (NTS) is known to play an important role in the central control of the cardiovascuiar system [27,49]. This is based primarily on a large body of electrophysiological [6,12.22,23,28,29,47] and neuroanatomical [5,6.8,10,11,24,46,54,56] evidence demonstrating that afferent fibers originating in systemic baroreceptors and chemoreceptors terminate within the caudal two-thirds of the NTS. In addition, activation of these systemic receptors alters the discharge rate of NTS neurons. Furthermore, stimulation of NTS neurons with the excitatory amino acid L-glutamic acid has been shown to elicit cardiovascular responses similar to those evoked

’ Corresponding author. [email protected]

and to bradycardiu responses as a resuh

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during activation of baroreceptors and chemoreceptors [ 13,351. Recently, there has been a considerable amount of experimental data presented suggesting that the tridecapeptide neurotensin (NT) may be involved in central pathways controlling the circulation. NT, first isolated and characterized chemically by Carraway and Leeman 14) from hypothalamic extracts, has been shown to have an extension distribution throughout the central nervous system [20]. Within the caudal NTS. cell bodies and axonal processes immunoreactive to NT have been observed [ 18,20,21,30,55]. In addition, Higgins et al. [ 181 have demonstrated that within the caudal NTS region the greatest density of NT immunoreactivity was found where aortic baroreceptor afferent fibers have been shown to terminate. Furthermore, these regions of NTS have been shown to have specific high affinity binding sites for NT [25,40,58], and iontophoretic application of neurotensin has been shown to excite neurons in the NTS region and to

0006-8993/97/$17.00 Copyright 0 1997 Elsevier Science B.V. All rights reserved. PII SOOO6-8993(96)01 176-6

evoke apneustic breathing [34]. Finally, intracerebroventricular injections of NT have also been shown to elicit either hypotension [39,43] or hypertension WI. In addition, microinjection of NT into the NTS region has been reported to enhance the depressor and bradycardic responses to aortic nerve stimulation [26]. Taken together. this evidence suggests that NT may exert an effect on NTS neurons that control the cardiovascular system. Therefore. the present study was done to systematically explore the region of NT’S for cardiovascular responsive sites to NT microinjection and to determine the components of the autonomic nervous system that mediated the cardiovascular responses. Furthermore, the ability of a NT fragment (NT t-8) [39] to elicit cardiovascular responses from similar site was tested. Finally. the effect of a potential NT antagonist ([D-Trp” ]NT) [39] on the cardiovascular responses to NT was investigated.

2. Methods and materials Experiments were done in 61 male Wistar rats (250-350 g) anesthetized with alpha-chloralose (60 mg/kg, iv. and additional doses of lo-30 pg/kg every I-2 11)after initial induction with Equithesin (0.3 ml/ 100 g. i.p.). The trachea was cannulated and the animals were paralyzed with pancuronium bromide (Pavulon, Organon Toronto, Canada: 0.8 ml/kg, i.v. initially and additional doses of 0.5 mg/kg when necessary), and they were artificially ventilated with 95% oxygen and S% room air using a rodent respirator (Harvard Apparatus, South Natick, MA; model 683). In three rats. the paralyzing agent was uot administered so that the e: I;ct of the pancuronium bromide on the magnitude of the cardiovascular responses could be investigated. Polyethylene catheters were inserted into the right femoral artery and vein for the recording of arterial pressure (AP) and the administration of drugs, respectively. AP was recorded through a Statham transducer (model P23D6) and a 7P4H Grass tachograph triggered by the AP pulse was used to monitor heart rate (HR). Both AP and HR were continuously recorded on a Grass model 79D polygraph. Body temperature was monitored and maintained at 37 + 0.2OC by a heating pad controlled from a Yellow Springs 73A temperature controller. The animals were placed into a Kopf stereotaxic apparatus and access to the dorsal brainstem was obtained by a partial occipital craniotomy. The dura was removed and the caudal floor of the fourth ventricle was exposed by gently removing the vermis of the cerebellum by suction. The region of the NTS was systematically explored in a grid pattern using obex as the point of reference and with penetmtions of the micropipette 300 pm apart. The stereotaxic coordinates for the NTS were obtained from an atlas

of the rat brain (381. Solutions were microinjected into the region of the NTS using double-barrelled or triple-barrelied glass micropipettes pulled from Socorex 5 PI capit-

lary tubing (Mississauga. Canada) with tip diameters of 30-45 lm. The injected volumes (S-25 nl) were measured by direct observation of the fluid meniscus in the micropipettes by usin,0 a microscope fitted with an ocular scale that allowed a resolution of 1 nl. The micropipettes were connected via PE-20 tubing to a Medical Systems pneumatic pressure pump (model PPM-2) for the microinjection of the solutions into the NTS region. In some experiments (IO), each of the barrels of the triple-barrelled pipettes was filled with one of a different amount of NT (0.5-15 pmol/nl dissolved in 0.9% saline, pH 7.2-7.4; Sigma Chemical, St. Louis. MO) and stereotaxically lowered into the caudal NTS region. Injections of the lower amount of NT were done first followed by the microinjection of the higher amount 45 min after the initial injection at the same site. As it was found that 10 pmol 01 NT elicited approximately the largest cardiovascular responses, the NTS region was explored for NT sensitive sites using this amount of NT. In additional experiments, one of the barrels of triplebarrelled micropipettes was filled with NT (Sigma Chemical, St. Louis, MO; 0.5 pmol/nl) in 0.9% saline (pH 7.2-7.4) and the other barrels contained the vehicle 0.9% saline for control injections or the NT fragment (NT l-8; 0.5 pmol/nl) [39] or the NT analogue/antagonist [DTrp” ]NT (0.5 pmol/nl: Sigma) [39]. In five additional animals. the effect of the prior injection of [D-T@ I]NT on the cardiovascular response to NT ( 10 pmol) was investigated. A cardiovascular responsive site was initially identified with NT and after I h an equimolar amount of the antagonist was injected into the smc site. Five to IO min after the antagonist injection, NT was re-injected into the same site. In 13 animals, the spinal cord was exposed at ihc level of C l-C2 and transected using a hot spatula. In nine additional animals, the cervical vagi were identified following a ventral midline incision in the neck, isolated from the cervical sympathetic and aortic depressor nerves, and transected bilaterally. In six of these animals with spinal cord transection, the vagus nerves were also cut bilaterally. In eight additional animals, when a cardiovascular responsive site to NT was identified in the NTS. the muscarinic receptor blocker, atropine methyl bromide (I mg/kg. i.v.), the nicotinic receptor blocker, hexamethonium bromide (20 mg/kg. i.v.) or both were administered and the same site in NTS re-stimulated. As hexamethonium administration causes a precipitous fall in AP, the alpha receptor agonist phenylephrine (2- 10 mg/kg/min) was given i.v. using a Harvard infusion pump (model 22) to maintain a stable and normal level of AP before the re-stimulation of an NTS site. This was done to eliminate the possibility that the lack of response to central injection after the hexamethonium bromide, was due to brain tissue damage as a result of low perfusion pressure. At the completion of an experiment, the micropipette was withdrawn and without removing the micropipette

from the stcreotaxic frame the saline barrel was filled by capillary action with India ink or Pontaminc sky blue. The micropipette was lowered stereotuxicully to the last site that elicited ;I response following the microin_jection of NT or its analogucs and marked by an injection (20-30 111)ot either the India ink or Pontrmline sky blue to mark the center of the injection site. Animals were then perfused transcardially with SO ml of 0.9% saline followed by 50 ml of a IO% buffered formaline-saline solution. The brains were removed and stored in fixative for a~ least 72 h before sectioning on a cryostat. Frozen, transverse (SO pm) sections of the medulla were cut, mounted onto gelatinized slides and stained with either thionine or Neutral red for the identification of the injection sites. The location of the sites of injections was plotted onto a series of representative sections of the dorsomedial medulla modified from a stereotaxic atlas of the rat brain [38]. Mean AP (MAP) was calculated by adding one-third of the pulse pressure to the diastolic pressure. Control levels of MAP and HR were calculated immediately before the injection. The peak of the responses was considered to be the time at which maximal changes in MAP or HR had occurred. A response was defined as a change of greater than 5 mm Hg for MAP and greater than 5 beats/min for HR. Comparisons of MAP and HR were made using Duncan’s multiple range test after an ANOVA indicated statistical significance. A P value of less than 0.05 ~~3s taken to indicate statistical significance.

sponses to NT administration cL,oked ;I small dscrcasr it\ RIAP f -4 + 2 mm Hg) and HR ( ---I + 2 beats/min). These IKII

changes were not significuntly fluctuak~ns

in baseline

MAP

different

and HR.

from

nor-

A representa-

HR response to microinjection of NT into in Fig. 2. Note that the AP pressure YTS ih shown response consisted of a decrease in both systolic and diastolic pressures that reached a peak approximately 1% SO s after the injection. The HR response followed a similar time course. To determine whether the NT fragment NT 1-8 and the potential antagonist/agonist [D-TI$‘]NT elicited depressor and bradycardic responses similar to those evoked by microinjection of NT into the NTS, equimolar (10 pmol) injections of the compounds were made into the NTS. As summarized in Fig. 3. microinjections of the NT fragment NT l-8 elicited depressor responses ( - I4 & 3 mm H$ that were significantly smaller than those elicited bq NT. However. the magnitude of the bradycardia ( - 22 + 4 beats/min) response to NT l-8 was not different from that elicited by NT. On the other hand, microinjection of tive AP

and

Necrotensn tpmol)

-25 l\

3. Results The region of the NTS was systematically cxplorcd fur sites that elicited cardiovascular responses during microinjections of NT ( IO pmol). Baseline MAP and HR were 131 + IS mm Hg and 397 + 37 beats/min. Fig. I shows the effect of varying the amount of NT on the magnitude of the cardiovascular responses elicited ii-0111the NTS. Both MAP and HR responses appeared to reach maximum when IO pmol of NT were microinjected. Further increases in the amount of NT microinjected into the NTS ( 15-300 pmol) did not significantly increase the magnitude of the MAP or HR response. NT stimulation ( 10 pmol) of 1 I5 histologically verified sites in the caudal NTS region of I9 animals elicited decreases in MAP (mean, - 34 + 3 mm Hg; range, - 5 to - 85 mm Hg) and HR (mean, - 28 12 beats/min; range. -5 to - 80 beats/min). On occasion sites were found that elicited either the AP or the HR response alone. At all sites. cardiovascular responses 01 similar magnitude could be reproduced following a second microinjection of NT when the injections were made at least 45 min apart. The magnitude of the MAP or HR responses was not affected by the paralyzing agent used in these studies (II = 10 sites: MAP, - 28 + 2 mm Hg; HR. -33 17 beats/min). Microinjection (20-25 nl) of the vehicle (0.9% saline) at the same sites that elicited re-

.lOO

I

BoB

F\(Tin the microinjcction into NTS MAP(A) iUldHR (B) responses. Each point i\

Fig. I. Effect of vluying the amount of on the magnitude of the the mean of

S-7 responses.

38

a

INTACT 200

ARTERIAL PRESSURE (mm Ws) “:;g (bpm)

flcfl 40$ --I 350 I

b -

\. ______---

BILATERAL

---

---.---

-

VAGOTOMY

“lzrc Ol 400 350 c

c

.-.

.

.-

Cl -SPINAL

-

TRANSECTION

200 r

t

OL 350

_.

-_.

300 1

+ Fig. 2. Representative cardiovascular responses after injections of NT ( IS pmol) into the commissural nucleus of NTS vclgotomizcd (b) and spin&lrunsecred

in an intctct W.

bilarcrally

(cl animal. Note that rhc magni-

tudc of the AP is not idtcrcd by bilateral vrrgotomy. but is abolished by spinal cord trimsection. On the other himd. the HR rcsponsc is attcnuntcd hy both surgical proccdurcs. Arrow shows time of injection\. Cirlibriltion mark reprcscnta I min.

the [o-Trp” ]NTdid not elicit significant cardiovascular responses from NTS sites. As [II-Trp" ]NThas been shown to have antagonistic

A

properties in the peripheral vasculature for NT [39], the effect of prior injection of this compound into 10 NTS sites shown to elicit cardiovascular responses to NT was tested on the responses to NT injections. Although the MAP ( - 25 + 10 mm Hg) and HR ( - 17 + IO beats/min) responses were lower than those elicited by NT alone (31 + 9 mm Hg; HR, 26 f 5 beats/min), they were found not to be significantly different. To investigate the contribution of the different components of the autonomic nervous system to the MAP and HR responses elicited after NT microinjections into the NTS, experiments were done in animals in which either the spinal cord was transected at the level of C l-C2 ( rz = 13 animals), the vagi were cut bilaterally (II = 9 animals), or both (n = 6 animals) and in animals (II = 8 animals) that received i.v. administration of atropine methyl bromide, hexamethonium bromide, or both. Microinjection of NT in spinal-transected animals elicited a small decrease in HR (- I2 f 3 beats/min) and no MAP response. The HR response in these animals was significantly smaller than that in intact animals (Table 1). Intravenous administration of the muscarinic receptor blocker abolished the remaining HR response. On the other hand, microinjection of NT into the NTS of animals that were only bilaterally vagotomized elicited a depressor response ( - 35 + 5 mm Hg) that was not significantly different from that elicited in intact animals, and a decrease in HR ( - I9 f 2 beats/min) which was significantly lower than that observed in the intact animals, but greater than that observed in spinal-transected animals. Administration of the nicotinic receptor blocker abolished both the MAP and HR responses to microinjection of NT into the NTS. Microinjcction of NT into the NTS in animals that were both spinally transected and bilaterally vagotomized did not elicit any cardiovascular responses. Similarly, microinjection of NT into the NTS in animals with total autonomic

B NT

0

NT l-8

ID-$

‘]-NT

NT ,‘.‘_‘*‘.‘d L’.‘.‘.‘.‘* c’.‘.‘.‘.‘, , . . . . . . . . . t ::::. b’.‘.‘.:.:; ,‘..... . , by::.‘:, ;::::, )~.~.~.‘.‘, C’.‘.‘:.‘* ;::::, :::::. :.::::: ._. . . . 4 ;:. .,._, ;::. ._, C’...‘.. , ..F..*_.z....L.

NT 1-8

ID-Tr$ ‘I-NT

b * .:::: Cm:*::. b.... . .., b’.‘.‘.‘. , ;.‘:::, . . . . . . 4 :.::: .*., ,.*. *:. . .., ,..::. . ::.:.: ‘::.- . . . . ;.:.:.I. . \ :::::. : ‘_....‘, ;: :::, i. 4...:..\...

l_._. ..&y

(291

(351 ~100~

Fig. 3.

Barcharts showingthe magnitudeof

in parentheses are the number of

the changes in MAP (A) and HR (B) after microinjection of NT. NT

sites stimulated.

I -8 and [~Trp”

INTinto

NTS.

Numbers

Mean _t SE.; 11 value represents the number of cardiovascular responsive hites in NTS. Any two means within a row with different letters arc significantly different (P magnitude

< 0.05)

and any two means with the same letters are not significlrntly

of the rtispoiisc from wntrol

lc\rl~.

Hex. hrxamethonium

12M

-I

r4vopinc.

utropine methyl bromide administration.

complex, in the lateral and medial subnuclei of the NTS. Additionally, a number of responsive sites were found in the subnucleus gelatinous, just ventrolateral to the area postrema. A few sites that elicited moderate cardiovascular responses were found in the medial subnucleus of the NTS complex rostra1 to the area postrema. Notably at this level, a few sites which elicited only cardiac slowing were found in a region imme&ately ventral to the lateral tip of the dorsal motor nucleus of the vagus in the reticular forma-

HEART

PRESSURE

’

Numbers in parentheses arc the calculated percentages for the

bromide administration:

blockade did not elicit cardiovascular responses. The results of these experiments are summarlzecl in Table 1. Fig. 4 and Fig. 5 show the location of the cardiovascular responsive sites in the region of the NTS. As shown in Fig. 4 for animals with an intact central nervous system, sites that elicited the largest changes in MAP and HR were located in the NTS from the level of the area postrema caudally to the obex (Fig. 4). These sites were localized to the dorsal and dorsolateral aspects of the NTS

ARTERIAL

different.

RATE

l

Fig. 4. Transverse hemisections through the rdt dorsomedial medulla showing the location of sites producing (*hlmgc:!,m AP (a-c) and HR a’-c’) after change of S-20 mm Hg or beats/min: 0. changeof injection of NT ( IO pmol) into the NTS region in intact animals. 0, no responsein MAPor HR;

v.

l2M. hypoglossal nucleus; Ap, uea postrrmu; cc, central canal; Corn. commissural subnucleus of NT!?: Cu. cuneate MdD, medial dorsal medullae oblongata centralis: SC. dorsal motor nucleus of the vagus: fg. faxiculus gracilis; Gr, gracilis nucleus;

greater than 20 mm Hg or beats/min. nucleus; DMV.

central subnucleus of NTS; Sl. lateral subnucleus of NTS; Sm. medial subnucleus of NT!? Sv. vcntrul subnucleus of NT!? Svl. ventrolaterul subnuclcuh of NTS; T. tractus solitarius. Calibration mark in c and c’ represents 0.2 mm and applies to a-c and ii-c’.

respectively.

Fig. 5. Trunsvrrsc pmol) into the NTS

hcmiscctions through the rut dorsal medial mdulhl

showing the location of sites eliciting HR responses Ax

region in bilutcrd vegotomizcd (u-c) and spinal-tmnsected (a’-~‘) animals. R&r

tion. No cardiovascular rcsponsivc sites

were found in the area postrcma, dorsal motor nucleus of the vagus, nucleus gracilis or nucleus cuncatus, areas all adjacent to the cardiovascular responsive region in the NTS. The distribution pattern of cardiovascular responsive sites in the vagotomized and spinal-transected animals was essentially the same, although no HR responsive sites were observed rostral to the area postrema in spinal-transected animals (Fig. 5).

4. Discussion These data have demonstrated that microinjections of NT into a restricted region of the NTS in the chloraloseanesthetized, paralyzed and artificially ventilated rat elicit arterial hypotension and bradycardia. The finding that one of the central effects of this neuropeptide is to cause a decrease in MAP is consistent with the earlier observations in the urethane-anesthetized rat that intracistemal administration of NT evokes a dose-dependent depressor response [39,43] and that microinjection of NT into the NTS region potentiates the aortic nerve depressor and bradycardic responses 1261.However, these findings are at variance with a report in the conscious rat that showed that intracistemal

microinjeclic~n ol NT ( IO

tcr Fig. 4 for additional Jctail\.

in_jcctions ot’ NT elicits a prcssor response with either no direct or reflex effect on HR [Xl.Although

the reasons for these discrepancies are not known. they may likely be due to different sites of injection, the effect of the anesthetics on the central neuronal circuits mediating the cardiovascular responses, the possibility of activation of different brainstem structures in the conscious animal, or that the pressor responses may have been secondary to muscular movements [St] or respiration in the conscious rat [34]. It may be argued that the effects of NT injections into the NTS were due to local distortion of neuronal processes [53], damage of the NTS neuropil, or the result of activation of NTS neurons by the NaCl contained in the vehicle used for injection of the neuropeptide [19]. However, these possibilities are unlikely as microinjections of the same volume of 0.9% saline into the same NTS site where NT exerted an effect on the cardiovascular system, did not elicit significant changes in AP or HR. Rather, the effect of NT on NTS neurons appears to be due to activation of NT receptors as the magnitude of the pressor and bradycardia responses were decreased during the microinjection of the less potent NT fragment NT 1-8 [39] and were absent during the microinjection of [D-TI$ ’ ]NT [39]. This was not unexpected as the absence of arginine in the amino acid sequence and the C-terminus ending would hinder the

binding of NT to its rcccptor [39]. As this unaloguc of NT has been suggested to have antagonistic propcrtics in the peripheral circulation. although agonistic effects have been reported in the central nervcus system 1.391.the cffcct 01’ this compound on the NT evoked rcsponscs was invcstioated. This NT analoguc did not significantly ;Iltcr the E cardiovascular response to injection of NT at the same NTS site. although a trend towards a reduction in the cardiovascular responses was evident. The apparent attenuaticjn may not be due to its antagonist properties, but possibly to tachyphylaxis to the multiple neuropeptide injections. Activation of NT receptors has ala 3 been shown to evoke a slow onset, long dur:.tion cld.polar zation of the neuronal membrane [X).57]. This &&II 2ation has been shown in myenteric neurons to bc associated with an increase in input resistance [57]. The slowness and long duration of the responses may be due to the activation 01 secondary intracellular mechanisms and to the deactivation of these mechanisms that follow the stimulation of NT receptors [ 171. In spinal neurons, the depolarization after activation of NT receptors has been shown to last for more than 7 min [50]. The suggestion that NTS neurons are excited by microinjections of NT is also supported by the observation that iontophoretic application of NT to NTS respiratory neurons causes a long lasting increase in the discharge frequency of the neurons [34]. Activation of these NTS inspiratory neurons has been shown to decrease phrenic nerve activity as a result of increased inspirulory drive [34]. These findings [34], along with those obtained in this study, were not ullexpcctcd as the NTS regions traditionally associated with respiratory and cardiovascular rcgulalion [49] have been shown to contain ;I dense network of’ NT itnmunorcactivc fibers and presumptive terminal boutons [l&30,55]. The location of immunorcactive NT in NTS is also paralleled with that of specific affinity binding sites for [ 3~]~~ [25,40.%]. Higgins et al. [ 181 have also shown the coexistence of aortic nerve afferent fibers and NT immunoreactivity in the dorsal NTS complex, where in this study both AP and HR responses were elicited by NT injections. These observations, taken together with those of the present study, suggest that NT mechanisms in the NTS may be involved in the facilitation of the baroreceptor reflex [26] by possibly enhancing transmission between first order primary baroreceptor afferents and NTS neurons. Such a role for NT has been suggested for synaptic transmission in the stellate ganglion [3]. Bachoo and Polosa [3] have shown that the cardioacceleration mediated exclusively by ganglionic nicotinic transmission was enhanced by intraganglionic administration of NT. NT is thought to be co-localized in and released by the axon terminals ot preganglionic sympathetic neurons [?I. However. NT may also act in NTS to inhibit the effect of another inhibitory neurotransmitter, as it has been shown to do for the

midbrain dopaminergic system [ 16.36.48j which then ~r~u]d ;IIIoM~;Lg-cater cxprcssion of the baroreccptor reflex. The site of origin of NT projections to the NTS inresponses is not known. \~r~l\~cd in these cardiovasculur However. NT projections to the NTS have been shown to originate in the ~tmygdala [ 151, bed nucleus of the strip terminalis [ 151 and wiGn the NTS itself [I&20.2 :,30.55]. Activation of these central structures has been shown to elicit cardiovascular responses qualitatively similar to those observed in this study [9,14,35]. It was shown in this study that the AP response was abolished by spinal cord transection or after the administration of the nicotinic receptor blocker hexamethonium bromide, whereas the HR response was abolished after both spinal cord transection and vagotomy or total autonomic blockade. Th~c:? data suggest that the AP response w’!s mediated via an ;!ihibitory effect on the sympathetic nervous system. On the other hand, the HR response was mediated by an inhibitory effect on the sympathetic ner~W.ISsystem and an excitatory effect on the vagal system. The likely pathways include a projection from the caudal NTS to the caudal ventrolateral medulla and the nucleus ambiguus [32,45]. The caudal ventrolateral medulla is thought to inhibit sympatho-excitatory neurons in the rostral ventrolateral medulla [I ,3 I] that project directly to the intermediolateral cell column [44], where preganglionic sympathetic neurons are located [4l]. The nucleus ambiguus has been shown to contain vagal cardiomotor neurons [7.32.33,37]. In summary, these data have demonstrated that microinjections of NT into NTS elicit arterial hypotension and bradycardia and suggest that NT may act as a neurotransmiltcr or modulator in ncuronul circuits controlling the Circ~Uli1tiOn.

Acknowledgements The excellent technical assistance of Z.M. Zhang and J. Nichols is gratefully acknowledged. This work was supported by the Heart and Stroke Foundation of Ontario.

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