Aortic baroreceptor reflex pathway: a functional mapping using [3H]2-deoxyglucose autoradiography in the rat

Journal of the Autonomic Nervous System, 8 (1983) 111-128

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Elsevier

Aortic baroreceptor reflex pathway: a functional mapping using [3H]2- deoxyglucose autoradiography in the rat J o h n Ciriello ~, Charles V. R o h l i c e k a n d C a n i o Polosa Department of Physiology, McGill University, Montreal Que. H3G 1Y6, Canada

(ReceivedMarch 29th, 1982) (Revised versionreceivedJanuary 11th, 1983) (AcceptedJanuary 18th, 1983)

Key words: aortic baroreceptor pathways--cardiovascular reflex pathways--

2-deoxyglucose autoradiography--functional neuroanatomy

Abstract The organization of pathways within the central nervous system which are activated by aortic baroreceptor input was studied in the urethane anesthetized rat using the 2-deoxyglucose method. [3H]2-deoxyglucose was administered i.v. while either the aortic nerve was electrically stimulated or aortic baroreceptors were physiologically activated by pulse increases in arterial pressure in animals with bilateral denervation of the carotid sinus. Autoradiographs of transverse sections of the central nervous system were developed and analyzed for changes in metabolic activity in discrete regions compared to control animals, as indicated by the density of the photographic emulsion. Electrical stimulation of the aortic nerve resulted in all animals in an increase in the uptake of deoxyglucose in a number of sites throughout the central nervous system, primarily ipsilateral to the site of stimulation. In the brainstem, structures previously implicated in cardiovascular reflexes were labeled. These included the nucleus of the solitary tract, the solitary tract, the dorsal motor nucleus of the vagus, and the nucleus ambiguus. In addition, the inferior olivary nucleus, the parabrachial nuclei and the ventrolateral reticular formation showed increased labeling. In the hypothalamus, increased labeling was observed only in the paraventricular and supraoptic nuclei.

To whom correspondenceshould be addressed at: Department of Physiology,University of Western Ontario, London, Ont. N6A 5C1, Canada. 0165-1838/83/$03.00 © 1983 ElsevierSciencePublishers B.V.

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Physiological stimulation of aortic baroreceptors resulted in increased bilateral labeling of the same structures, with the addition of the posterior, anterior and periventricular hypothalamic areas, the suprachiasmatic nucleus and the amygdala.

Introduction

The aortic nerve relays baroreceptor afferent information from the aortic arch to the central nervous system [13,48,77]. On the basis of a large body of neuroanatomical and neurophysiological evidence it has been demonstrated that the site of termination of primary aortic nerve afferent fibers is the nucleus of the solitary tract [15,20,21,22,26,42,43,45,50,80]. Additionally, it has been suggested that the region of the dorsal motor nucleus of the vagus [22,42,72], the external cuneate nucleus [26] and the paramedian reticular nucleus [26] receive direct inputs from the aortic nerve. On the other hand, the distribution of second order neurons in the aortic baroreceptor reflex arc has been more difficult to study because of the widespread connections of the nucleus of the solitary tract [3,17,53,60,67]. Nevertheless, stimulation of the aortic nerve has been shown to alter the activity of structures within the medullary reticular formation such as the nucleus ambiguus, the lateral reticular nucleus, the nucleus gigantocellularis, and the nucleus medullae oblongatae centralis [4,12,15,50]. Projections to supramedullary structures from the aortic nerve have been demonstrated electrophysiologically to the anterior hypothalamus, the subthalamus, and the paraventricular and supraoptic nuclei [9,10,11,18,46]. Although these studies have demonstrated aortic nerve afferent projections to these medullary and supramedullary structures, it cannot be unequivocally concluded that aortic baroreceptor afferent fibers project to these central areas. This is primarily due to the fact that in the species previously studied with the notable exception of the rabbit, both baroreceptor and chemoreceptor afferent fibers are found in the aortic nerves, and it is not possible to selectively activate one group of afferent fibers by electrical stimulation as both types of receptors have been shown to have axons in a similar size range [31]. Therefore, in the present study advantage was taken of the fact that aortic baroreceptor afferent fibers can be activated selectively in the rat by electrical stimulation of the aortic nerve, which in this species carries only baroreceptor afferent fibers [69,70], and that the 2-deoxyglucose method [76] can provide a comprehensive functional map of the central nervous system areas which receive aortic baroreceptor afferent information. The 2-deoxyglucose technique has been shown to provide a functional neuroanatomical map of sensory pathways by the visualization of areas in the central nervous system with increased metabolic activity as a result of stimulation of the appropriate sensory receptors [27,39,40,47,66,74]. Materials and Methods

Surgical and stimulation procedures Experiments were done on male Wistar rats weighing 250-300 g under urethane

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anesthesia (1.5 g / k g , i.p.). The trachea was cannulated and the animals breathed spontaneously. Polyethylene catheters were inserted into the femoral artery to record arterial pressure, and into the femoral vein for the administration of the isotope and drugs. Arterial pressure measured with a Statham P23Dd transducer was continuously recorded on a Grass model 7 polygraph. Rectal temperature was monitored and maintained between 36 and 37°C by radiant heat. In 7 animals the aortic nerve was approached by a ventral midline incision in the neck, identified near its junction with the superior laryngeal nerve, dissected free from the vagus and cervical sympathetic nerves for a few cm and crushed distally. The central end of the isolated nerve was placed on bipolar silver stimulating electrodes and covered with warm mineral oil to prevent drying. The isolated nerve was stimulated every 20 s with a 10 s train of rectangular pulses at a frequency of 25 Hz, a pulse duration of 0.5 ms and an intensity of 2.5-15 V, for 45 min. A decrease in mean arterial pressure of 25-30 mm Hg during each train of pulses was taken as evidence that aortic baroreceptor afferent fibers were being activated. In two additional experimental animals the glossopharyngeal and the carotid sinus nerves were exposed and cut before their entry into the cranium at the level of the tympanic bulla. In these animals care was taken to avoid damage to the aortic nerves. Aortic baroreceptors were physiologically activated by the elevated arterial pressure resulting from the i.v. infusion of phenylephrine (2 /~g/kg) in 0.1 ml of saline over a period of 5 s delivered once every minute using a Harvard infusion pump for 45 min. The reflex decrease in heart rate elicited by the increase in arterial pressure was taken to indicate aortic baroreceptor activation. Fig. 1 shows examples of the systemic arterial pressure changes associated with electrical stimulation of the aortic nerve and the bolus injections of pressor doses of phenylephrine from the two different experimental preparations.

1rain

Fig. 1. Arterial pressure responses elicited in two different experiments by either intermittent electrical stimulation of the aortic nerve (A) or by the i.v. bolus injections of phenylephrine (B) in an animal with

bilateral denervation of the carotid sinus. In each panel, top is arterial pressure, and bottom is marker indicating stimulus application. Administration of [3H]2-deoxyglucoseat arrow. Break in records lasts 41 min in A and 33 rain in B.

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2-Deoxyglucose autoradiography The isotope solution was prepared by the evaporation of the commercially available tritium-labeled 2-deoxyglucose (New England Nuclear, 2-[1,2-3]-deoxy-Dglucose, spec. act. 30-60 C i / m m o l ) to dryness under a nitrogen gas stream. The dry residue was reconstituted in physiological saline to a concentration of 1-2 mCi/ml. Once stimulation was seen to produce a consistent response, an injection of the isotope was given intravenously (300 # C i / 1 0 0 g body weight). Stimulation was continued for 45 min, during which time the arterial pressure responses were monitored for effectiveness of the stimulation. This duration of stimulation has been suggested to ensure essentially the complete uptake and phosphorylation of the 2-deoxyglucose [76]. After this period of time the animals were decapitated, the brains immediately removed and frozen on dry-ice. Serial transverse sections of the brain, extending from the olfactory bulbs to the spinomedullary junction were cut at 8/~m in a Harris cryostat at - 2 6 ° C . In every series of 10, 3 successive sections were picked up on coverglass kept at room temperature and rapidly dried on a hot plate at 70°C. Two series of sections were assembled on pieces of cardboard, placed in contact with a tritium sensitive film (Ultrofilm, LKB, Sweden) in an X-ray cassette, and exposed at 4°C for 3-9 weeks. The films were then developed according to the manufacturers instructions. An immediately adjacent series of sections was stained with Neutral red to histologically differentiate nuclear groups.

Control preparations To examine the basal uptake of 2-deoxyglucose within the central nervous system in rats under urethane anesthesia, experiments were done in 5 animals. All animals underwent the same routine surgical procedures as the experimental rats. In 3 animals no additional surgery or stimulation was done. In two animals, to also examine the possibility that some of the resting uptake of 2-deoxyglucose in the non-stimulated animal was due to tonic baroreceptor input, the rats were bilaterally sino-aortic denervated. These control animals were also decapitated 45 min after the administration of the same dosage of the isotope as the experimental rats, their brains quickly removed and frozen, and the tissue similarly processed for autoradiography.

Data analysis The autoradiographs were first examined by eye after magnification with a hand lens for evidence of discrete regions of enhanced 2-deoxyglucose uptake. By visual inspection it was possible to identify areas of increased density and localize them on immediately adjacent stained histological sections. These areas were then identified by comparing the sections to an atlas of the rat brain [64]. Selected autoradiographs of brain sections were also analyzed by a computer-based interactive image processing system [65]. The autoradiographs were digitized to 64 levels of optical density over a 576 × 576 point image raster using a Zeiss Axiomat microscope and a Quantimet 720 D microdensitometer custom interfaced to a

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Digital Equipment, PDP-11/40 computer system. The resulting digital images were then averaged using a 4 point × 4 point image window to produce 144 point by 144 point 7-bit optical density based digital images which were used in the actual analysis. To minimize the effects of non-uniform illumination of the microscope and the inherent shading of the television microdensitometer, shading correction factors were computed for every image point from 8 blank fields. The average of the 8 shading fields was used to correct all digitized images before computing the normalized displays. These were in turn processed by digital computer to create image displays on a CRT normalized to a reference white matter structure (i.e. corpus caUosum and/or spinal trigeminal tract). By presenting the data in this manner interanimal differences in optical densities resulting from variations in the dosage of the isotope, the period the autoradiographs were exposed, in the apposition of the films to the tissue containing the isotope, and in the development of the films were minimized. Several representative autoradiographs from different levels of the neuraxis were further reconstructed in pseudocolour by computerized densitometry and image processing, a process which transforms optical density values into different colours, [33]. This was done to improve the viewers ability to perceive differences in optical density and to determine relative levels of 2-deoxyglucose uptake.

Results

Areas activated by stimulation of aortic baroreceptors Autoradiographs of the animals in which the aortic nerve was electrically stimulated exhibited pronounced increases in optical density in brainstem and hypothalamic structures compared to control cases. In this report only those structures which consistently showed a greater optical density in the stimulated cases (alternatively referred to as 'activation') will be described in detail and are summarized in Table I. These structures were either ipsilateral to the stimulated nerve or bilateral. Increases in the optical density of central structures were most evident at the level of the brainstem. As shown in Fig. 2A, a zone of darkening was observed to extend throughout the dorsocaudal medulla, bilaterally. This region of increased optical density was primarily confined to the nucleus of the solitary tract. In the rostral medulla this area of increased activity was found more laterally (Fig. 2B) and included the region of the solitary tract on the ipsilateral side (Fig. 2A). Additionally, the region of the ipsilateral dorsal motor nucleus of the vagus was activated (Fig. 2A). Activation, primarily ipsilateral to the side of stimulation was also observed in the regions of the nucleus ambiguus (Fig. 2A), the inferior olivary complex (Fig. 2A) and the ventrolateral medullary reticular formation (Fig. 2B). A moderate activation of these structures was also observed contralaterally. Additionally, a less well-defined area in the reticular formation connecting the regions of the solitary complex and that of the nucleus ambiguus was activated ipsilaterally (Fig. 2A; region of lateral

117 TABLE I DENSITOMETRIC RATIOS OF [3H]2-DEOXYGLUCOSE UPTAKE AFTER AORTIC BARORECEPTOR A C T I V A T I O N Only ratios showing a greater than 20% increase are considered significant and included in the table. Ratios have been calculated as follows: density of activated structure/density of corpus callosum or spinal trigeminal tract in stimulated animals ] density of structure in control animals/density of corpus callosum or spinal trigeminal tract in control animals

l

- , structures which are not activated. Mean values of ratios of the combined ipsilateral and contralateral sides are given. Refer to legends of Figs 2-5 for list of abbreviations, lpsi, ipsilateral to aortic nerve stimulation; Contra, contralateral to aortic nerve stimulation. Central structure

Unilateral aortic nerve stimulation

Bilateral aortic baroreceptor activation

Sinoaortic denervation

Ipsi

Contra

NTS DMV AMB ION PBN

2.03 2.01 1.91 2.24 2.01

1.85 1.80 1.76 2.06 1.93

2.28 2.28 2.57 2.70 2.22

-

RFvl AH SON PVH SC AMG PHA PVA

2.31 1.20 1.39 -

2.25 1.36 -

2.72 1.75 1.99 1.69 1.98 2.44 1.45 1.82

0.79 1.94 2.01 0.80 -

tegmental field). Within the region of the nucleus ambiguus several sites of high optical density were observed (Fig. 2A). These sites corresponded to areas in which clusters of neurons were observed in histologically processed adjacent sections. A mild increase in activity was also observed in the region of the contralateral nucleus ambiguus (Fig. 2A). In the inferior olivary complex the area of increased activity was

Fig. 2. Computer analyzed deoxyglucose autoradiographs of transverse sections of the medulla taken at a level approximately 0.5 mm (A) and 2 mm (B) rostral to the obex of an anesthetized rat after stimulation of the right aortic nerve. Note the increase in density of the autoradiographs in the regions of the ipsilateral nucleus and tract of the solitary complex (A and B), the ipsilateral lateral aspect of the dorsal motor nucleus of the vagus (A), the ipsilateral nucleus ambiguus (A), and the ipsilateral ventrolateral reticular formation (B). 5SP, spinal trigeminal nucleus; 5ST, spinal trigeminal tract; AMB, nucleus ambiguus; Cr, cerebellum; CuL, lateral cuneate nucleus; FTL, lateral tegmental field; lcp, inferior cerebellar peduncle; ION, inferior olivary nucleus; NF, fastigial nucleus; NTS, nucleus of the solitary tract; P, pyramidal tract; RFvl, ventrolateral reticular formation; S, solitary tract; v, 4th ventricle; VN, vestibular nucleus; X, dorsal motor nucleus of the vagus; XII, hypoglossal nucleus. Calibration mark in A of I mm applies to both sections.

118 located in the caudal portion of the nucleus a n d confined to the dorsal aspect of the dorsal medial olivary nucleus (Fig. 2A). The region of e n h a n c e d activity in the ventrolateral medullary reticular formation of the rostral medulla extended along the ventral surface to a depth of approximately one millimeter and from the pyramidal tract to the spinal trigeminal tract (Fig. 2B). In the rostral brainstem, a mild increase in the density of the region of the ipsilateral parabrachial nucleus was also observed. Two principal structures were activated in the region of the h y p o t h a l a m u s in the stimulated animals: the paraventricular a n d supraoptic nuclei. A l t h o u g h the activation of these structures was p r e d o m i n a n t l y ipsilateral, a n increase in activity was also observed in the contralateral paraventricular nucleus (Fig. 3A and B). The nuclei were activated homogeneously throughout their rostrocaudal extent. In addition, an

Fig. 3. Computer-analyzeddeoxyglucose autoradiographs of transverse sections through the region of the suprachiasmatic nucleus (A and C) and the paraventricular nucleus (B and D) of the hypothalamus of the anesthetized rat. A and B: left aortic nerve stimulated rat, C and D: bilateral aortic baroreceptor stimulation with pressor drug in carotid sinus denervated rat. Bb corresponds to area outlined in B. Note the predominant ipsilateral increase in density in the SC, PVH and SON in A and B, and the bilateral activation of these structures in C and D. AAA, anterior amygdala; AHA, anterior hypothalamic area; CC, corpus callosum; CI, internal capsule; CO, optic chiasm; Fx, fornix; MT, mammillothalamic tract; NOT, nucleus of the olfactory tract; PVH, paraventricular nucleus; SC, suprachiasmatic nucleus; SM, stria medullaris; SON, supraoptic nucleus; V, 3rd ventricle. Calibration mark of 2 mm in B applies to all sections except Bb where it represents 1 mm.

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area of high density was also clearly apparent in the dorsolateral and medial portions of the paraventricular nucleus (Fig. 3B and Bb). The autoradiographs obtained in the case in which aortic baroreceptors were

A 1

B Cr

1

TS ,I

i

u

|

Fig. 4. Computer-analyzed deoxyglucose autoradiographs of transverse sections of the brainstem of a non-stimulated intact rat (AI_3) and of a carotid sinus denervated animal in which aortic baroreceptors were bilaterally activated with a pressor drug (Bl_3). Autoradiographs with similar numbers were taken from sections of approximately the same rostrocaudal level. PBN, parabrachial nucleus. Refer to Fig. 2 for additional details and abbreviations. Calibration mark of 2 m m applies to all sections illustrated.

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bilaterally activated by an increase in systemic arterial pressure were essentially similar to those o b t a i n e d during electrical s t i m u l a t i o n of the aortic nerve. However, in these cases the structures were equally activated bilaterally a n d in general the relative optical densities of the structures were greater. Fig. 4B shows a series of representative autoradiographs taken from the b r a i n s t e m of one animal in which aortic baroreceptors were physiologically activated. In the hypothalamus, in addition to increased activity in the same structures activated by stimulation of the aortic nerve (Fig. 3C a n d D), there was also increased activity in the anterior (Fig. 5C~), periventricular (Fig. 5C2), a n d posterior h y p o t h a l a m i c areas a n d the suprachiasmatic nucleus. F u r t h e r m o r e , a n increase in activity of the amygdala (Fig. 5C~) was observed.

i

,

Fig. 5. Computer-analyzed deoxyglucose autoradiographs of transverse sections through the hypothalamus of 3 experimental cases. A: sinoaortic denervated non-stimulated rat. B: non-stimulated intact rat. C: bilateral aortic baroreceptor stimulated carotid sinus denervated rat. Sections with similar numbers were taken at approximately the same rostrocaudal level in all animals. Note the different levels of activity in hypothalamic nuclei during the complete removal of baroreceptor input (A) and during the bilateral activation of aortic baroreceptors (C). Calibration mark applies to all sections illustrated, 2 mm. Refer to Figs. 3 and 4 for details and abbreviations. AMG, amygdala; OT, optic tract; PHA, posterior hypothalamic area.

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Control preparations In general, in both groups of control animals the gray matter had a relatively high optical density on the autoradiograph, with some structures being more dense than others. In the brainstem, of importance in this study, the nucleus of the solitary tract, the nucleus ambiguus region, the caudal inferior olivary nucleus and the lateral parabrachial nucleus appeared metabolically active in the urethane anesthetized rat. In the forebrain, the amygdala, posterior hypothalamic area and suprachiasmatic nucleus also exhibited an increased density above background levels (Fig. 5B). In the sinoaortic denervated rat the dorsolateral aspect of the paraventricular and the supraoptic nuclei (Fig. 5A2) showed increased activation above that of control intact animals (Fig. 5B2), but below that of aortic baroreceptor stimulated animals. In addition, the anterior hypothalamus and the suprachiasmatic nucleus showed decreased activity (Table I). The white matter in all groups of animals, except for the solitary tract on the ipsilateral side of stimulation of the aortic nerve (Fig. 2A, on the right side) and bilaterally in animals in which aortic baroreceptors were physiologically activated, was characterized by a relatively low optical density on the autoradiograph. This resulted in fiber bundles being well outlined (i.e. Fig. 2A, on the left side).

Discussion

Before the introduction of the 2-deoxyglucose autoradiographic method the available neuroanatomical and electrophysiological techniques only allowed the study of one synapse at a time in a modality-specific sensory pathway. The development of the 2-deoxyglucose method, which has been used in the determination of regional levels of metabolic activity in the central nervous system [76], has offered the possibility for the simultaneous visualization of the neuronal circuitry associated with specific sensory receptors. In the present investigation the central structures involved in mediating aortic baroreceptor afferent information were identified with this technique. Tritium labeled deoxyglucose was used as it has been shown to significantly improve the resolution of the autoradiographs compared to the radioactive carbon labeled sugar [32]. In this study, primary aortic baroreceptor afferent fibers were activated either by electrical stimulation of the aortic nerve [69,70] or physiologically by the increase in arterial pressure caused by the administration of a pressor drug [41]. As activation of this afferent system resulted in the increased uptake of deoxyglucose in several brainstem and forebrain structures, the possible anatomical and functional significance of increased activity in the structures merits discussion. The most prominently labeled structure in the medulla was the nucleus of the solitary tract. This was not an unexpected finding as this structure has been shown to be the primary site of termination of aortic nerve fibers [15,20-22,26,42,43]. The results of the present study have demonstrated that activation of aortic baroreceptors increased the uptake of deoxyglucose throughout the ipsilateral nucleus and solitary tract and in the

122 caudal portion of the contralateral nucleus. These findings are in agreement with a recent neuroanatomical study in the rat showing that aortic baroreceptor afferent fibers after entering the rostral medulla become incorporated in the ipsilateral solitary tract and give off fibers that terminate throughout the ipsilateral nucleus. Some of the fibers, just caudal to the obex, cross the midline and terminate in the caudal portion of the contralateral nucleus [21]. The region of the ipsilateral dorsal motor nucleus of the vagus and the nucleus ambiguus were activated during stimulation of the aortic nerve. The role of these two medullary structures in cardiovascular reflex regulation has been well documented [13]. Electrical stimulation of these structures has been shown to elicit vagal bradycardia [15,16,78] and these structures have been shown to contain preganglionic vagal neurons which innervate the heart [16,57,61,72]. In addition, single unit activity in both nuclei is altered by electrical stimulation of aortic afferent fibers [15,16,20,50,51,72], and lesions of the region of the nucleus ambiguus have been shown to selectively abolish the reflex vagal bradycardia elicited by stimulation of the aortic nerve [15]. A less well-defined area which appeared to connect the region of the solitary complex with the nucleus ambiguus and ventrolateral medulla was activated in the medullary reticular formation during stimulation of aortic baroreceptors. This area corresponds well to the location of sites shown to elicit bradycardia during electrical stimulation [16], which is thought to be due to the activation of fiber tracts originating in the region of the nucleus of the solitary tract and projecting to the region of the nucleus ambiguus and ventrolateral medulla [53,60,63,67]. Therefore it is likely that the activity observed in this region reflects the activation of fiber tracts mediating aortic baroreceptor afferent information which connect these medullary structures. The increased activity was also observed to extend into the region of the ventrolateral medulla. This region has been implicated in cardiovascular regulation [30,34,52], respiratory regulation [52] and vasopressin release [6]. Furthermore, this region contains neurons which project directly to the supraoptic and paraventricular nuclei [54,56], areas previously implicated in cardiovascular control [19] and in the release of vasopressin [7,28], and to the intermediolateral nucleus of the thoracic cord [1,55,68]. The finding of an increased activity in the ventral medulla oblongata suggests that this region functions in the relaying of aortic baroreceptor afferent information to cardiovascular regulating areas of the hypothalamus and the spinal cord. An unexpected finding was the increased activity of the caudal portion of the inferior olivary complex. Within this complex, the region of maximum activation was located in the ipsilateral dorsal medial olivary nucleus. This portion of the inferior olivary complex has been shown to play a role in central cardiovascular regulation [16,75]. In addition, a direct projection from the nucleus of the solitary tract to the dorsal medial olivary nucleus has been demonstrated [53] and single units in this region have been shown to alter their activity during stimulation of carotid sinus and aortic afferent fibers [ 12]. Furthermore, it is interesting to note that this portion of the inferior olivary complex sends direct climbing fiber input to the fastigial nucleus [25], a cerebellar relay nucleus known to alter baroreceptor reflex

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responses and sympathetic activity to heart and blood vessels [58]. It therefore seems reasonable to suggest on the basis of this evidence and the demonstration that the cerebellum provides a tonic excitatory input on sympathetic activity to heart [79], that the inferior olivary complex is part of a reflex arc by which aortic baroreceptor afferent information regulates the level of tonic activity exerted by the cerebellum on the sympathetic nervous system. Activation of the parabrachial nucleus was also observed during stimulation of aortic baroreceptor afferent fibers. Recent studies have demonstrated that electrical stimulation of this nucleus elicits changes in arterial pressure and heart rate [35,59]. Furthermore, electrical stimulation of the aortic nerve has been shown to alter the activity of single units in this nucleus [35]. The finding that this nucleus is interconnected with many other central structures which are themselves important in control of the circulation [54,69] suggests that one of its functions is to relay baroreceptor information from the aortic arch to other central cardiovascular regulating centers. Several hypothalamic regions were also found to increase their metabolic activity: the paraventricular, supraoptic and suprachiasmatic nuclei, and the anterior, posterior and periventricular hypothalamic areas. This was not unexpected as all these areas, with the notable exception of the suprachiasmatic nucleus, have been implicated in cardiovascular regulation [ 10,13,19,77]. The role of the suprachiasmatic nucleus in the control of the circulation is unknown, but recent evidence has shown that lesions of this nucleus prevent the elevation of arterial pressure in the salt-sensitive Dahl rat [2]. The paraventricular and supraoptic nuclei were observed to be activated both during stimulation of baroreceptor fibers and in animals which were bilaterally sinoaortic denervated. This finding can be explained by assuming two different neural mechanisms causing increased metabolic activity of these nuclei. Activation of baroreceptor afferents has been shown to have a dual action on these nuclei, inhibiting the activity of vasopressin containing neurons [44,49,81], and exciting a large number of other neurons [11]. It therefore seems reasonable to suggest that the increased metabolic activity during stimulation of aortic baroreceptors observed in this study represents the activity of inhibitory interneurons that synapse on vasopressinergic neurons. On the other hand, bilateral aortic nerve section results in an increase in vasopressin secretion [8], suggesting that the activation of these nuclei observed after sinoaortic denervation represents the increased metabolic activity of vasopressin-producing neurons. The final area observed to increase its activity was the amygdala. Electrical stimulation of this structure has been shown to alter cardiovascular variables [29,36,37]. In addition, this nucleus receives direct inputs from the nucleus of the solitary tract, the parabrachial nucleus and the paraventricular nucleus [24,62,67], structures also shown in the present study to increase their activity during stimulation of aortic baroreceptor afferent fibers. Furthermore, electrical stimulation of the aortic and carotid sinus nerves have been shown to alter the activity of single units in the amygdala [ 14]. In conclusion, the components of central neuronal circuits relaying sensory information from baroreceptors located in the region of the aortic arch have been

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AORT#C ~ | ~ e BARO- "~-RECEPTORSI

•

• • _] NTS "[ :1 • -]

RFvI

:]

1

Fig. 6. Schematic diagram indicating the major projection sites of aortic baroreceptor afferent fibers demonstrated by the present study. The connections indicated are based on data in the literature (see Discussion). PVA, periventricular area of the hypothalamus. Refer to the previous figures for list of abbreviations. ?, The number of synapses in these pathways are not known.

identified with the 2-deoxyglucose autoradiographic technique. Fig. 6 is a schematic of the structures which were activated. Although the involvement of many of the central structures described in this report could have been predicted on the basis of the current literature, these data have provided direct evidence that they are involved in the integration or relaying of aortic baroreceptor afferent information. The results of the present study provide no information on the number of synapses involved in this neuronal circuit, nor the order in which different structures are activated. However, on the basis of previous neuroanatomical and electrophysiological data it is suggested that this method has revealed changes in metabolic activity of structures at least two synapses away from the stimulus (Fig. 6). It must be pointed out that although effects on neuron systems controlling cardiovascular functions are most prominent with the activation of baroreceptors, baroreceptor afferents have also been shown to influence other neuron systems: respiratory neurons, gamma motoneurons, pyramidal tract neurons, and autonomic neurons controlling other effectors [5,23,38,73]. Therefore it is possible that some of the structures activated in the present study are related to the latter systems rather than to neurons controlling cardiovascular effectors.

Acknowledgements The excellent technical assistance of Dr. R.S. Poulsen with the computer analysis of the autoradiographs and the critical comments by Dr. F.R. Calaresu in the preparation of the manuscript are gratefully acknowledged. This work was sup-

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ported by the Quebec and Ontario Heart Foundations and Medical Research Council of Canada. J.C. is a Canadian Heart Foundation Scholar.

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