Neuroanatomical localization of substance P: Implications for central cardiovascular control

Peptides, Vol. 3, pp. 479-483, 1982. Printed in the U,S.A.

Neuroanatomical Localization of Substance P: Implications for Central Cardiovascular Control C I N D A J. H E L K E

Department of Pharmacology, Uniformed Services University of the Health Sciences 4301 Jones Bridge Road, Bethesda, MD 20814

HELKE, C. J. Neuroanatomical localization of substance P: Implicationsfor central cardiovascularcontrol. PEPTIDES 3(3)479-483, 1982.--The presence of substance P (SP) neurons in pathways known to be involved in the central control of the cardiovascular system has been studied with neuroanatomical and neurochemical techniques. SP-immunoreaetive (SP-I) neurons are found in afferent baro- and chemoreceptor pathways which transmit information from peripheral receptors to the nucleus tractus solitarius. In addition, SP-I neurons located in the nucleus interfascicularis hypogiossi of the ventral medulla innervate the intermediolateral cell column, the site of origin of preganglionic sympthetic nerves. The role of these SP-I neurons in cardiovascular control remains to be determined. Substance P Viscerosensory afferents Intermediolateral cell column

Nucleus tractus solitarius

T H E undecapeptide, substance P (SP), is widely distributed within the central and peripheral nervous systems. The ability of SP to alter blood pressure was first reported by von Euler and Gaddum over 50 years ago [50]. They found that the intravenous administration of a crude extract of SP decreased the blood pressure of an anesthetized rabbit. Since then it has been established that SP is one of the most potent peripheral vasodilators known. SP dilates arteries in several vascular beds [4,7] probably via a direct action on specific SP receptors [5, 7, 13]. Conversely, SP appears to have no direct effects on cardiac function [3,4]. The cardiovascular effects of centrally administered SP have also been described. Centrally administered (either intracerebroventricular or intracisternal) SP evokes a pressor reSponse [15, 21, 48] which is mediated by the sympathetic nervous system [42,48]. Centrally administered SP has variable effects on heart rate. In most strains of rats, tachycardia is Seen with intracerebroventricular or intracisternal administration of SP [21, 48, 49]. However bradycardia is noted in Wistar Kyoto rats and in rabbits [42,49]. Following sinoaortic denervation the bradycardia is no longer seen, instead a tachycardic response is elicited by SP [42,49], Thus the bradycardia appears to be reflex in origin and the sensitivity of the centrally mediated tachycardic response to override the reflex varies among species and strains of animals. While much has been learned about SP and central cardiovascular control, the central site of action and the role of SP in mediating or modulating neuronal activity which affects cardiovascular function remain unclear. Our approach to answering these questions was to determine whether or not SP is utilized by neural pathways known to be involved

Ventral medulla

in cardiovascular control. Our focus was on medullary pathways involved in vagal and sympathetic reflexes. The afferent limb of these reflexes arises from baro- and chemoreceptors located in the carotid sinus and aortic arch. The cell bodies of these viscerosensory afferent neurons are located in the petrosal and nodose ganglia. The fibers enter the CNS in the IX and Xth cranial nerves [40] and terminate primarily in the nucleus tractus solitarius (NTS) [8,41] (see Fig. 1). Integration of this afferent information occurs in the NTS. Projections from the NTS are distributed in numerous forebrain and hindbrain nuclei, including those nuclei which contain preganglionic vagal neurons or cell bodies which project to the preganglionic sympathetic neurons in the intermediolateral column of the spinal cord [33]. Neurons which innervate the intermediolateral cell column are located in several hindbrain and forebrain nuclei [1, 10, 33, 35, 40]. Particularly pertinent to this discussion of CNS SP containing neuronal pathways and cardiovascular control are the primary baro- and chemoreceptor afferent fibers and the efferent projections from the ventral medulla oblongata to the spinal cord. SP-I IN VISCEROSENSORYAFFERENTS Because afferent baro- and chemoreceptor fibers terminate in the NTS, agents likely to be neurotransmitters in this system must be present in this nucleus. The immunohistochemical studies of Ljungdahl and colleagues [31] and Cuello and Kanazawa [9] found a high density of SP positive nerve terminals in the NTS. In addition, SP-immunoreactive (SP-I) fibers were seen in the IXth cranial nerve as it entered

479

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HELKE

I G.

FIG. 1. Schematic drawing of SP-I pathways which are located in strategic sites for central control of the cardiovascular system. Coronal sections of the medulla and thoracic spinal cord are adapted from Ljungdahl e t al. [31]. SP-I nerve terminals and axons are drawn on the right side (densities indicated by the relative density of the dots) and cell bodies (asterisks) on the left side. Abbreviations: amb ap iml nih nts ntv nX nXil P rl ro rp TS

nucleus ambiguus area postrema intermediolateral cell column nucleus interfascicularis hypoglossi nucleus tractus solitarius nucleus tractus spinalis nervi trigemini nucleus originis dorsalis nervi vagi nucleus originis nervi hypoglossi tractus corticospinalis nucleus reticularis lateralis nucleus raphe obscurus nucleus raphe pallidus tractus solitarius

the brain stem [9], and the nodose ganglia and vagus (Xth cranial nerve) nerve were reported to contain SP-I [16,36]. However, in order to classify SP as a neurotransmitter in viscerosensory afferent fibers, we wanted to establish that SP-I is located in all portions of the afferent neural pathway and that the SP of each portion was contained in afferent neurons. To do these anatomical studies we used several procedures which included immunohistochemistry, denervation studies and radioimmunoassay of SP-I, and the combined use of fluorescent retrograde neuronal labeling and immunohistochemistry. In the first study, we investigated the distribution of SP-I in the various regions of the NTS and the effect of unilateral intracranial deafferentation of the IXth and Xth cranial nerves on the SP-I content of the NTS. To do this we microdissected the NTS of cats with the micropunch technique of Palkovits [39] and assayed the samples for SP-I with a specific and sensitive radioimmunoassay [18,25]. We found that the parts of the feline NTS where baro- and chemoreceptor fibers terminate, namely, the commissural and intermediate areas, contained significantly more SP-I than either the caudal or rostral areas (see Table 1). Both the petrosal and nodose ganglia were also found to contain SP-I [18].

Animals subjected to unilateral intracranial deafferentation had less SP-I in the intermediate region of the NTS from the denervated side of the medulla whereas less SP-I was found in both the denervated and intact sides of the commissural region of the NTS (Table 1). These findings are consistent with previous anatomical studies which demonstrated little or no crossover of afferent fibers in the intermediate part but marked crossing in the commissural part of the NTS [2,41]. Denervation had no effect on the SP-I content in the portions of the NTS which do not receive input from baroand chemoreceptor afferents. We also quantitated the SP-I content of several regions of the NTS in Sprague-Dawley rats with micropunch dissection and radioimmunoassay and investigated the effect of unilateral nodose ganglionectomy [25]. SP-I was detected in the nucleus commissuralis and three regions of the NTS in the rat. The highest concentration was in the intermediate portion (i.e., the area immediately rostral to the obex) (Table 2). Seven to 8 days after left nodose ganglionectomy, a decline of SP-I was found in the left caudal and intermediate areas of the NTS, areas which in the rat are primarily unilaterally innervated by baro- and chemoreceptor afferent fibers [47]. The rostral portion of the NTS, an area which receives gustatory afferents from the VIIth cranial nerve [2] was unaffected by the denervation procedure (Table 2). Sectioning the vagus and superior laryngeal nerves caudal to the nodose ganglion was also done as a control for loss of impulse flow to the NTS in the absence of nerve terminal degeneration. This procedure did not alter the NTS SP-I content 7-8 days after surgery [25]. Thus, the decrease in SP-I after denervation required nerve terminal degeneration and, therefore, was not likely to be a transynaptic regulatory phenomenon occurring in the NTS subsequent to the loss of afferent impulses. The above-cited studies told us that at least a portion of the SP-I in the NTS arose from peripheral afferent sources. In addition, the distribution in the NTS was consistent with the known termination of baro- and chemoreceptor afterents. However, we also needed to demonstrate that SP-I was present in the peripheral receptive areas and their associated nerves. To do this we used indirect immunohistochemistry for SP in peripheral tissues from paraformaldehyde perfused rats [22, 25, 27]. SP-I was observed within discrete varicose nerve fibers, nerve bundles and within neuronal perikarya. Discrete varicose nerve fibers with SP-I were found in the peripheral regions known to contain baro- and chemoreceptor afferent nerves, i.e., the tunica adventitia of the aortic arch and the carotid sinus region [25], and among the glomus cells of the carotid body [27]. In addition, SP-I fibers were found in nerve fascicles and trunks in proximity to the aortic arch [25] and in the carotid sinus nerve [27]. Furthermore, numerous SP-I neurons were found clustered at the rostral pole of the nodose ganglion whereas singly dispersed SP-I cell bodies were found throughout the ganglion [22,25]. In order to demonstrate that the SP-I seen in the peripheral receptive areas was contained in viscerosensory afferent fibers, we used the technique of retrograde transport of a fluorescent marker combined with immunohistochemistry for SP. To do this, the fluorescent marker "true blue" was placed on the central cut end of the aortic nerve of Wistar Kyoto rats [22]. This nerve contains viscerosensory afferent nerves from aortic arch receptors, the neuronal cell bodies are located in the nodose ganglion [29]. Three days after the application of "true blue" to the aortic nerve, numerous intensely fluorescent neurons were seen in the nodose gan-

SUBSTANCE P AND CARDIOVASCULAR CONTROL

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TABLE 1 SUBSTANCEP-IMMUNOREACTIVITY(pg/p.gprotein) IN THE NUCLEUSTRACTUS SOLITARIUSOF THE CAT: EFFECTOF INTRACRANIALSECTIONOF IXth AND Xth CRANIALNERVES* Sham NTS region Caudal Commissural Intermediate Rostral

Right 4.26 7.47 6.68 2.45

± 0.88 _-_ 1.33 ± 1.53 ± 1.07

Unilateral Denervation Left

Intact

3.49 ± 0.61 7.17 ± 1.15 6.92 ___1.80 4.02 ± 0.47

2.29 3.52 6.18 2.28

_ 0.49 ± 0.62t _+ 1.35 ___0.58

Denervated 2.75 +-- 0.76 3.97 ± 0.90t 3.67 +- 0.87~: 2.46 _+ 0.67

*Adapted from Gillis et al. [18]. tp<0.05 Compared to corresponding value of sham operated controls. ¢p<0.05 Compared to corresponding value of intact side.

TABLE 2 SUBSTANCEP-IMMUNOREACTIVITY(pg//xgprotein)IN THE NUCLEUSTRACTUS SOLITARIUSOF THE RAT: EFFECTOF LEFT NODOSE GANGLIONECTOMY* Sham

Left nodose ganglionectomy

NTS region

Right

Left

Right

Left

Caudal Intermediate Rostral

2.78 _ 0.12 5.59 ± 0.24 2.11 _+ 0.29

2.67 _ 0.16 6.32 _ 0.37 2.47 ± 0.27

2.81 - 0.25 6.19 --- 0.42 1.82 -+ 0.21

2.16 - 0.13# 4.80 --- 0.37t 1.88 +- 0.59

*Adapted from Helke et al. [25]. tp<0.05 Compared to corresponding value of the right NTS.

glion. Some of these same cells also stained for SP-I [22]. These results suggested that certain of the afferent fibers in the aortic nerve and thus the aortic arch afferents are SP-I nerves. Therefore, anatomical data supports the idea of SP containing baro- and chemoreceptor afferent nerves (Fig. 1). Other types of studies have also investigated whether the SP-I in the NTS is of viscerosensory origin and if it functions as a neurotransmitter in baro- and/or chemoreceptor reflexes. One of the criteria for neurotransmitter status of a chemical is that the agent be released in a calcium-dependent manner from nerve terminals upon their depolarization [38]. Thus, we assessed whether SP-I is released from NTS tissue slices in vitro with potassium depolarization [23]. We found that addition of a medium containing 50 mM potassium increased the release of endogenous SP-I 2.5 fold above the average basal level. With removal of the depolarizing amounts of potassium from the buffer, the SP-I in the medium returned to prestimulation values. In addition, because the homovanillylamide derivative, capsalcin, has been demonstrated to evoke SP-I effiux from regions receiving sensory afferents (the dorsal horn of the spinal cord) but ineffective in other nonsensory regions (hypothalamus and substantia nigra) [17,46], we also assessed the ability of capsaicin to release SP-I from the NTS and the hypothalamus in vitro [23]. We found that capsalcin dose dependently (0.33, 3.3, 33 /zM) increased the efflux of SP-I from NTS slices.

Thirty three/xM capsaicin resulted in a 224% increase in the release of SP-I from NTS slices. Hypothalamic tissue slices were unresponsive to the SP-I releasing actions of capsaicin but were responsive to potassium-induced depolarization. Omission of calcium from the incubation buffer completely prevented the potassium or capsalcin-induced elevation of the release of SP-I from the NTS [23]. Thus, SP-I is released from the NTS in a calcium dependent manner. In addition, the fact that capsaicin can also evoke SP-I release suggests that at least a portion of the peptide released is from afferent sensory fibers. To date, studies of the function or physiologic role of SP in baro- and chemoreceptor reflexes consist of those using local application of SP to the NTS and the monitoring of cardiovascular function. Conflicting results have been obtained with this approach. Whereas, Granata and Woodruff [19] report an increase in blood pressure and heart rate, Haeusler and Osterwalder [21] report a dose-dependent decrease in these parameters when SP is either injected into or topically applied to the NTS region of cats and rats. In contrast, Talman and Reis [45] report that microinjection of SP into the NTS does not affect arterial pressure, heart rate or phenylephrine-induced reflex bradycardia. However, a functional relationship between central SP systems and these cardiovascular reflexes is suggested by the findings that sectioning the vagus nerve proximal to the nodose ganglion [15] or chronic bilateral sinoaortic denervation [42] enhances the pressor response to intracisternal SP.

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Additional studies need to be done to further investigate the role of the SP in afferent nerves in baroreceptor reflexes, to study SP systems in chemoreceptor reflexes and to learn of interactions between SP and other potential afferent neurotransmitters in this viscerosensory system. sP-I IN VENTRALMEDULLARYPROJECTIONSTO THE INTERMEDIOLATERALCELL COLUMN Another SP neuronal projection which may be important in the regulation of cardiovascular function originates in the ventral medulla. The ventral surface of the medulla has been implicated in a wide range of visceral and endocrine functions, including cardiovascular control [14, 20, 32]. Electrical stimulation of this area raises blood pressure [10,32] and destruction lowers blood pressure [10,20]. Topical application of various putative neurotransmitters including acetylcholine, GABA and glutamate can change blood pressure [10, 12, 51]. The ventral medulla is also thought to contain the chemosensitive neurons which mediate the pressor response to cerebral ischemia [11]. Recent anatomical studies show that fibers originating in nuclei of the ventral medulla (i.e., the nucleus interfascicularis hypoglossi and nucleus reticularis gigantocellularis, pars a) provide a major innervation of the intermediolateral cell column of the ~pinal cord [1,35]. Cell bodies containing SP, thyrotropin releasing hormone, enkephalin, catecholamines and serotonin are present [26, 28, 31]. In addition, Loewy and McKellar [34] report that these serotonergic neurons project to the intermediolateral cell column (IML). Because certain of the SP-I neurons in the ventral medulla are also serotonergic [6, 26, 28] and because SP-I terminals are found in the intermediolateral cell column [31] we studied the SP neurons to determine whether they also project to the IML and if so whether the neurons contain both SP-I and serotonin. To do this we destroyed various hindbrain areas with electrolytic lesions and measured the SP-I content of microdissected areas of the thoracic spinal cord [24].

We found that a unilateral ventral medullary lesion, which included the nucleus interfascicularis hypoglossi, bilaterally reduced the SP-I content of the IML by 38%. The more rostral portions of the ventral medulla do not appear to contribute to the SP-I innervation of the spinal cord because lesions of the raphe magnus which included the nucleus reticularis gigantocellularis, pars ct, did not alter the SP-I content of the IML [24]. Likewise, midbrain hemisections did not alter the spinal cord SP-I content. This ventral medullary SP-I projection to the IML (Fig. l) does not appear to be contained in serotonergic nerves because destruction of serotonergic nerves with 5,7dihydroxytryptamine administered into the lateral ventricles and cisterna magna reduced spinal cord serotonin but did not alter the content of SP-I in the IML [24]. Whereas, nonspecific destruction of ventral medullary neurons either with electrolytic lesions or the neurotoxin, kainic acid, decreases resting blood pressure [10, 20, 37], destruction of serotonergic neurons does not consistently alter cardiovascular function in a normotensive animal [30]. This suggests that non-serotonergic neurons in the area may in part be responsible for the tonic maintenance of blood pressure. Further studies will clarify the role of this bulbospinal SP-I projection in cardiovascular control. In summary, SP-I neurons are contained in pathways which influence CNS and autonomic neural activity to the cardiovascular system. In addition, SP has direct peripheral cardiovascular effects. That a neurochemical may influence the cardiovascular system via separate central and peripheral mechanisms has also been reported for such classical neurotransmitters as norepinephrine [44], serotonin [30], and acetylcholine [43]. This interrelationship between central and peripheral functions may also occur with neuropeptides such as SP. ACKNOWLEDGEMENTS Supported by USPHS grant #HL-26849.

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