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Evidence against a hemodynamic role for serotonin in the dorsal motor nucleus of the vagus
BrainResearchBulletin,Vol. 37, No. 5, pp. 457-462, 1995 Copyright© 1995ElsevierScienceLtd Printedin the USA.All rightsreserved 0361-9230/95 $9.50 + .00
Pergamon 0361-9230(95)00024-0
Evidence Against a Hemodynamic Role for Serotonin in the Dorsal Motor Nucleus of the Vagus PETER D. FELDMAN 1 AND FLOYD J. GALIANO
Department of Pharmacology and Experimental Therapeutics, Louisiana State University Medical Center, 1901 Perdido Street, New Orleans, LA 70112-1393 [Received 11 April 1994; Accepted 17 November 1994] ABSTRACT: This study was performed to investigate the potential role of serotonin (5-HT) in the dorsal motor nucleus of the vagus (dmnX) in regulating peripheral hemodynamics. Microinjections (5 or 25 nmol in 50 hi) of the monoaminergic neurotransmitter were made into the dorsomedial medulla of the urethaneanesthetized rat during continuous recording of femoral arterial blond pressure. Heart rate was extracted electronically from the pressure waveform. Discrete injections of 5-HT placed directly in the dmnX were found to be entirely without effect on peripheral hemodynamics, in contrast, injections placed in the solitary tract nucleus, lying immediately above the dmnX, were found to have profound depressor and bredycardic affects, while the iramediately subjacent hypoglossal nucleus appeared to contain both depressor and unresponsive sites. These findings cast doubt on the involvement of serotonin in the dmnX in the regulation of cardiovascular hemodynamics.
dures have revealed the existence in the nucleus of binding sites for serotonergic receptor ligands [3,28]. A physiological role for serotonergic systems in the dmnX has been suggested based on the results of experiments involving microinjection of 5-HT or its analogs directly into the nucleus. Injection of minute volumes of 5-HT into the dmnX of the rat produces increases of both gastric motility and tone, and these effects can be prevented by postganglionic blockade with methylatropine [30]. Moreover, 5-HT appears to play a facilitatory role in the production of gastric acid, rather than a stimulatory one, as it augments the effects of microinjection of thyrotropinreleasing hormone into the nucleus [32]. In contrast to these clear sets of results, the effects of 5-HT on cardiovascular hemodynamics remains a point of controversy. One group that administered 500 nl volumes of 5-HT into the dorsomedial medulla [52] found that such treatment led to marked elevations of blood pressure, with variable effects on heart rate. A more recent team of investigators, however, found that somewhat more discrete volumes of selective 5-HT~A receptor agonists can evoke bradycardia, with no accompanying change of blood pressure [45]. In view of this controversy, the present study was designed to investigate further the potential cardiovascular role of 5-HT in the dmnX, with particular attention being paid to the anatomical specificity of action of the amine in bringing about changes of heart rate and arterial blood pressure.
KEY WORDS: Autonomic, Cardiovascular, Hypoglossai, Intercaletua, Microinjection, Parasympathetic, Reticular, Serotonin, Solitarius, Vagus.
INTRODUCTION The dorsal motor nucleus of the vagus (dnmX), located in the dorsal medulla, is a major source of the parasympathetic motor pathways that course through the tenth cranial nerve and ultimately innervate the viscera of the thorax and abdominal cavity [26,29,36,41]. Both electrical [11,17,35] and chemical [45] stimulation of the dnmX of the rat, dog, or rabbit can activate chronotropic cardioinhibitory motoneurons, leading to a profound primary bradycardia. Stimulation of the nucleus has also been shown to induce bronchoconstriction [29] and an increase in the force of gastric contractions [37] and levels of gastric acid secretion [32,53]. Anatomical and physiological studies have revealed that a number of neurotransmitters may have an important function as mediators of the afferent input to the dmnX. Among the list of candidates is the monoaminergic transmitter serotonin (5-HT). Investigators using either the formaldehyde-induced histofluorescence technique [10,14] or immunocytochemistry [6,48] have demonstrated that serotonergic nerve terminals are densely distributed within the nucleus. Postsynaptic sites of serotonergic action are also evident in the dmnX, as autoradiographic proce-
METHOD
Animals and Surgery Male Wistar rats weighing between 200 and 450 g were anesthetized with urethane ( 1.2-1.5 g/kg b.wt., IP; Sigma Chemical Co.). The femoral artery was cannulated with a polyethylene catheter (PE-50; Intramedic) to obtain continuous recordings of mean and instantaneous arterial blood pressure with a Gould Statham P23XL pressure transducer linked to a Coulbourne Instruments $72-25 strain gauge coupler and $77-34 blood pressure processor. Heart rate was extracted electronically from the pressure wave (Coulbourne Instruments $77-26 tachometer). Rats were tracheotomized and artificially ventilated with room air (Kent Scientific RSPI002 small animal respirator), and blood gases were regulated by adjusting respiration rate and inspiratory volume and by supplementing the inspired air with 100% 02.
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Animals were placed in a stereotaxic apparatus (David Kopf Instruments) with their heads ventroflexed at a 45 ° angle. The muscles of the neck were surgically retracted, and a limited occipital craniotomy was performed.
Microinjection Three-barreled microinjection assemblies were constructed from 6 - i n c h glass microcapillary tubing (o.d. 1.0 mm, i.d. 0.58 mm; A-M Systems). One barrel was backtilled with a 2% solution of Pontamine Sky blue 6BX dye (Bio/medical Specialties) in 0.5 M sodium acetate, and another barrel was backfilled with 5-HT (t00 or 500 mM; Sigma Chemical Co.) dissolved in 0.9% sodium chloride and adjusted to a pH of 7.2-7.4. The third barrel was backfilled with a control solution of saline vehicle (0.9%). Following data collection at each injection site, Pontamine dye was ejected (50 nl) to mark the site for subsequent histological identification. Serotonin was injected in a volume of 50 nl. The amount of injected solution was controlled manually with either a micropneumophoresis system (Medical Systems Corp. BH-2) or by manual compression of a hand-held syringe while monitoring the movement of the fluid meniscus with a Nikon SMZ-2B dissecting microscope during the pressurization of the injection barrel. To control for the potential physiological effects of the vehicle itself, 50-nl microinjections of 0.9% saline were made, and their effects compared to the effects of 5-HT. Microinjections of the agonist or of vehicle were performed several times at a given site to verify that an initially observed response, or lack thereoL could be replicated, and injections were spaced temporally by at least 15 min to minimize the potential effects of receptor desensitization.
5 within the nucleus of the tractus solitarius (nTS), 5 primarily within the hypoglossal nucleus (nXII), and 3 in the dorsal reticular regions, ventral and lateral to the dmnX and nXII. Following microinjection of 5-HT, sites could be characterized on the basis of the induced changes of blood pressure and heart rate as depressor/bradycardic, depressor only, pressor, or unresponsive. When all sites were classified in this manner, a map correlating hemodynamic responses with specific structures showed a distinct regional variability in the ability of 5-HT to induce changes of hemodynamics (Fig. 1). In view of previous reports of hemodynamic responses to infusion of 5-HT or its analogs into the region of the dorsal vagal complex, one particularly striking finding in this study was that the dmnX was essentially unresponsive to infusion of the amine. Twenty-four of the 27 sites in the dmnX that were studied were characterized as completely unresponsive to application of 5-HT. Sites in the dmnX were tested with microinjections of either 5 nmol 5-HT (n = 23) or a higher dose of 25 nmol (n = 4). As depicted by the bar graph in Fig. 2, no significant change of either blood pressure (top) or heart rate (bottom) could be detected at
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Histology and Statistical Analysis At the end of each experiment, rats were perfused transcardially with phosphate-buffered 10% formalin, and brains were removed and fixed in formalin for 2 - 6 days. Sections (40 #m) of the medulla were cut with a cryostatic microtome (International Equipment Co.) and counterstained with 1% neutral red (Sigma Chemical Co.). The rostrocaudal level of the section where a dye spot appeared to be centered was calculated by counting the number of 40 #m thick sections between it and the section where the obex (defined as the opening of the central canal into the Fourth Ventricle) could be visually identified. Student's paired and Cochran's unpaired t-tests were used to determine the statistical significance of changes of heart rate and blood pressure. It had been proposed that the chi-square test be used to determine by statistical methods if correlations existed between anatomical structures and the responsiveness of the sites studied within those structures. This proved impractical, however, as the number of sites studied in some structures was too low for meaningful application of the test. Pooled data are expressed in the text as the mean _+ SE. Differences were considered significant at a p-value of 0.05 or less. RESULTS The histological staining performed in this study was of such a clarity that differentiation could easily be made between medullary structures, and the delineation of nuclear borders readily performed. Moreover, the dye spots marking the locations that were studied were quite discrete, with diameters of spread typically around 100 #m, which permitted the clear identification of the structures involved. A total of 28 rats was used in this study, yielding unambiguous data from 27 confirmed sites in the dmnX,
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FIG. I. Schematic representation of the locations of microinjections of 5-HT into the dorsomedial medulla. The sites of microinjection are interpolated from the centers of dye spots deposited by pneumophoresis of Pontamine sky blue from one barrel of the microinjection assembly. Locations are shown projected onto four representative transverse sections, with the rostrocaudal level, relative to obex, shown in microns at the left of each section. Associated changes of blood pressure are shown in the left series, changes of heart rate on the right. Decreases are depicted as filled circles, increases as filled triangles, and unresponsive sites are marked with open circles, ap--area postrema; cc--central canal; dmnX--dorsal motor nucleus of the vagus; DRF--dorsal reticular formation; nTS--nucleus of the tractus solitarius; nXll--hypoglossal nucleus.
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indicate a regional variability within the nXII, considerable further investigation will be required before such a postulate may be proposed. The dorsal reticular field has had a long-standing reputation for a role in cardiovascular regulation [1,27]. Moreover, a significant density of 5-HTrlike binding sites has been detected in the region [49,50]. It was therefore an unexpected finding that injections of 5-HT (5 nmol) placed in the area were without effect (see Fig. 1). Again, however, due to the limited number of sites studied in the region, no conclusion can be drawn regarding the hemodynamic role of 5-HT in the medullary reticular formation.
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FIG. 2. Hemodynamic effects of microinjection of two doses of 5-HT into the dmnX, 5 nmol (n = 23) and 25 nmol (n = 4). Pooled data are shown for changes of blood pressure (top) and heart rate (bottom). N.S.--p > 0.05. either dose, even with the inclusion of the low-dose data from the three responsive sites. Of the latter, two sites overlapped considerably with the nTS, lying immediately dorsal to the dmnX. One of these sites also may have included the nXII, as indicated by the spread of dye, and was associated with a mild elevation of blood pressure (+8 to +12 mmHg) and a decrease of heart rate ( - 4 0 to - 6 0 bpm). The chronotropic change was presumed to be baroreflexive in nature, although a vagotomy was not performed to confirm this possibility. The other site was characterized by mild depressor responses ( - 1 0 to - 2 8 mmHg) with no accompanying changes of heart rate. The third responsive site overlapped considerably with the the subjacent nXII, and was characterized by a mild increase of blood pressure and decrease of heart rate. In contrast to the unresponsiveness of the dmnX, sites entirely within the nTS were consistently found to be associated with large decreases of both blood pressure and heart rate (Fig. 3). Pooling the data from the five sites tested for the effects of 5-HT (5 nmol) revealed a mean decrease in blood pressure of 25 _ 3.4 mmHg and a drop in heart rate of 47.5 + 5.6 bpm. Both effects were highly significant (p < 0.01), and could be obtained from both the commissural levels of the nucleus and more rostrally at the level of the area postrema (see Fig. 1). Responses to injection of 5-HT (5 nmol) into the five sites primarily within the nXII proved to be somewhat variable. Three sites were associated with significant drops of blood pressure (-23.8 +__7.2 mmHg) and a decrease of heart rate (-37.5 + 15.9 bpm) that collectively approached significance (0.05 < p < 0.1), whereas the other two sites were unresponsive. Both responsive and unresponsive sites were found at all rostrocaudal levels (see Fig. 1). Although it is tempting to suggest that these findings may
The major finding of the present study was that the dmnX does not appear to support 5-HT-induced changes of peripheral hemodynamics. This contrasts with the results of previous studies that suggest that activation of 5-HT~, receptors in the dmnX with very low concentrations of highly selective agonists such as 8hydroxy-dipropylaminotetralin(8-OH-DPAT) can induce a transient bradycardia [45]. It is particularly intriguing that the more selective compound was effective in the previous experiments, whereas the broad-spectrum autacoid used in the present study was ineffective. One possible explanation that might be offered is that 8-OH-DPAT is generally far more potent that 5-HT, which would imply that the 5-HT used in the present study was not given at a dose high enough to affect 5-HTIA receptors. This argument is unsatisfactory, however, in view of in vitro data that indicate that 5-HT has a Kd of jUSt 2 nM at 5-HT]A receptors [19,20]. With the 50 nl volumes used in the present study, this corresponds to a dose of just 100 fmol, whereas the 5 and 25 nmol doses used here are well above that figure. An alternative explanation is that the difference in results arises from the different volumes of microinjectate being used in the two sets of studies, as the previous study employed 100 nl volumes, whereas the present study involved the use of 50 nl injections. It is conceivable that the larger volume may be associated with a sphere of drug action sufficiently large enough to affect neurons in the nTS, which the present study demonstrates is capable of mediating profound 5-HT-induced decreases of both heart rate and arterial pressure. The dmnX has long been known to have a role in cardiomotor chronotropic regulation. Although the projection from the nucleus ambiguus to the myocardium is much heavier than that from the dmnX [46,47], it has nevertheless been demonstrated repeatedly that a significant component of the cardiac branch of
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the vagus nerve arises from the dmnX in the cat [16,26], dog [5], rabbit [11,24], and rat [35,46]. However, previous anatomical investigations indicate that vagal cardiomotor preganglionics located within the nuclear boundaries of the dmnX do not receive serotonergic input [22]. The data from the present study provide physiological support for this notion. One objection that might be raised to this interpretation, however, is that many of the motorneurons of the dmnX have long, ramifying dendrites that extend dorsally into the nTS [31], and that the 5-HT-induced alterations of hemodynamic activity attributed here to the nTS may, in fact, be due to a direct effect on neurons in the dmnX. This possibility seems remote, however, based on several points. First, the changes of cardiovascular function that were induced with nTS microinjections included decreases of blood pressure. It has been demonstrated that such changes are not secondary to decreases of heart rate, as bilateral transection of the vagus nerves can eliminate the bradycardia, but not the vasodepression, associated with 5-HT injection into the nTS [13]. Although electrical stimulation of the dmnX can activate vagal motor pathways that produce bradycardia [35], primary vasodepressor responses have never been obtained from the dmnX. Thus, we may be reasonably sure that the cardiovascular effects seen here with microinjection of 5-HT into the nTS are, in fact, due to an effect on neurons in that structure, and not those in the dmnX. A second counterargument that could be made is based on our current understanding of the electrophysiological effects of 5-HT in the dorsal vagal complex. Recent studies performed in this laboratory with physiologically-relevant concentrations of 5-HT [12] suggest that the monoamine primarily exerts a 5-HT~ receptor-mediated postsynaptic inhibition in the nTS. It is a commonly accepted generality that receptors that mediate inhibitory influences on neuronal activity are usually located on cell bodies, rather than on dendrites. Unfortunately, conclusive evidence is lacking regarding the electrophysiology of 5-HT in the dmnX. It therefore appears that, based on the knowledge obtained to date, the function of 5-HT in the dmnX is restricted to regulation of gastric function. In this regard, McCann and co-workers [30] have demonstrated that the nucleus is, in fact, exquisitely sensitive to 5-HT, as microinjection of minuscule volumes of extremely low concentrations of the amine into the dmnX induces vagally-mediated increases of gastric pressure and motility. There is considerable interaction between 5-HT and a neuropeptide, thyrotropin-releasing hormone (TRH). "Conditioning" applications of TRH to the surface of the dorsomedial medulla augment the effects of 5-HT on gastric motility and tone [30]; conversely, prior application of 5-HT augments the stimulatory effect of TRH on secretion of gastric acid [32]. It is not entirely clear what structures contribute to the serotonergic innervation of the dmnX, but leading candidates include the raphe nuclei [4,7], the nTS [8,42] and the primary visceral sensory afferents, themselves, from the vagus [ ! 5] or carotid sinus [51 ] nerves. For their part, TRH-containing afferents to the dmnX are known to originate in the medullary raphe [38]. A strong possibility therefore exists that 5-HT and TRH are colocalized in the synaptic vesicles of afferents from the raphe, where the two transmitters have been found within the same neuron [23,25]. As with the dmnX, a role in digestive regulation has been proposed for 5-HT in the nTS based on the observation that deglutitory movements of the pharynx can be elicited through what appears to be a 5-HTz receptor-mediated mechanism [ 18]. In contrast to the dmnX, however, it is now clear that 5-HT also has a prominent role in cardiovascular regulation in the nTS. In the present study, microinjection of 5-HT into the nucleus led to profound decreases of both blood pressure and heart rate, changes that resembled those seen during activation of the baroreceptor
FELDMAN AND G A L I A N O
reflex. This supports previous findings in a number of laboratories that suggest a 5-HT2 receptor-mediated parasympathoexcitation and sympathoinhibition [21,44]. Despite these indications of the presence of 5-HT2 receptors in the nTS, however, surprisingly little anatomical evidence has been available for the presence of this receptor subtype in the nucleus. A high density of 5-HT~A-like sites has been described [28,40], and a lower level of 5 - H T ~ r l i k e binding is also present [28,49], but autoradiographic studies with 5-HTz-selective ligands seem to indicate little, if any, 5-HT2-1ike binding in the nTS [3,39]. Nevertheless, more recent experiments using antibodies to the 5-HT2 receptor indicate that there may be some very light 5-HTz-like binding in a discrete region of the medial subnucleus at levels rostral to the obex [33]. Interestingly, the area in question correlates precisely with the site of termination of subdiaphragmatic esophageal afferents [2], rather than of baroreceptive or pharyngeal afferents [2,91. At this point, it would be premature to propose a cardiovascular role for 5-HT in the nXlI. Three of the five sites studied in the present investigation appeared to support 5-HT-induced changes of heart rate and blood pressure, and, judging from the locations and diameters of spread of the dye spots that were left at these sites, the effective locations did not overlap with the nTS. Nevertheless, these data are limited in number. Moreover, it is altogether quite possible that the rostraimost of the effective locations, which was located rostral to the obex, may have included such perihypoglossal structures as the nucleus intercalatus, which has been demonstrated to contain dendrites of vagal preganglionic neurons and to receive considerable afferent input from forebrain autonomic structures [34,43], as well as being the site of very high densities of 5-HTIA- and 5 - H T ~ r l i k e binding sites [49,50]. Resolution of this issue will require considerable further investigation, using even smaller microinjection volumes than were used in the present study. To summarize, this set of experiments was performed to investigate the potential involvement of 5-HT in the dmnX in regulating cardiovascular hemodynamics. No evidence for such a role was found, as microinjection of the amine into the nucleus failed to evoke any changes of either heart rate or blood pressure. On the other hand, microinjection of 5-HT into the overlying nTS led to profound decreases of both blood pressure and heart rate, changes similar to those seen during activation of the baroreceptor reflex. It is always more difficult to disprove the existence of something than to prove its existence. Therefore, perhaps more work is needed to rule out the contribution of serotonergic systems in the dmnX towards the regulation of peripheral hemodynamics. Nevertheless, the present findings cast serious doubt on this possibility. By contrast, these data support the involvement of serotonergic systems in the nTS in the regulation of cardiovascular hemodynamics, suggesting that they contribute to mechanisms that activate parasympathetic cardiomotor pathways and inhibit sympathetic outflow. ACKNOWLEDGEMENT This work was supported by NIH Grant R29-NS29458.
REFERENCES 1. Alexander, R. S. Tonic and reflex functions of medullary sympathetic cardiovascular centers. J. Neurophysiol. 9:205-217; 1946. 2. Altschuler, S. M.; Bat, X.; Bieger, D.; Hopkins, D. A.; Miselis, R. R. Viscerotopic representations of the upper alimentary tract in the rat: Sensory ganglia and nuclei of the solitary and spinal trigeminal tracts. J. Comp. Neurol. 283:248-268; 1989.
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3. Appel, N. M.; Mitchell, W. M.; Garlick, R. K.; Glennon, R. A.; Titeler, M.; De Souza, E. B. Autoradiographic characterization of (_+)- l-(2,5-dimethoxy-4-[~2sI]iodophenyl)-2-aminopropane (['25I]DOI) binding to 5-HT2 and 5-HT~c receptors in rat brain. J. Pharmacol. Exp. Ther. 255:843-857; 1990. 4. Basbaum, A. I.; Clanton, C. H.; Fields, H. L. Three bulbospinal pathways from the rostral medulla of the cat: An autoradiographic study of pain modulatory systems. J. Comp. Neurol. 178:209-224; 1978. 5. Bennett, J. A.; Kidd, C.; Latif, A. B.; McWilliam, P. N. A horseradish peroxidase study of vagal motoneurones with axons in cardiac and pulmonary branches of the cat and dog. Quart. J. Exp. Physiol. 66:145-154; 1981. 6. Berk, M. L.; Smith, S. E.; Karten, H. J. Nucleus of the solitary tract and dorsal motor nucleus of the vagus nerve of the pigeon: Localization of peptide and 5-hydroxytryptamine immunoreactive fibers. J. Comp. Neurol. 338:521-548; 1993. 7. Bobillier, P.; Seguin, S.; Petitjean, F.; Salvert, D.; Touret, M.; Jouvet, M. The raphe nuclei of the cat brainstem: A topographical atlas of their efferent projections as revealed by autoradiography. Brain Res. 113:449-486; 1976. 8. Calz& L.; Giardino, L.; Grimaldi, R.; Rigoli, M.; Steinbusch, H. W. M.; Tiengo, M. Presence of 5-HT-positive neurons in the medial nuclei of the solitary tract. Brain Res. 347:135-139; 1985. 9. Ciriello, J. Brainstem projections of aortic baroreceptor afferent fibers in the rat. Neurosci. Lett. 36:37-42; 1983. 10. Dahlstr/~m, A. B.; Fuxe, K. Localization of monoamines in the lower brain stem. Experientia 20:398-399; 1964. 11. Ellenberger, H. H.; Haselton, J. R.; Liskowsky, D. R.; Schneiderman, N. The location of chronotropic cardioinhibitory vagal motoneurons in the medulla of the rabbit. J. Auton. Nerv. Syst. 9:513529; 1983. 12. Feldman, P. D. Electrophysioiogical effects of serotonin in the solitary tract nucleus of the rat. Naunyn Schmiedebergs Arch. Pharmacol. 349:447-454; 1994 13. Feldman, P. D.; Galiano, F. J. Hemodynamic effects of serotonin in the nucleus tractus solitarius. Am. J. Physiol. (in press). 14. Fuxe, K. The distribution of monoamine terminals in the central nervous system. Acta Physiol. Scand. 64(Suppl. 247):38-85; 1965. 15. Gaudin-Chazal, G.; Portalier, P4 Barrit, M. C.; Puizillout, J.-J. Serotonin-like immunoreactivity in paraffin-sections of the nodose ganglia of the cat. Neurosci. Lett. 33:169-172; 1982. 16. Geis, G. S.; Wurster, R. D. Horseradish peroxidase localization of cardiac vagal preganglionic somata. Brain Res. 182:19-30; 1980. 17. Gunn, C. G.; Sevelius, G.; Puiggari, M. J.; Myers, F. K. Vagal cardiomotor mechanisms in the hindbrain of the dog and cat. Am. J. Physiol. 214:258-262; 1968. 18. Hashim, M. A.; Bieger, D. Excitatory action of 5-HT on deglutitive substrates in the rat solitary complex. Brain Res. Bull. 18:355-363; 1987. 19. Hoyer, D.; Engel, G.; Kalkman, H. O. Molecular pharmacology of 5-HTj and 5-HT2 recognition sites in rat and pig brain membranes: Radioligand binding studies with [3H]5-HT, [3H]8-OH-DPAT, (-)[J25I]iodocyanopindolol, [3H]mesulergine and [3H]ketanserin. Eur. J. Pharmacol. 118:13-23; 1985. 20. lsmaiel, A. M.; Titeler, M.; Miller, K. J.; Smith, T. S.; Glennon, R. A. 5-HT~ and 5-HT2 binding profiles of the serotonergic agents a-methylserotonin and 2-methylserotonin. J. Med. Chem. 33:755758; 1990. 21. ltoh, H.; Bufiag, R. D. Cardiovascular and sympathetic effects of injecting serotonin into the nucleus tractus solitarius in rats. J. Pharmacol. Exp. Ther. 256:1147-1153; 1991. 22. lzzo, P. N.; Deuchars, J.; Spyer, K. M. Localization of cardiac preganglionic motoneurones in the rat: Immunocytochemical evidence of synaptic inputs containing 5-hydroxytryptamine. J. Comp. Neurol. 327:572-583; 1993. 23. Johansson, O.; Htikfelt, T.; Pernow, B.; Jeffcoate, S. L.; White, N.; Steinbusch, H. W. M.; Verhofstad, A. A. J.; Emson, P. C.; Spindel, E. Immunohistochemical support for three putative transmitters in one neuron: Coexistence of 5-hydroxytryptamine, substance P- and thyrotropin releasing hormone-like immunoreactivity in medullary
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42.
43.
neurons projecting to the spinal cord. Neuroscience 6:1857-1881 ; 1981. Jordan, D.; Khalid, M. E. M.; Schneiderman, N.; Spyer, K. M. The location and properties of preganglionic vagal cardiomotor neurones in the rabbit. Pfliigers Arch. 395:244-250; 1982. Kachidian, P.; Poulat, P.; Marlier, L.; Privat, A. Immunohistochemical evidence for the coexistence of substance P, thyrotropin-releasing hormone, GABA, methionin-enkephalin, and leucin-enkephalin in the serotonergic neurons of the caudal raphe nuclei: A dual labeling in the rat. J. Neurosci. Res. 30:521-530; 1991. Kalia, M. Brain stem localization of vagal preganglionic neurons. J. Auton. Nerv. Syst. 3:451-481; 1981. Magoun, H. W.; Rhines, R. An inhibitory mechanism in the bulboreticular formation. J. Neurophysiol. 9:165-171 ; 1946. Manaker, S.; Verderame, H. M. Organization of serotonin IA and I B receptors in the nucleus of the solitary tract. J. Comp. Neurol. 301:535-553; 1990. McAllen, R. M.; Spyer, K. M. Two types of vagal preganglionic motoneurones projecting to the heart and lungs. J. Physiol. (Lond.) 282:353-364; 1978. McCann, M. J.; Hermann, G. E.; Rogers, R. C. Dorsal medullary serotonin and gastric motility: Enhancement of effects by thyrotropin-releasing hormone. J. Auton. Nerv. Syst. 25:35-40; 1988. McLean, J. H.; Hopkins, D. A. Ultrastructure of the dorsal motor nucleus of the vagus nerve in monkey with comparison of synaptology in monkey and cat. J. Comp. Neurol. 231:162-174; 1985. McTigue, D. M.; Rogers, R. C.; Stephens, R. L., Jr. Thyrotropinreleasing hormone analogue and serotonin interact within the dorsal vagal complex to augment gastric acid secretion. Neurosci. Lett. 144:61-64; 1992. Morilak, D. A.; Garlow, S. J.; Ciaranello, R. D. Immunocytochemical localization and description of neurons expressing serotonin2 receptors in the rat brain. Neuroscience 54:701-717; 1993. Norgren, R. E.; Smith, G. P. Central distribution of subdiaphragmatic vagal branches in the rat. J. Comp. Neurol. 273:207-223; 1988. Nosaka, S.; Yamamoto, T.; Yasunaga, K. Localization of vagal cardioinhibitory preganglionic neurons within rat brain. J. Comp. Neurol. 186:79-92; 1979. Ortega-Villalobos, M,; Garcia-Baz~,n, M.; Solano-Flores, L. P.; Ninomiya-Alarc~m, J. G.; Guevara-Guzm~.n, R.; Wayner, M. J. Vagus nerve afferent and efferent innervation of the rat uterus: An electrophysiological and HRP study. Brain Res. Bull. 25:365-371; 1990. Pagani, F. D.; Norman, W. P.; Kasbekar, D. K.; Gillis, R. A. Localization of sites within the dorsal motor nucleus of the vagus that affect gastric motility. Am. J. Physiol. 249:G73-G84; 1985. Palkovits, M.; Mezey, t~.; Eskay, R. L.; Brownstein, M. J. Innervation of the nucleus of the solitary tract and the dorsal vagal nucleus by thyrotropin-releasing hormone-containing raphe neurons. Brain Res. 373:246-251 ; 1986. Pazos, A.; Cort6s, R.; Palacios, J. M. Quantitative autoradiographic mapping of serotonin receptors in the rat brain. II. Serotonin-2 receptors. Brain Res. 346:231-249; 1985. Pompeiano, M.; Palacios, J. M.; Mengod, G. Distribution and cellular localization of mRNA coding for 5-HT~A receptor in the rat brain: Correlation with receptor binding. J. Neurosci. 12:440-453; 1992. Rinaman, L. M.; Miselis, R. R. The organization of vagal innervation of rat pancreas using cholera toxin-horseradish peroxidase conjugate. J. Auton. Nerv. Syst. 21:109-125; 1987. Rogers, R. C.; Kita, H.; Butcher, L. L.; Novin, D. Afferent projections of the dorsal motor nucleus of the vagus. Brain Res. Bull. 5:365-373; 1980. Shapiro, R. E.; Miselis, R. R. The central organization of the vagus nerve innervating the stomach of the rat. J. Comp. Neurol. 238:473488; 1985.
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44. Shvaloff, A.; Laguzzi, R. Serotonin receptor in the rat nucleus tractus solitarii and cardiovascular regulation. Eur. J. Pharmacol. 132:283288; 1986. 45. Sporton, C. E.; Shepheard, S. L.; Jordan, D.; Ramage, A. G. Microinjections of 5-HT~A agonists into the dorsal motor vagal nucleus produce a bradycardia in the atenolol-pretreated anesthetized rat. Br. J. Pharmacol. 104:466-470; 1991. 46. Stuesse, S. L. Origins of cardiac vagal preganglionic fibers: A retrograde transport study. Brain Res. 236:15-25; 1982. 47. Stuesse, S. L.; Powell, K. S. Cardiac vagal preganglionic fibers in neonatal rats: A comparison with cervical vagal components. Neurosci. Lett. 34:7-12; 1982. 48. Tallaksen-Greene, S. J.; Elde, R. J.; Wessendorf, M. W. Regional distribution of serotonin and substance P co-existing in nerve fibers and terminals in the brainstem of the rat. Neuroscience 53:1127 1142; 1993.
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49. Thor, K. B.; Blitz-Siebert, A.; Helke, C. J. Autoradiographic localization of 5-HTL binding sites in the medulla oblongata of the rat. Synapse 10:185-205; 1992. 51). Thor, K. B.; Blitz-Siebert, A.; Helke, C. J. Autoradiographic localization of 5-HT~ binding sites in autonomic areas of the rat dorsomedial medulla oblongata. Synapse 10:217-227; 1992. 51. Wang, Z.-Z.; Stensaas, L. J.; Dinger, B. G.; Fidone, S. J. The co-existence of biogenic amines and neuropeptides in the type I cells of the cat carotid body. Neuroscience 47:473-480; 1992. 52. Wolf, W. A.; Kuhn, D. M.; Lovenberg, W. M. Blood pressure responses to local application of serotonergic agents in the nucleus tractus solitarii. Eur. J. Pharmacol. 69:291-299; 1981. 53. Wyrwicka, W.; Garcia, R. Effect of electrical stimulation of the dorsal nucleus of the vagus nerve on gastric acid secretion in cats. Exp. Neurol. 65:315-325; 1979.
Pergamon 0361-9230(95)00024-0
Evidence Against a Hemodynamic Role for Serotonin in the Dorsal Motor Nucleus of the Vagus PETER D. FELDMAN 1 AND FLOYD J. GALIANO
Department of Pharmacology and Experimental Therapeutics, Louisiana State University Medical Center, 1901 Perdido Street, New Orleans, LA 70112-1393 [Received 11 April 1994; Accepted 17 November 1994] ABSTRACT: This study was performed to investigate the potential role of serotonin (5-HT) in the dorsal motor nucleus of the vagus (dmnX) in regulating peripheral hemodynamics. Microinjections (5 or 25 nmol in 50 hi) of the monoaminergic neurotransmitter were made into the dorsomedial medulla of the urethaneanesthetized rat during continuous recording of femoral arterial blond pressure. Heart rate was extracted electronically from the pressure waveform. Discrete injections of 5-HT placed directly in the dmnX were found to be entirely without effect on peripheral hemodynamics, in contrast, injections placed in the solitary tract nucleus, lying immediately above the dmnX, were found to have profound depressor and bredycardic affects, while the iramediately subjacent hypoglossal nucleus appeared to contain both depressor and unresponsive sites. These findings cast doubt on the involvement of serotonin in the dmnX in the regulation of cardiovascular hemodynamics.
dures have revealed the existence in the nucleus of binding sites for serotonergic receptor ligands [3,28]. A physiological role for serotonergic systems in the dmnX has been suggested based on the results of experiments involving microinjection of 5-HT or its analogs directly into the nucleus. Injection of minute volumes of 5-HT into the dmnX of the rat produces increases of both gastric motility and tone, and these effects can be prevented by postganglionic blockade with methylatropine [30]. Moreover, 5-HT appears to play a facilitatory role in the production of gastric acid, rather than a stimulatory one, as it augments the effects of microinjection of thyrotropinreleasing hormone into the nucleus [32]. In contrast to these clear sets of results, the effects of 5-HT on cardiovascular hemodynamics remains a point of controversy. One group that administered 500 nl volumes of 5-HT into the dorsomedial medulla [52] found that such treatment led to marked elevations of blood pressure, with variable effects on heart rate. A more recent team of investigators, however, found that somewhat more discrete volumes of selective 5-HT~A receptor agonists can evoke bradycardia, with no accompanying change of blood pressure [45]. In view of this controversy, the present study was designed to investigate further the potential cardiovascular role of 5-HT in the dmnX, with particular attention being paid to the anatomical specificity of action of the amine in bringing about changes of heart rate and arterial blood pressure.
KEY WORDS: Autonomic, Cardiovascular, Hypoglossai, Intercaletua, Microinjection, Parasympathetic, Reticular, Serotonin, Solitarius, Vagus.
INTRODUCTION The dorsal motor nucleus of the vagus (dnmX), located in the dorsal medulla, is a major source of the parasympathetic motor pathways that course through the tenth cranial nerve and ultimately innervate the viscera of the thorax and abdominal cavity [26,29,36,41]. Both electrical [11,17,35] and chemical [45] stimulation of the dnmX of the rat, dog, or rabbit can activate chronotropic cardioinhibitory motoneurons, leading to a profound primary bradycardia. Stimulation of the nucleus has also been shown to induce bronchoconstriction [29] and an increase in the force of gastric contractions [37] and levels of gastric acid secretion [32,53]. Anatomical and physiological studies have revealed that a number of neurotransmitters may have an important function as mediators of the afferent input to the dmnX. Among the list of candidates is the monoaminergic transmitter serotonin (5-HT). Investigators using either the formaldehyde-induced histofluorescence technique [10,14] or immunocytochemistry [6,48] have demonstrated that serotonergic nerve terminals are densely distributed within the nucleus. Postsynaptic sites of serotonergic action are also evident in the dmnX, as autoradiographic proce-
METHOD
Animals and Surgery Male Wistar rats weighing between 200 and 450 g were anesthetized with urethane ( 1.2-1.5 g/kg b.wt., IP; Sigma Chemical Co.). The femoral artery was cannulated with a polyethylene catheter (PE-50; Intramedic) to obtain continuous recordings of mean and instantaneous arterial blood pressure with a Gould Statham P23XL pressure transducer linked to a Coulbourne Instruments $72-25 strain gauge coupler and $77-34 blood pressure processor. Heart rate was extracted electronically from the pressure wave (Coulbourne Instruments $77-26 tachometer). Rats were tracheotomized and artificially ventilated with room air (Kent Scientific RSPI002 small animal respirator), and blood gases were regulated by adjusting respiration rate and inspiratory volume and by supplementing the inspired air with 100% 02.
To whom requests for reprints should be addressed. 457
458
F E L D M A N AND G A L I A N O
Animals were placed in a stereotaxic apparatus (David Kopf Instruments) with their heads ventroflexed at a 45 ° angle. The muscles of the neck were surgically retracted, and a limited occipital craniotomy was performed.
Microinjection Three-barreled microinjection assemblies were constructed from 6 - i n c h glass microcapillary tubing (o.d. 1.0 mm, i.d. 0.58 mm; A-M Systems). One barrel was backtilled with a 2% solution of Pontamine Sky blue 6BX dye (Bio/medical Specialties) in 0.5 M sodium acetate, and another barrel was backfilled with 5-HT (t00 or 500 mM; Sigma Chemical Co.) dissolved in 0.9% sodium chloride and adjusted to a pH of 7.2-7.4. The third barrel was backfilled with a control solution of saline vehicle (0.9%). Following data collection at each injection site, Pontamine dye was ejected (50 nl) to mark the site for subsequent histological identification. Serotonin was injected in a volume of 50 nl. The amount of injected solution was controlled manually with either a micropneumophoresis system (Medical Systems Corp. BH-2) or by manual compression of a hand-held syringe while monitoring the movement of the fluid meniscus with a Nikon SMZ-2B dissecting microscope during the pressurization of the injection barrel. To control for the potential physiological effects of the vehicle itself, 50-nl microinjections of 0.9% saline were made, and their effects compared to the effects of 5-HT. Microinjections of the agonist or of vehicle were performed several times at a given site to verify that an initially observed response, or lack thereoL could be replicated, and injections were spaced temporally by at least 15 min to minimize the potential effects of receptor desensitization.
5 within the nucleus of the tractus solitarius (nTS), 5 primarily within the hypoglossal nucleus (nXII), and 3 in the dorsal reticular regions, ventral and lateral to the dmnX and nXII. Following microinjection of 5-HT, sites could be characterized on the basis of the induced changes of blood pressure and heart rate as depressor/bradycardic, depressor only, pressor, or unresponsive. When all sites were classified in this manner, a map correlating hemodynamic responses with specific structures showed a distinct regional variability in the ability of 5-HT to induce changes of hemodynamics (Fig. 1). In view of previous reports of hemodynamic responses to infusion of 5-HT or its analogs into the region of the dorsal vagal complex, one particularly striking finding in this study was that the dmnX was essentially unresponsive to infusion of the amine. Twenty-four of the 27 sites in the dmnX that were studied were characterized as completely unresponsive to application of 5-HT. Sites in the dmnX were tested with microinjections of either 5 nmol 5-HT (n = 23) or a higher dose of 25 nmol (n = 4). As depicted by the bar graph in Fig. 2, no significant change of either blood pressure (top) or heart rate (bottom) could be detected at
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Histology and Statistical Analysis At the end of each experiment, rats were perfused transcardially with phosphate-buffered 10% formalin, and brains were removed and fixed in formalin for 2 - 6 days. Sections (40 #m) of the medulla were cut with a cryostatic microtome (International Equipment Co.) and counterstained with 1% neutral red (Sigma Chemical Co.). The rostrocaudal level of the section where a dye spot appeared to be centered was calculated by counting the number of 40 #m thick sections between it and the section where the obex (defined as the opening of the central canal into the Fourth Ventricle) could be visually identified. Student's paired and Cochran's unpaired t-tests were used to determine the statistical significance of changes of heart rate and blood pressure. It had been proposed that the chi-square test be used to determine by statistical methods if correlations existed between anatomical structures and the responsiveness of the sites studied within those structures. This proved impractical, however, as the number of sites studied in some structures was too low for meaningful application of the test. Pooled data are expressed in the text as the mean _+ SE. Differences were considered significant at a p-value of 0.05 or less. RESULTS The histological staining performed in this study was of such a clarity that differentiation could easily be made between medullary structures, and the delineation of nuclear borders readily performed. Moreover, the dye spots marking the locations that were studied were quite discrete, with diameters of spread typically around 100 #m, which permitted the clear identification of the structures involved. A total of 28 rats was used in this study, yielding unambiguous data from 27 confirmed sites in the dmnX,
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FIG. I. Schematic representation of the locations of microinjections of 5-HT into the dorsomedial medulla. The sites of microinjection are interpolated from the centers of dye spots deposited by pneumophoresis of Pontamine sky blue from one barrel of the microinjection assembly. Locations are shown projected onto four representative transverse sections, with the rostrocaudal level, relative to obex, shown in microns at the left of each section. Associated changes of blood pressure are shown in the left series, changes of heart rate on the right. Decreases are depicted as filled circles, increases as filled triangles, and unresponsive sites are marked with open circles, ap--area postrema; cc--central canal; dmnX--dorsal motor nucleus of the vagus; DRF--dorsal reticular formation; nTS--nucleus of the tractus solitarius; nXll--hypoglossal nucleus.
SEROTONIN IN THE DMNX
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indicate a regional variability within the nXII, considerable further investigation will be required before such a postulate may be proposed. The dorsal reticular field has had a long-standing reputation for a role in cardiovascular regulation [1,27]. Moreover, a significant density of 5-HTrlike binding sites has been detected in the region [49,50]. It was therefore an unexpected finding that injections of 5-HT (5 nmol) placed in the area were without effect (see Fig. 1). Again, however, due to the limited number of sites studied in the region, no conclusion can be drawn regarding the hemodynamic role of 5-HT in the medullary reticular formation.
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FIG. 2. Hemodynamic effects of microinjection of two doses of 5-HT into the dmnX, 5 nmol (n = 23) and 25 nmol (n = 4). Pooled data are shown for changes of blood pressure (top) and heart rate (bottom). N.S.--p > 0.05. either dose, even with the inclusion of the low-dose data from the three responsive sites. Of the latter, two sites overlapped considerably with the nTS, lying immediately dorsal to the dmnX. One of these sites also may have included the nXII, as indicated by the spread of dye, and was associated with a mild elevation of blood pressure (+8 to +12 mmHg) and a decrease of heart rate ( - 4 0 to - 6 0 bpm). The chronotropic change was presumed to be baroreflexive in nature, although a vagotomy was not performed to confirm this possibility. The other site was characterized by mild depressor responses ( - 1 0 to - 2 8 mmHg) with no accompanying changes of heart rate. The third responsive site overlapped considerably with the the subjacent nXII, and was characterized by a mild increase of blood pressure and decrease of heart rate. In contrast to the unresponsiveness of the dmnX, sites entirely within the nTS were consistently found to be associated with large decreases of both blood pressure and heart rate (Fig. 3). Pooling the data from the five sites tested for the effects of 5-HT (5 nmol) revealed a mean decrease in blood pressure of 25 _ 3.4 mmHg and a drop in heart rate of 47.5 + 5.6 bpm. Both effects were highly significant (p < 0.01), and could be obtained from both the commissural levels of the nucleus and more rostrally at the level of the area postrema (see Fig. 1). Responses to injection of 5-HT (5 nmol) into the five sites primarily within the nXII proved to be somewhat variable. Three sites were associated with significant drops of blood pressure (-23.8 +__7.2 mmHg) and a decrease of heart rate (-37.5 + 15.9 bpm) that collectively approached significance (0.05 < p < 0.1), whereas the other two sites were unresponsive. Both responsive and unresponsive sites were found at all rostrocaudal levels (see Fig. 1). Although it is tempting to suggest that these findings may
The major finding of the present study was that the dmnX does not appear to support 5-HT-induced changes of peripheral hemodynamics. This contrasts with the results of previous studies that suggest that activation of 5-HT~, receptors in the dmnX with very low concentrations of highly selective agonists such as 8hydroxy-dipropylaminotetralin(8-OH-DPAT) can induce a transient bradycardia [45]. It is particularly intriguing that the more selective compound was effective in the previous experiments, whereas the broad-spectrum autacoid used in the present study was ineffective. One possible explanation that might be offered is that 8-OH-DPAT is generally far more potent that 5-HT, which would imply that the 5-HT used in the present study was not given at a dose high enough to affect 5-HTIA receptors. This argument is unsatisfactory, however, in view of in vitro data that indicate that 5-HT has a Kd of jUSt 2 nM at 5-HT]A receptors [19,20]. With the 50 nl volumes used in the present study, this corresponds to a dose of just 100 fmol, whereas the 5 and 25 nmol doses used here are well above that figure. An alternative explanation is that the difference in results arises from the different volumes of microinjectate being used in the two sets of studies, as the previous study employed 100 nl volumes, whereas the present study involved the use of 50 nl injections. It is conceivable that the larger volume may be associated with a sphere of drug action sufficiently large enough to affect neurons in the nTS, which the present study demonstrates is capable of mediating profound 5-HT-induced decreases of both heart rate and arterial pressure. The dmnX has long been known to have a role in cardiomotor chronotropic regulation. Although the projection from the nucleus ambiguus to the myocardium is much heavier than that from the dmnX [46,47], it has nevertheless been demonstrated repeatedly that a significant component of the cardiac branch of
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FIG. 3. Hemodynamic effects of microinjection of 5-HT (5 nmol) into the nTS (n = 5). Pooled data are shown for changes of blood pressure (left) and heart rate (right).
460
the vagus nerve arises from the dmnX in the cat [16,26], dog [5], rabbit [11,24], and rat [35,46]. However, previous anatomical investigations indicate that vagal cardiomotor preganglionics located within the nuclear boundaries of the dmnX do not receive serotonergic input [22]. The data from the present study provide physiological support for this notion. One objection that might be raised to this interpretation, however, is that many of the motorneurons of the dmnX have long, ramifying dendrites that extend dorsally into the nTS [31], and that the 5-HT-induced alterations of hemodynamic activity attributed here to the nTS may, in fact, be due to a direct effect on neurons in the dmnX. This possibility seems remote, however, based on several points. First, the changes of cardiovascular function that were induced with nTS microinjections included decreases of blood pressure. It has been demonstrated that such changes are not secondary to decreases of heart rate, as bilateral transection of the vagus nerves can eliminate the bradycardia, but not the vasodepression, associated with 5-HT injection into the nTS [13]. Although electrical stimulation of the dmnX can activate vagal motor pathways that produce bradycardia [35], primary vasodepressor responses have never been obtained from the dmnX. Thus, we may be reasonably sure that the cardiovascular effects seen here with microinjection of 5-HT into the nTS are, in fact, due to an effect on neurons in that structure, and not those in the dmnX. A second counterargument that could be made is based on our current understanding of the electrophysiological effects of 5-HT in the dorsal vagal complex. Recent studies performed in this laboratory with physiologically-relevant concentrations of 5-HT [12] suggest that the monoamine primarily exerts a 5-HT~ receptor-mediated postsynaptic inhibition in the nTS. It is a commonly accepted generality that receptors that mediate inhibitory influences on neuronal activity are usually located on cell bodies, rather than on dendrites. Unfortunately, conclusive evidence is lacking regarding the electrophysiology of 5-HT in the dmnX. It therefore appears that, based on the knowledge obtained to date, the function of 5-HT in the dmnX is restricted to regulation of gastric function. In this regard, McCann and co-workers [30] have demonstrated that the nucleus is, in fact, exquisitely sensitive to 5-HT, as microinjection of minuscule volumes of extremely low concentrations of the amine into the dmnX induces vagally-mediated increases of gastric pressure and motility. There is considerable interaction between 5-HT and a neuropeptide, thyrotropin-releasing hormone (TRH). "Conditioning" applications of TRH to the surface of the dorsomedial medulla augment the effects of 5-HT on gastric motility and tone [30]; conversely, prior application of 5-HT augments the stimulatory effect of TRH on secretion of gastric acid [32]. It is not entirely clear what structures contribute to the serotonergic innervation of the dmnX, but leading candidates include the raphe nuclei [4,7], the nTS [8,42] and the primary visceral sensory afferents, themselves, from the vagus [ ! 5] or carotid sinus [51 ] nerves. For their part, TRH-containing afferents to the dmnX are known to originate in the medullary raphe [38]. A strong possibility therefore exists that 5-HT and TRH are colocalized in the synaptic vesicles of afferents from the raphe, where the two transmitters have been found within the same neuron [23,25]. As with the dmnX, a role in digestive regulation has been proposed for 5-HT in the nTS based on the observation that deglutitory movements of the pharynx can be elicited through what appears to be a 5-HTz receptor-mediated mechanism [ 18]. In contrast to the dmnX, however, it is now clear that 5-HT also has a prominent role in cardiovascular regulation in the nTS. In the present study, microinjection of 5-HT into the nucleus led to profound decreases of both blood pressure and heart rate, changes that resembled those seen during activation of the baroreceptor
FELDMAN AND G A L I A N O
reflex. This supports previous findings in a number of laboratories that suggest a 5-HT2 receptor-mediated parasympathoexcitation and sympathoinhibition [21,44]. Despite these indications of the presence of 5-HT2 receptors in the nTS, however, surprisingly little anatomical evidence has been available for the presence of this receptor subtype in the nucleus. A high density of 5-HT~A-like sites has been described [28,40], and a lower level of 5 - H T ~ r l i k e binding is also present [28,49], but autoradiographic studies with 5-HTz-selective ligands seem to indicate little, if any, 5-HT2-1ike binding in the nTS [3,39]. Nevertheless, more recent experiments using antibodies to the 5-HT2 receptor indicate that there may be some very light 5-HTz-like binding in a discrete region of the medial subnucleus at levels rostral to the obex [33]. Interestingly, the area in question correlates precisely with the site of termination of subdiaphragmatic esophageal afferents [2], rather than of baroreceptive or pharyngeal afferents [2,91. At this point, it would be premature to propose a cardiovascular role for 5-HT in the nXlI. Three of the five sites studied in the present investigation appeared to support 5-HT-induced changes of heart rate and blood pressure, and, judging from the locations and diameters of spread of the dye spots that were left at these sites, the effective locations did not overlap with the nTS. Nevertheless, these data are limited in number. Moreover, it is altogether quite possible that the rostraimost of the effective locations, which was located rostral to the obex, may have included such perihypoglossal structures as the nucleus intercalatus, which has been demonstrated to contain dendrites of vagal preganglionic neurons and to receive considerable afferent input from forebrain autonomic structures [34,43], as well as being the site of very high densities of 5-HTIA- and 5 - H T ~ r l i k e binding sites [49,50]. Resolution of this issue will require considerable further investigation, using even smaller microinjection volumes than were used in the present study. To summarize, this set of experiments was performed to investigate the potential involvement of 5-HT in the dmnX in regulating cardiovascular hemodynamics. No evidence for such a role was found, as microinjection of the amine into the nucleus failed to evoke any changes of either heart rate or blood pressure. On the other hand, microinjection of 5-HT into the overlying nTS led to profound decreases of both blood pressure and heart rate, changes similar to those seen during activation of the baroreceptor reflex. It is always more difficult to disprove the existence of something than to prove its existence. Therefore, perhaps more work is needed to rule out the contribution of serotonergic systems in the dmnX towards the regulation of peripheral hemodynamics. Nevertheless, the present findings cast serious doubt on this possibility. By contrast, these data support the involvement of serotonergic systems in the nTS in the regulation of cardiovascular hemodynamics, suggesting that they contribute to mechanisms that activate parasympathetic cardiomotor pathways and inhibit sympathetic outflow. ACKNOWLEDGEMENT This work was supported by NIH Grant R29-NS29458.
REFERENCES 1. Alexander, R. S. Tonic and reflex functions of medullary sympathetic cardiovascular centers. J. Neurophysiol. 9:205-217; 1946. 2. Altschuler, S. M.; Bat, X.; Bieger, D.; Hopkins, D. A.; Miselis, R. R. Viscerotopic representations of the upper alimentary tract in the rat: Sensory ganglia and nuclei of the solitary and spinal trigeminal tracts. J. Comp. Neurol. 283:248-268; 1989.
SEROTONIN IN THE DMNX
3. Appel, N. M.; Mitchell, W. M.; Garlick, R. K.; Glennon, R. A.; Titeler, M.; De Souza, E. B. Autoradiographic characterization of (_+)- l-(2,5-dimethoxy-4-[~2sI]iodophenyl)-2-aminopropane (['25I]DOI) binding to 5-HT2 and 5-HT~c receptors in rat brain. J. Pharmacol. Exp. Ther. 255:843-857; 1990. 4. Basbaum, A. I.; Clanton, C. H.; Fields, H. L. Three bulbospinal pathways from the rostral medulla of the cat: An autoradiographic study of pain modulatory systems. J. Comp. Neurol. 178:209-224; 1978. 5. Bennett, J. A.; Kidd, C.; Latif, A. B.; McWilliam, P. N. A horseradish peroxidase study of vagal motoneurones with axons in cardiac and pulmonary branches of the cat and dog. Quart. J. Exp. Physiol. 66:145-154; 1981. 6. Berk, M. L.; Smith, S. E.; Karten, H. J. Nucleus of the solitary tract and dorsal motor nucleus of the vagus nerve of the pigeon: Localization of peptide and 5-hydroxytryptamine immunoreactive fibers. J. Comp. Neurol. 338:521-548; 1993. 7. Bobillier, P.; Seguin, S.; Petitjean, F.; Salvert, D.; Touret, M.; Jouvet, M. The raphe nuclei of the cat brainstem: A topographical atlas of their efferent projections as revealed by autoradiography. Brain Res. 113:449-486; 1976. 8. Calz& L.; Giardino, L.; Grimaldi, R.; Rigoli, M.; Steinbusch, H. W. M.; Tiengo, M. Presence of 5-HT-positive neurons in the medial nuclei of the solitary tract. Brain Res. 347:135-139; 1985. 9. Ciriello, J. Brainstem projections of aortic baroreceptor afferent fibers in the rat. Neurosci. Lett. 36:37-42; 1983. 10. Dahlstr/~m, A. B.; Fuxe, K. Localization of monoamines in the lower brain stem. Experientia 20:398-399; 1964. 11. Ellenberger, H. H.; Haselton, J. R.; Liskowsky, D. R.; Schneiderman, N. The location of chronotropic cardioinhibitory vagal motoneurons in the medulla of the rabbit. J. Auton. Nerv. Syst. 9:513529; 1983. 12. Feldman, P. D. Electrophysioiogical effects of serotonin in the solitary tract nucleus of the rat. Naunyn Schmiedebergs Arch. Pharmacol. 349:447-454; 1994 13. Feldman, P. D.; Galiano, F. J. Hemodynamic effects of serotonin in the nucleus tractus solitarius. Am. J. Physiol. (in press). 14. Fuxe, K. The distribution of monoamine terminals in the central nervous system. Acta Physiol. Scand. 64(Suppl. 247):38-85; 1965. 15. Gaudin-Chazal, G.; Portalier, P4 Barrit, M. C.; Puizillout, J.-J. Serotonin-like immunoreactivity in paraffin-sections of the nodose ganglia of the cat. Neurosci. Lett. 33:169-172; 1982. 16. Geis, G. S.; Wurster, R. D. Horseradish peroxidase localization of cardiac vagal preganglionic somata. Brain Res. 182:19-30; 1980. 17. Gunn, C. G.; Sevelius, G.; Puiggari, M. J.; Myers, F. K. Vagal cardiomotor mechanisms in the hindbrain of the dog and cat. Am. J. Physiol. 214:258-262; 1968. 18. Hashim, M. A.; Bieger, D. Excitatory action of 5-HT on deglutitive substrates in the rat solitary complex. Brain Res. Bull. 18:355-363; 1987. 19. Hoyer, D.; Engel, G.; Kalkman, H. O. Molecular pharmacology of 5-HTj and 5-HT2 recognition sites in rat and pig brain membranes: Radioligand binding studies with [3H]5-HT, [3H]8-OH-DPAT, (-)[J25I]iodocyanopindolol, [3H]mesulergine and [3H]ketanserin. Eur. J. Pharmacol. 118:13-23; 1985. 20. lsmaiel, A. M.; Titeler, M.; Miller, K. J.; Smith, T. S.; Glennon, R. A. 5-HT~ and 5-HT2 binding profiles of the serotonergic agents a-methylserotonin and 2-methylserotonin. J. Med. Chem. 33:755758; 1990. 21. ltoh, H.; Bufiag, R. D. Cardiovascular and sympathetic effects of injecting serotonin into the nucleus tractus solitarius in rats. J. Pharmacol. Exp. Ther. 256:1147-1153; 1991. 22. lzzo, P. N.; Deuchars, J.; Spyer, K. M. Localization of cardiac preganglionic motoneurones in the rat: Immunocytochemical evidence of synaptic inputs containing 5-hydroxytryptamine. J. Comp. Neurol. 327:572-583; 1993. 23. Johansson, O.; Htikfelt, T.; Pernow, B.; Jeffcoate, S. L.; White, N.; Steinbusch, H. W. M.; Verhofstad, A. A. J.; Emson, P. C.; Spindel, E. Immunohistochemical support for three putative transmitters in one neuron: Coexistence of 5-hydroxytryptamine, substance P- and thyrotropin releasing hormone-like immunoreactivity in medullary
461
24.
25.
26. 27. 28.
29.
30.
31.
32.
33.
34.
35.
36.
37.
38.
39.
40.
41.
42.
43.
neurons projecting to the spinal cord. Neuroscience 6:1857-1881 ; 1981. Jordan, D.; Khalid, M. E. M.; Schneiderman, N.; Spyer, K. M. The location and properties of preganglionic vagal cardiomotor neurones in the rabbit. Pfliigers Arch. 395:244-250; 1982. Kachidian, P.; Poulat, P.; Marlier, L.; Privat, A. Immunohistochemical evidence for the coexistence of substance P, thyrotropin-releasing hormone, GABA, methionin-enkephalin, and leucin-enkephalin in the serotonergic neurons of the caudal raphe nuclei: A dual labeling in the rat. J. Neurosci. Res. 30:521-530; 1991. Kalia, M. Brain stem localization of vagal preganglionic neurons. J. Auton. Nerv. Syst. 3:451-481; 1981. Magoun, H. W.; Rhines, R. An inhibitory mechanism in the bulboreticular formation. J. Neurophysiol. 9:165-171 ; 1946. Manaker, S.; Verderame, H. M. Organization of serotonin IA and I B receptors in the nucleus of the solitary tract. J. Comp. Neurol. 301:535-553; 1990. McAllen, R. M.; Spyer, K. M. Two types of vagal preganglionic motoneurones projecting to the heart and lungs. J. Physiol. (Lond.) 282:353-364; 1978. McCann, M. J.; Hermann, G. E.; Rogers, R. C. Dorsal medullary serotonin and gastric motility: Enhancement of effects by thyrotropin-releasing hormone. J. Auton. Nerv. Syst. 25:35-40; 1988. McLean, J. H.; Hopkins, D. A. Ultrastructure of the dorsal motor nucleus of the vagus nerve in monkey with comparison of synaptology in monkey and cat. J. Comp. Neurol. 231:162-174; 1985. McTigue, D. M.; Rogers, R. C.; Stephens, R. L., Jr. Thyrotropinreleasing hormone analogue and serotonin interact within the dorsal vagal complex to augment gastric acid secretion. Neurosci. Lett. 144:61-64; 1992. Morilak, D. A.; Garlow, S. J.; Ciaranello, R. D. Immunocytochemical localization and description of neurons expressing serotonin2 receptors in the rat brain. Neuroscience 54:701-717; 1993. Norgren, R. E.; Smith, G. P. Central distribution of subdiaphragmatic vagal branches in the rat. J. Comp. Neurol. 273:207-223; 1988. Nosaka, S.; Yamamoto, T.; Yasunaga, K. Localization of vagal cardioinhibitory preganglionic neurons within rat brain. J. Comp. Neurol. 186:79-92; 1979. Ortega-Villalobos, M,; Garcia-Baz~,n, M.; Solano-Flores, L. P.; Ninomiya-Alarc~m, J. G.; Guevara-Guzm~.n, R.; Wayner, M. J. Vagus nerve afferent and efferent innervation of the rat uterus: An electrophysiological and HRP study. Brain Res. Bull. 25:365-371; 1990. Pagani, F. D.; Norman, W. P.; Kasbekar, D. K.; Gillis, R. A. Localization of sites within the dorsal motor nucleus of the vagus that affect gastric motility. Am. J. Physiol. 249:G73-G84; 1985. Palkovits, M.; Mezey, t~.; Eskay, R. L.; Brownstein, M. J. Innervation of the nucleus of the solitary tract and the dorsal vagal nucleus by thyrotropin-releasing hormone-containing raphe neurons. Brain Res. 373:246-251 ; 1986. Pazos, A.; Cort6s, R.; Palacios, J. M. Quantitative autoradiographic mapping of serotonin receptors in the rat brain. II. Serotonin-2 receptors. Brain Res. 346:231-249; 1985. Pompeiano, M.; Palacios, J. M.; Mengod, G. Distribution and cellular localization of mRNA coding for 5-HT~A receptor in the rat brain: Correlation with receptor binding. J. Neurosci. 12:440-453; 1992. Rinaman, L. M.; Miselis, R. R. The organization of vagal innervation of rat pancreas using cholera toxin-horseradish peroxidase conjugate. J. Auton. Nerv. Syst. 21:109-125; 1987. Rogers, R. C.; Kita, H.; Butcher, L. L.; Novin, D. Afferent projections of the dorsal motor nucleus of the vagus. Brain Res. Bull. 5:365-373; 1980. Shapiro, R. E.; Miselis, R. R. The central organization of the vagus nerve innervating the stomach of the rat. J. Comp. Neurol. 238:473488; 1985.
462
44. Shvaloff, A.; Laguzzi, R. Serotonin receptor in the rat nucleus tractus solitarii and cardiovascular regulation. Eur. J. Pharmacol. 132:283288; 1986. 45. Sporton, C. E.; Shepheard, S. L.; Jordan, D.; Ramage, A. G. Microinjections of 5-HT~A agonists into the dorsal motor vagal nucleus produce a bradycardia in the atenolol-pretreated anesthetized rat. Br. J. Pharmacol. 104:466-470; 1991. 46. Stuesse, S. L. Origins of cardiac vagal preganglionic fibers: A retrograde transport study. Brain Res. 236:15-25; 1982. 47. Stuesse, S. L.; Powell, K. S. Cardiac vagal preganglionic fibers in neonatal rats: A comparison with cervical vagal components. Neurosci. Lett. 34:7-12; 1982. 48. Tallaksen-Greene, S. J.; Elde, R. J.; Wessendorf, M. W. Regional distribution of serotonin and substance P co-existing in nerve fibers and terminals in the brainstem of the rat. Neuroscience 53:1127 1142; 1993.
FELDMAN AND GALIANO
49. Thor, K. B.; Blitz-Siebert, A.; Helke, C. J. Autoradiographic localization of 5-HTL binding sites in the medulla oblongata of the rat. Synapse 10:185-205; 1992. 51). Thor, K. B.; Blitz-Siebert, A.; Helke, C. J. Autoradiographic localization of 5-HT~ binding sites in autonomic areas of the rat dorsomedial medulla oblongata. Synapse 10:217-227; 1992. 51. Wang, Z.-Z.; Stensaas, L. J.; Dinger, B. G.; Fidone, S. J. The co-existence of biogenic amines and neuropeptides in the type I cells of the cat carotid body. Neuroscience 47:473-480; 1992. 52. Wolf, W. A.; Kuhn, D. M.; Lovenberg, W. M. Blood pressure responses to local application of serotonergic agents in the nucleus tractus solitarii. Eur. J. Pharmacol. 69:291-299; 1981. 53. Wyrwicka, W.; Garcia, R. Effect of electrical stimulation of the dorsal nucleus of the vagus nerve on gastric acid secretion in cats. Exp. Neurol. 65:315-325; 1979.