SYNAPSE 10:217-227 (1992)
Autoradiographic Localization of 5HT, Binding Sites in Autonomic Areas of the Rat Dorsomedial Medulla Oblongata KARL B. THOR, ALISA BLITZ-SIEBERT,AND CINDA J. HELKE Department of Pharmacology, Uniformed Services University of the Health Sciences, Bethesda, Maryland 20814 (K.B.T.,A.B.-S.,C.J.H.); Division of CNS Research, Lilly Research Laboratories, Eli t i l l y and Co., Indianapolis, Indiana 46285 (K.B.T.)
KEY WORDS
Serotonin receptors, Receptor autoradiography, Medulla oblongata, Nucleus tractus solitarius, Nucleus ambiguus, Dorsal motor nucleus of the vagus nerve, Nucleus intercalatus, 3H-8-OH-dipropyl-amino-tetraline, 1251-iodocyanopindolol
ABSTRACT Serotonin (5HT) binding sites in autonomic portions of the dorsomedial medulla oblongata of the rat were localized using autoradiographic techniques with radioactive ligands that express high affinity for the 5HT1 (3H-5HT),5HTlA (3H-80HDPAT), or 5HTlB (1251-cYPwith isoproterenol) receptor subtypes. 5HTlA sites were densely distributed in the nucleus tractus solitarius (NTS), with the highest densities localized to the interstitial subnucleus and the central subnucleus. 5HTlB sites were also found in the NTS, with the highest densities localized to the substantia gelatinosa subnucleus. The dorsal motor nucleus of the vagus nerve and nucleus ambiguus exhibited low densities of 5HTIBsites. However, the nucleus intercalatus, a cerebellar relay nucleus that also contains dendrites of vagal parasympathetic preganglionic neurons and receives autonomic forebrain afferent input, showed very dense 5HTlB sites. The promontorium, paratrigeminal islands, and the dorsomedial portion of the trigeminal nucleus (DM5), which are areas of viscerosomatic integration, exhibited high densities of both 5HTIAand 5HTlB sites. The area postrema contained low levels of both 5HTIAand 5HTlB sites. Visceral deafferentation via cervical vagotomy or nodose ganglionectomy caused a significant decrease in 5HTlAsites in the interstitial subnucleus of the NTS ipsilateral to the lesion. No changes were seen in 5HTIBsites. These studies suggest that 5HT,, and 5HTlB sites are involved in the processing of visceral sensory information in the NTS and associated areas. Based upon viscerotopic organization of the NTS, 5HTlA sites appear preferentially distributed in portions of the NTS that are associated with the coordination of swallowing, respiration, and cardiovascular function, while 5HTlB sites appear preferentially distributed in areas of the NTS associated with gastrointestinal, hepatic, pancreatic, and cardiovascular function. However, since these associations were not absolute and there was a great deal of overlap between the two sites, speculation regarding their specific functions in autonomic control must await pharmacological examination.
INTRODUCTION The dorsomedial medulla oblongata, comprised primarily of the NTS and dorsal motor nucleus of the vagus nerve (DMV), is a vital area for control of a number of autonomic functions. The NTS is the primary region of termination for visceral primary afferent fibers contained in the vagus and glossopharyngeal nerves, while the DMV, as well as the nucleus ambiguus, are the origin for the efferent component of these nerves (Kalia and Sullivan, 1982; Norgren and Smith, 1988; Altschuler et al., 1989). 0 1992 WILEY-LISS, INC.
These autonomic areas of the dorsomedial medulla receive dense innervation from medullary serotonergic neurons (Thor and Helke, 19871, and various physiological and pharmacological studies have indicated a role for serotonin (5-hydroxytryptamine,5HT) in the control of autonomic function in the medulla. For example, medullary serotonergic systems have been implicated in regulation of swallowing (Kessler and Jean, 1985,1986; Lucier and Sessle, 1981; Hashim and Bieger, 19871, Received December 21,1990;accepted in revised form July 26,1991
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respiration (Millhorn et al., 1983; Lalley, 1986; Sessle and Henry, 1985; Holtman et al., 1986, 1987; Gillis et al., 19891,and cardiovascular function (McCall et al., 1987;Gillis et al., 1989).Many of the effects of serotonin on these systems have been complex, and the mechanisms and anatomical sites of action have not been determined. The complexity of serotonin's effects might be explained by the fact that there are multiple subtypes of receptors for serotonin (Peroutka, 1988) and that serotonin might have multiple sites of action along autonomic reflex pathways. To determine possible medullary sites of action for the various serotonin receptor subtypes, receptor autoradiography techniques were applied to sections of rat medulla, and the densities of the various serotonin receptor subtypes were examined 1) in areas of visceral afferent termination (Astrom, 19531, i.e., the NTS, the promontorium, the paratrigeminal islands, the area postrema, and the dorsomedial portion of the trigeminal nucleus and 2) in areas containing vagal efferent neurons, i.e., the DNIV and nucleus ambiguus.
unilateral cervical vagotomy or nodose ganglionectomy on serotonergic binding. Rats were anesthetized with chloral hydrate and the vagus nerve was isolated. In five rats, the vagus nerve was cut 10 mm from its entrance into the tenth cranial nerve foramen (i.e., distal to the nodose ganglion). In five rats, the vagus nerve was retracted to expose the nodose ganglion in the foramen and the ganglion was excised. In five shamoperated rats, the vagus nerve was exposed but not cut. After 10 days, the animals were sacrificed, and the medullae were processed as described above. Nuclear subdivisions of the NTS were based upon the descriptions of Kalia and Sullivan (1982).
RESULTS 3H-5HTbinding In general, 3H-5HTbinding reflected the combination of binding seen with 3H-8-OH-DPAT and 1251-CYP, which are described below in detail. The relative distribution of 3H-5HTbinding among the various autonomic areas of the dorsomedial medulla are listed in Table I.
3H-8-OH-DPATbinding
METHODS
The highest density of 3H-8-OH-DPATbinding in the NTS was in the interstitial (Fig. 1B to 1D) and central Detailed methods can be found in Thor et al. (1992). subnuclei (Fig. 1D to 1F). Binding in the commissural Briefly, adult male Sprague-Dawley rats were decapitated, and the brains were rapidly removed and frozen subnucleus of the NTS (Kalia and Sullivan, 1982) was on powdered dry ice. Frozen sections (20 pm thick) were intermediate in density, while other portions of the NTS cut on a cryostat, placed on subbed slides, desiccated in exhibited lower levels of binding (Fig. 1, Table I). At a vacuum overnight at 4"C, and stored at -70°C until rostral levels of the medulla, 3H-8-OH-DPATbinding in incubation. 5HT, sites were indiscriminately labelled the NTS was moderately dense, with an increase in using 31H1-5HT,5HT,, sites were labelled using 3[Hl-8- density medially to laterally (Fig. 1,Table I). The dense OH-dipropyl aminotetraline (3H]80H-DPAT), and binding in the lateral NTS at rostral levels was contig5HTIBsites were labelled using 125[I]-iodocyanopindolol uous with dense binding in the dorsomedial subnucleus (l"I-CYP) with 30 pM isoproterenol to block beta- of the trigeminal complex. Cervical vagotomy and nodose ganglionectomy each adrenergic binding sites. Nonspecific binding of ligands was assessed using 1 pM 5HT. Radioactive standards caused a decrease (49 8%and 57 5 6%, respectively; and correction for lipid quenching of the 3H signal have n = 5 for each) in 5HTlAbinding sites in the interstitial subnucleus (Fig. 2) ipsilateral t o the lesion, indicating been described in Thor et al. (1992). Fifteen rats were studied to determine the effects of an association of these sites with visceral afferent pathways. No other changes were detected in these lesioned animals. 3H-8-OH-DPAT binding was sparse in the DMV (Fig. 1) and NA (Table I; cf. Fig. 4, Thor et al., 1992). Abbreviations However, the lateral portion of the nucleus intercalatus 10 dorsal motor nucleus of the vagus nerve (NU,which lies between the DMV and the hypoglossal 12 hypoglossal nucleus Ap area postrema nucleus, exhibited substantial binding (Fig. 1A-B, TaCEN central subnucleus of the NTS ble I). Binding in the area postrema was very low DM5 dorsomedial portion of the trigeminal nucleus IST interstitial nucleus of the NTS (Fig. lC, Table I). NA nucleus ambiguus Areas of the medulla that are not in the NTS proper NC nucleus cuneatus NG nucleus gracilis but also receive visceral primary afferent input (AsNI nucleus intercalatus trom, 1953) also exhibited substantial 3H-8-OH-DPAT NTS nucleus tractus solitarius binding. The promontorium, which caps the dorsomePr promontorium PrH prehypoglossal nucleus dial aspect of the trigeminal nucleus at caudal levels, PTI paratrigeminal islands the paratrigeminal islands, and the dorsomedial subnupyramidal tract, decussation PY>PYX SG substantia gelatinosa cleus of the trigeminal nucleus oralis each exhibited so superior olives substantial binding (Table I; cf. Figs. 4 and 5 , Thor spinal tract of the trigeminal nerve SP5 ts tractus solitarius et al., 1992).
*
~~
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MEDULLARY AUTONOMIC SEROTONERGIC BINDING SITES TABLE I. Specific binding (expressed as femtomoles/mg wet weight) of 3H-5HT,3H-8-OH-DPAT,and 1251-CYPin subnuclei of the NTS, areas of the medulla that are closely associated with the NTS, and in uagal efferent nuclei Ligand Concentration
3H-5HT (5 nM)
Caudal/intermediate NTS subnuclei Commissural 124.7 f 11.2 Dorsal 85.1 f 6.0 Dorsolateral 118.2 f 7.6 Medial 98.5 f 6.9 Interstitial 147.2 f 7.3 Central 150.1 f 9.9 Intermediate 126.0 f 9.1 Substantia gelatinosa 137.8 f 8.7 Ventrolateral 121.6 =k 8.2 Ventral 129.7 f 6.5 Rostra1 NTS 165.2 f 8.7 Medial Lateral 174.9 f 7.6 Associated areas Promontorium 164.3 f 9.7 Paratrigeminal 108.7 f 8.6 DM5 82.4 f 7.6 Area postrema 38.5 f 5.7 Vagal motor nuclei and nucleus intercalatus 119.8 f 12.2 DMV, DMV, 115.4 10.1 Nucleus intercalatus 185.1 18.3 Nucleus ambiguus 79.3 f 8.2
*
*
3H-8-OH-DPAT (2 nM)
'25I-CYP (50 PM)
34.5 f 1.9 21.4 f 1.7 24.5 f 2.3 28.0 f 1.8 58.7 f 3.0 55.6 f 2.2 33.9 f 1.9 25.8 f 2.0 27.0 f 2.3 27.9 f 2.9
6.98 f 0.46 4.84 f 0.41 5.63 k 0.39 6.34 f 0.42 6.01 f 0.21 5.11 f 0.32 5.30 f 0.28 8.18 f 0.69 7.41 f 0.64 7.23 f 0.57
42.3 f 3.1 49.2 f 2.8
8.63 f 0.49 8.93 f 0.32
59.7 31.8 33.8 9.7
f 5.6 f 4.9 f 2.2 f 1.3
5.89 f 0.47 5.31 f 0.51 4.91 f 0.42 2.08 f 0.11
18.9 f 1.1 19.7 f 1.4 38.0 f 2.6 18.6 f 1.4
6.89 f 0.43 5.32 f 0.51 9.93 f 0.58 3.20 f 0.27
DM5 = dorsomedial portion of the trigeminal nucleus; DMV = dorsal motor nucleus of the vagus nerve.
1251-CYPbinding
binding in the dorsal portion of nucleus reticularis parvocellularis. At caudal levels, binding in the DMV proper was moderately dense, being homogeneous with that seen dorsally in the commissural subnucleus of the NTS (Fig. 3A-C, Table I). However, at intermediate levels, the DMV became distinguished by its sparse binding, compared to the denser binding seen in the nucleus intercalatus ventrally and the substantia gelatinosa dorsally (Fig. 3D and E). Similar to the sparse binding that distinguished the DMV at intermediate levels, the nucleus ambiguus (NA) was also distinguished by exhibiting less binding than the surrounding neuropil (Fig. 3G1, Fig. 4, Table I). The sparse binding in NA was conspicuous (Fig. 4C-F) from the caudal border of the seventh motor nucleus to the level of the nucleus reticularis lateralis. At this more caudal level (Fig. 4A and B), the pattern of sparse binding in NA became less distinct, presumably because adjacent structures, such as the nucleus reticularis lateralis, also exhibit a paucity of '251-CYPbinding and because neurons in NA at this level are distributed more diffusely than they are at rostral levels (Bieger and Hopkins, 1987). No changes were observed in 1251-CYPbinding following cervical vagotomy or nodose ganglionectomy.
The highest levels of 1251-CYPbinding were found in the rostral third of the NTS (Fig. 3 H 4 , Table I), with greater density in the lateral portion, which was contiguous with dense binding in the dorsomedial portion of the trigeminal nucleus. At intermediate levels, the NTS binding diminished medial to the tractus solitarius, except for a circular area in the dorsomedial quadrant of the NTS adjacent to the ventricle, where binding was denser (Fig. 3E-F; Table I).This area of denser binding in the NTS corresponds to the substantia gelatinosa subnucleus. Further caudally, this area of denser binding in the dorsomedial portion of the NTS became less distinct as binding in the commissural and medial subnuclei increased (Fig. 3A-D). Ventrolateral portions of the NTS exhibited a moderately dense level of 1251CYP binding at all levels. The relationship between 1251-CYP binding and the dorsal motor nucleus of the vagus nerve ( D W ) was complex (Fig. 3) due to the heterogeneous pattern of binding in this area. On the one hand, there was a highly distinguishable, thin (50 x 150 pm), horizontal strip of extremely dense binding that appeared to be localized along the boundary of the DMV and hypoglossal nucleus, i.e., in the nucleus intercalatus (Fig. 3B-E). This strip of dense binding, seen at both the intermediate and DISCUSSION caudal levels of the NTS, extended laterally from the The NTS receives a dense innervation from the medfloor of the IV ventricle and from the central canal, respectively (Fig. 3B-E), and was contiguous with dense ullary raphe and associated groups of serotonergic neu-
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Figure 1.
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Fig. 2. Loss of high-density 3H-80H-DPATbinding sites in the IST followingunilateral vagotomy (right side). Calibration bar = 250 pm.
rons (Thor and Helke, 1987). The purpose of the present study was t o anatomically describe the relative distributions of certain 5HT binding sites among the various portions of the medulla oblongata that are directly involved in autonomic control. The anatomical distributions of the serotonin binding sites will be discussed in the context of the viscerotopic organization of the NTS, as revealed by horseradish peroxidase (HRP) tracing studies of visceral primary afferent fibers, and with regards to the various physiological roles that serotonin has been proposed to play in autonomic control. The results indicate that both 5HTlA and 5HTlB sites are very specifically and differentially distributed among
Fig. 1. 'H-SOH-DPAT binding in the dorsomedial medulla (caudalrostral, A-H, respectively, all sections from a single animal). A-D: Binding is moderately dense in the commissural region of the NTS. At the level of the AP,dense binding can be seen in the IST. At levels rostral to the AP, dense binding occurs in the CEN as well. Binding in the AP is sparse. E-H: 3H-80H-DPAT binding remains dense in the CEN. At rostral levels, binding within the NTS is homogeneous and contiguous with binding in DM5. The calibration bar in the lower right of panel A = 200 km.
the various subnuclei of the NTS and the autonomic motor nuclei. The 5HTlA sites were most dense in the interstitial, intermediate, and central subnuclei of the NTS, with moderate binding in the commissural subnucleus. The interstitial and commissural subnuclei have been shown to receive primary afferent terminals from fibers contained in the aortic depressor and carotid sinus nerves of rats (Housley et al., 1987; Seiders and Steusse, 1984; Ciriello, 1983). Thus, 5HTIAreceptors in the NTS may have a role in regulation of baroreceptor and chemoreceptor inputs. In fact, direct application of 5HT into the NTS has been shown to alter blood pressure (Laguzzi et al., 1984). In addition, the interstitial and the central subnuclei of the NTS receive inputs from primary afferent fibers that innervate the larynx, pharynx, and esophagus (Hamilton and Norgren, 1984; Hisa et al., 1985; Altschuler et al., 19891, structures responsible for coordinating the act of swallowing. In fact, the distribution of 5HTIAbinding sites in this area of the NTS is remarkably similar to the distribution ofHRP labelling of upper
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Figure 3A-H.
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Fig. 3. '251-CYPbinding in the dorsomedial medulla (caudal-rostral, A-I, respectively, all sections from a single animal). A-D: Dense homogeneous binding occurs in the commissural subnucleus of the NTS. Moderately dense binding is seen in the dorsal column nuclei, NG and NC. A very dense band of binding occurs in NI (white arrows in panels B and C). Note that 10 contains less binding (see also Fig. 11). AF' contains sparse binding. In panel D, a circular area of denser
binding in the SG (white arrows) appears, which becomes more distinct rostrally (see panels E and F). E, F: 'z51-CYPbinding remains dense in the SG. G, H: At rostral levels ofthe medulla, where NAis compact and distinct, the sparse binding differentiates it from the surrounding neuropil. I, J: '"'I-CYP binding in the NTS remains dense at rostral levels. Calibration bar at bottom right of panel A = 1 mm.
alimentary tract afferent fibers (cf. Figs. 7-11, Altschuler et al., 1989). The anatomical association between 5HTlA sites and swallowing is strengthened by pharmacological studies that have shown that swallowing can be modulated by serotonergic drugs or electrical stimulation of raphe nuclei that contain serotonergic projections to the NTS (Bieger, 1981; Hashim and Bieger, 1987; Kessler and Jean, 1985,1986). A component of the serotonergic modulation of swallowing may occur via a direct presynaptic action on primary afferent terminals, since electrical stimulation of nucleus raphe magnus, which contains a large population of serotonergic neurons that project to the NTS (Thor and Helke, 1987), produces a depolarization of laryngeal primary afferent terminals (Lucier and Sessle, 1981).The possibility of a direct action of serotonin on primary afferent terminals in the NTS is supported by the present findings of a decrease in 5HTIA binding sites in the interstitial subnucleus following cervical vagotomy or nodose ganglionectomy. Since cervical vagotomy (which presumably does not destroy the central terminals of vagal afferent neurons) reduced 5HTlA binding to a level comparable to the reduction seen following nodose ganglionectomy (which does destroy the central terminals of vagal afferent neurons), one must assume that cervical vagotomy causes a "chromatolytic-like" decrease in the expression of 5HTlA receptors if these receptors are on primary afferent terminals. Otherwise, the receptors could be on interneurons, whose expression of the receptor site is dependent upon normal levels of vagal primary afferent activity. In conjunction with the localization of 5HTlA sites on vagal primary afferent terminals, it is interesting to
note that this receptor subtype has also been postulated to be on spinal-cord primary afferent terminals (Daval et al., 1987). Since 5,7-DHT treatment did not reduce 5HTlA sites in the dorsomedial medulla (Thor et al., 1992), it is unlikely that these receptors are located presynaptically on serotonergic terminals, which is consistent with pharmacological studies of serotonergic terminal autoreceptors being of the 5HTIB subtype (Maura et al., 1986; Palacios and Dietl, 1988). 5HTlB sites were fairly dense and uniformly distributed amongst the caudal NTS subnuclei. At intermediate levels of the NTS, however, dense binding was distinctively associated with the substantia gelatinosa area. The binding in this area correlates with the distribution of HRP-labeled primary afferent fibers that innervate the stomach (cf. Fig. 12,Altschuler et al., 1989; cf. Fig. 2B, Leslie et al., 1982; Gwyn et al., 1979; cf. Fig. 7B, Gwyn et al., 1985; cf. Fig. 1, Shapiro and Miselis, 1985; Kahlia and Mesulam, 1980), pancreas (Norgren and Smith, 19881, and liver (Rogers and Hermann, 1983; Norgren and Smith, 1988). However, it is doubtful that the 5HTl, binding sites are on primary afferent terminals, since this binding was not affected by cervical vagotomy or nodose ganglionectomy. Similarly, it is unlikely that a significant portion of the 5HTlB sites were located on serotonergic terminals, since 5,7-DHT pretreatment did not affect binding density (Thor et al., 1992). This latter finding is similar to previous studies that have been unable to show a loss of 5HT1, binding in most brain regions following 5,7-DHT lesions (Palacios and Dietl, 1988). 5HTlB binding in the commissural subnucleus may
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Fig. 4. Sparse lZ6I-CYPbinding in vagal motor nuclei (caudal-rostral, A-F, respectively, all sections from a single animal). At rostral levels of the medulla (C-F), where NA (white arrows) is compact, sparse binding makes it distinct. At caudal levels (A-B), where the motor neurons are more diffusely distributed, the binding is less distinct. 10 (white arrowheads) also shows a paucity of binding that is contrasted with the dense binding just ventral to it in NI and 12. Calibration bar at lower right of panel A = 1mm.
also suggest a role for this receptor subtype in cardiovascular regulation, since the commissural subnucleus also receives an input from primary afferent fibers in the aortic depressor and carotid sinus nerves (Ciriello, 1983; Housley et al., 1987). Areas that contained high densities of both 5HTIA and 5HT,, binding sites were the paratrigeminal islands, the promontorium, and the dorsomedial portion of the rostral trigeminal nucleus. These regions of the trigeminal system are unique in receiving convergent inputs from visceral and somatic afferent terminals
(Astrom, 1953; Contreras et al., 1982; Hamilton and Norgren, 1984; Menetrey and Basbaum, 1987; Altschuler et al., 1989). In addition, the promontorium and paratrigeminal islands contain neurons that project to the NTS (Menetrey and Basbaum, 1987; Thor, unpublished observations) and the parabrachial nucleus (Cechetto et al., 1985; Standaert et al., 19861, which is also important in central autonomic control. This association of 5HTlA and 5HTlB binding sites with areas of somatovisceral primary afferent overlap is maintained along the spinal cord, where 5HTlA and 5HTIBbinding
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is exceptionally dense in lamina I of the dorsal horn at the thoracolumbar and sacral levels (Thor and Helke, in progress), i.e., areas of overlap of visceral and somatic primary afferent fibers (Morgan et al., 1981; Thor et al., 1989). The anatomical association of serotonin binding sites with areas of overlap of terminals of somatic and visceral afferent neurons is strengthened by a recent pharmacological study that has implicated serotonin in the regulation of somatovisceral interactions in the spinal cord (Thor et al., 1990). A final area that contained high densities of both ~ H T and ~ A5HTlB sites was the rostra1 NTS, an area that receives primary afferent input from gustatory receptors (Hamilton and Norgren, 1984). Thus, it is tempting to speculate that serotonergic drugs are involved in the regulation of taste perception. Surprisingly, there was not a remarkable correlation between 5HT binding sites and vagal motor nuclei. The only portion that displayed moderate levels of binding was the DMV caudal to the area postrema where moderate 5HTIB binding was observed. Binding around other vagal efferent neurons was low. The lack of 5HT binding sites in the vagal nuclei proper might be interpreted to reflect a lack of involvement of 5HT in the control of visceral efferent activity. However, this interpretation may be erroneous in light of 5HTlAand 5HTlB binding in the nucleus intercalatus, which lies along the ventral border of the DMV and receives dendritic projections from vagal preganglionic neurons (Shapiro and Miselis, 1985; Norgren and Smith, 1988). In addition to containing dendrites of vagal preganglionic neurons, the nucleus intercalatus also contains neurons that project to the cerebellum (Kotchabhakdi et al., 1978)and the superior colliculus (Stechison et al., 1985). Furthermore, the nucleus intercalatus receives 1)central autonomic inputs from the prefrontal cortex, amygdala, and hypothalamus (van der Kooy et al., 1984; Schwaber et al., 1982; Hopkins and Holstege, 1978), 2) primary afferent inputs from the cervical dorsal root ganglion neurons (Stechison and Saint-Cyr, 1986), 3) proprioceptive inputs from the vestibular nuclei (Mergner et al., 1977), and 4) cerebellar inputs from the fastigial nucleus (Thomas et a]., 1957).Neurons in the fastigial nucleus can be activated by vagal (Perrin and Crousillat, 1985) and splanchnic (Perrin and Crousillat, 1979) visceral afferent stimulation, and electrical or chemical stimulation of the fastigial nucleus produces marked changes in cardiovascular function (Chida et al., 1986; Dormer et al., 1986). Thus, this area of the cerebellum is considered to be involved in autonomic control. In light of the other above-mentioned autonomic connections, it is tempting to speculate that the nucleus intercalatus may be involved in cerebellarautonomic integration and that 5HTIA and 5HTlB receptors may be involved in regulating this integration. Again, an area of visceral-somatic integration exhibits both 5HTlA and 5HTlB receptors. The restriction of 5HTIAsites to the lateral portion of the nucleus sug-
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gests that these sites may regulate primary afferent input from cervical dorsal-root ganglion neurons, since the terminal distribution from these neurons is also restricted to the lateral portion of the nucleus (Stechison and Saint-Cyr, 1986). A recent paper (Manaker and Verderame, 1990) also examined 5HTIAand 5HTlBbinding sites in the NTS in a highly quantitative manner. The results of the present study confirmed many of their findings but also revealed additional anatomical relationships, e.g., the dense 5HTlA binding in the interstitial nucleus and dense 5HTIB binding in the substantia gelatinosa, which can be vaguely discerned in their Figs. 4 and 13, respectively, but were not outstanding, as they appear in the present autoradiographs. In addition, the Manaker and Verderame (1990) paper concludes that the dorsal motor nucleus of the vagus nerve had dense 5HTlB binding, while the present paper concludes that this nucleus has sparse binding. This difference in conclusions is not due to differences in data, only to differences in interpretation of the autoradiograms. For example, from their own figures (cf. Figs. 11 and 12, Manaker and Verderame, 19901, it appears that the area of dense 5HTIB binding is ventral t o the dorsal motor nucleus of the vagus nerve (i.e., in the nucleus intercalatus, as described in the present paper), while the dorsal motor nucleus proper has sparse binding. Finally, the present paper additionally provides evidence l) against 5HTIB sites in the NTS being on serotonergic terminals, since the 5HT neurotoxin 5,7DHT did not significantly modify binding, and 2) for an association of 5HTIA binding sites with primary afferent inputs from the vagus nerve. The present study has shown a distinctive distribution of 5HTIAand 5HTIB sites among the various subnuclei of the NTS and related areas of the dorsomedial medulla oblongata. Recent studies have also shown that medullary 5HT3 receptor sites are localized in the NTS, with half of the binding associated with primary afferent fibers (Pratt and Bowery, 1989; Waeber et al., 1988; Barnes et al., 1990). Hopefully, future pharmacological studies will examine the specific role of each 5HT receptor subtype in determining the complex role of serotonin in the central medullary control of autonomic function. ACKNOWLEDGMENTS This work was supported by NIH grants NS20991 and NS24876 to CJH. REFERENCES Altschuler, S.M., Bao, X., Bieger, D., Hopkins, D.A., and Miselis, R.R. (1989) Viscerotopic representation of the upper alimentary tract in the rat: Sensory ganglia and nuclei of the solitary and spinal trigeminal tracts. J. Comp. Neurol., 283:24&268. Astrom, K.E. (1953) On the central course of afferent fibres in the trigeminal, facial, glossopharyngeal and vagal nerves and their nuclei in the mouse. Acta Physiol. Scand., 29(Suppl. 106):209-320. Barnes, J.M., Barnes, N.M., Costall, B., Naylor, I.L., Naylor, R.J., and Rudd, J.A. (1990) Topographical distribution of 5-HT, receptor
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bution of the cervical vagus nerve and gastric afferent and efferent projections in the rat. Brain Res. Bull., 8:3743. Lucier, G.E. and Sessle, B.J. (1981) Presynaptic excitability changes induced in the solitary tract endings of laryngeal primary afferents by stimulation of nucleus raphe magnus and locus coeruleus. Neurosci. Lett., 26:221-226. Manaker M. and Verdame H.M. (1990) Organization of serotonin-lA and serotonin-1B receptors in the nucleus of the solitary tract. J . Comp. Neurol., 301535-553. Maura, G., Roccatagliata, E., and Raiteri, M. (1986)Serotonin autoreceptor in rat hippocampus: Pharmacological characterization as a subtype of the 5-HT, receptor. Naunyn Schmiedebergs Arch. Pharmacol., 334:323-326. McCall, R.B., Patel, B.N., and Harris, L.T. (1987)Effects of serotonin, and serotonin, receptor agonists and antagonists on blood pressure, heart rate and sympathetic nerve activity. J. Pharmacol. Exp. Ther., 242~1152-1159. Menetrey, D. and Basbaum, A.I. (1987) Spinal and trigeminal projections to the nucleus of the solitary tract: A possible substrate for somatovisceral and viscerovisceral reflex activation. J . Comp. Neurol., 255:439450. Mergner, T., Pompeiano, O., and Corvaja, N. (1977)Vestibular projections to the nucleus intercalatus of Staderini mapped by retrograde transport of horseradish peroxidase. Neurosci. Lett., 5:309-313. Millhorn, D.E., Eldridge,F.L., Waldrop,T.G., andKlingler, L.E. (1983) Centrally and peripherally administered 5-HTP have opposite effects on respiration. Brain Res., 264:349-354. Morgan, C., Nadelhaft, I., and de Groat, W.C. (1981)The distribution of visceral primary afferents from the pelvic nerve to Lissauer’s tract and the spinal gray matter and its relationship to the sacral parasympathetic nucleus. J . Comp. Neurol., 201:415-440. Norgren, R. and Smith, G.P. (1988) Central distribution of subdiaphragmatic vagal branches in the rat. J . Comp. Neurol., 273:207223. Palacios, J.M. and Dietl, M.M. (1988) Autoradiographic studies of serotonin receptors. In: The Serotonin Receptors. E. Sanders-Bush, ed. Humana Press, Clifton, NJ, p. 89. Peroutka, S.J. (1988) 5-hydroxytryptamine receptor subtypes. Annu. Rev. Neurosci., 11:45-60. Perrin, J . and Crousillat, J. (1979) Effets de la stimulation des mecanorecepteurs splanchniques sur l’activite des cellules cerebelleuses de Purkinje. Exp. Brain Res., 37:405-416. Perrin, J. and Crousillat, J. (1985)The projection ofvagal afferents on the cerebellar vermis of the cat. J . Auton. Nerv. Syst., 13:175-177. Pratt, G.D. and Bowery, N.G. (1989) The 5-HT3 receptor ligand, [“HIBRL 43694, binds to presynaptic sites in the nucleus tractus solitarius of the rat. Neuropharmacology, 28(12):1367-1376. Rogers, R.C. and Hermann, G.E. (1983) Central connections of the hepatic branch of the vagus nerve: A horseradish peroxidase histochemical study. J . Auton. Nerv. Syst., 7:165-174. Schwaber, J.S., Kapp, B.S., Higgins, G.A., and Rapp, P.R. (1982) Amygdaloid and basal forebrain direct connections with the nucleus of the solitary tract and the dorsal motor nucleus. J . Neurosci., 2314261438. Seiders, E.P. and Stuesse, S.L. (1984)A horseradish peroxidase investigation of carotid sinus nerve components in the rat. Neurosci. Lett., 46:13-18. Sessle, B.J. and Henry, J.L. (1985)Effects of enkephalin and 5-hydroxytryptamine on solitary tract neurones involved in respiration and respiratory reflexes. Brain Res., 327:221-230. Shapiro, R.E. and Miselis, R.R. (1985) The central organization of the vagus nerve innervating the stomach of the rat. J . Comp. Neurol., 238:473488. Standaert, D.G., Watson, S.J., Houghten, R.A., and Saper, C.B. (1986) Opioid peptide immunoreactivity in spinal and trigeminal dorsal horn neurons projecting to the parabrachial nucleus in the rat. J. Neurosci., 6(5):1220-1226. Stechison, M.T. and Saint-Cyr, J.A. (1986) Organization of spinal inputs to the perihypoglossal complex in the cat. J. Comp. Neurol., 2461555667. Stechison, M.T., Saint-Cyr, J.A., and Spence, S.J. (1985) Projections from the nuclei prepositus hypoglossi and intercalatus to the superior colliculus in the cat: An anatomical study using WGA-HRP.Exp. Brain Res., 59:139-150. Thomas, D.M., Kaufman, R.P., Sprague, J.M., and Chambers, W.W. (1957) Experimental studies of the vermal cerebellar projections in the brain stem of the cat (fastigiobulbar tract). J. Anat., 90:371-385. Thor, K.B., Blitz-Siebert, A,, and Helke, C.J. (1992) Autoradiographic localization of 5HT, binding sites in the medulla oblongata of the rat. Synapse, 10:185-205.
MEDULLARY AUTONOMIC SEROTONERGIC BINDING SITES Thor, K.B. and Helke, C.J. (1987) Serotonin- and substance P-containing projections to the nucleus tractus solitarii of the rat. J . Comp. Neurol., 265:275-293. rhor, K,B,, Hisamitsu, T,, and de Groat, W.C. (1990)Unmasking of a neonatal somatovesical reflex in adult catsby the serotonin autoreagonist 5-methox~~N~N-dimethy*twptamine~ Dev. Brain Res., 54:35-42. Thor, K.B., Morgan, C., Nadelhaft, I., Houston, M., and de Groat, W.C.
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(1989) Organization of afferent and efferent pathways in the pudendal nerve of the female cat. J. Comp. Neurol., 288:263-279. van der Kooy, D., Koda, L.Y., McGinty, J.F., Gerfen, C.R., and Bloom, F.E. (1984) The Organization Of projections from the cortex, and hypothalamus to the Of the tract in rat. J. Comp. Neurol., 224:l-24. Waeber, C., Dixon, K., Hoyer, D., and Palacios, J.M. (1988)Localisation by autoradiography of neuronal5-HT3 receptors in the mouse CNS. Eur. J. Pharmacol., 151:351-352.
Autoradiographic Localization of 5HT, Binding Sites in Autonomic Areas of the Rat Dorsomedial Medulla Oblongata KARL B. THOR, ALISA BLITZ-SIEBERT,AND CINDA J. HELKE Department of Pharmacology, Uniformed Services University of the Health Sciences, Bethesda, Maryland 20814 (K.B.T.,A.B.-S.,C.J.H.); Division of CNS Research, Lilly Research Laboratories, Eli t i l l y and Co., Indianapolis, Indiana 46285 (K.B.T.)
KEY WORDS
Serotonin receptors, Receptor autoradiography, Medulla oblongata, Nucleus tractus solitarius, Nucleus ambiguus, Dorsal motor nucleus of the vagus nerve, Nucleus intercalatus, 3H-8-OH-dipropyl-amino-tetraline, 1251-iodocyanopindolol
ABSTRACT Serotonin (5HT) binding sites in autonomic portions of the dorsomedial medulla oblongata of the rat were localized using autoradiographic techniques with radioactive ligands that express high affinity for the 5HT1 (3H-5HT),5HTlA (3H-80HDPAT), or 5HTlB (1251-cYPwith isoproterenol) receptor subtypes. 5HTlA sites were densely distributed in the nucleus tractus solitarius (NTS), with the highest densities localized to the interstitial subnucleus and the central subnucleus. 5HTlB sites were also found in the NTS, with the highest densities localized to the substantia gelatinosa subnucleus. The dorsal motor nucleus of the vagus nerve and nucleus ambiguus exhibited low densities of 5HTIBsites. However, the nucleus intercalatus, a cerebellar relay nucleus that also contains dendrites of vagal parasympathetic preganglionic neurons and receives autonomic forebrain afferent input, showed very dense 5HTlB sites. The promontorium, paratrigeminal islands, and the dorsomedial portion of the trigeminal nucleus (DM5), which are areas of viscerosomatic integration, exhibited high densities of both 5HTIAand 5HTlB sites. The area postrema contained low levels of both 5HTIAand 5HTlB sites. Visceral deafferentation via cervical vagotomy or nodose ganglionectomy caused a significant decrease in 5HTlAsites in the interstitial subnucleus of the NTS ipsilateral to the lesion. No changes were seen in 5HTIBsites. These studies suggest that 5HT,, and 5HTlB sites are involved in the processing of visceral sensory information in the NTS and associated areas. Based upon viscerotopic organization of the NTS, 5HTlA sites appear preferentially distributed in portions of the NTS that are associated with the coordination of swallowing, respiration, and cardiovascular function, while 5HTlB sites appear preferentially distributed in areas of the NTS associated with gastrointestinal, hepatic, pancreatic, and cardiovascular function. However, since these associations were not absolute and there was a great deal of overlap between the two sites, speculation regarding their specific functions in autonomic control must await pharmacological examination.
INTRODUCTION The dorsomedial medulla oblongata, comprised primarily of the NTS and dorsal motor nucleus of the vagus nerve (DMV), is a vital area for control of a number of autonomic functions. The NTS is the primary region of termination for visceral primary afferent fibers contained in the vagus and glossopharyngeal nerves, while the DMV, as well as the nucleus ambiguus, are the origin for the efferent component of these nerves (Kalia and Sullivan, 1982; Norgren and Smith, 1988; Altschuler et al., 1989). 0 1992 WILEY-LISS, INC.
These autonomic areas of the dorsomedial medulla receive dense innervation from medullary serotonergic neurons (Thor and Helke, 19871, and various physiological and pharmacological studies have indicated a role for serotonin (5-hydroxytryptamine,5HT) in the control of autonomic function in the medulla. For example, medullary serotonergic systems have been implicated in regulation of swallowing (Kessler and Jean, 1985,1986; Lucier and Sessle, 1981; Hashim and Bieger, 19871, Received December 21,1990;accepted in revised form July 26,1991
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respiration (Millhorn et al., 1983; Lalley, 1986; Sessle and Henry, 1985; Holtman et al., 1986, 1987; Gillis et al., 19891,and cardiovascular function (McCall et al., 1987;Gillis et al., 1989).Many of the effects of serotonin on these systems have been complex, and the mechanisms and anatomical sites of action have not been determined. The complexity of serotonin's effects might be explained by the fact that there are multiple subtypes of receptors for serotonin (Peroutka, 1988) and that serotonin might have multiple sites of action along autonomic reflex pathways. To determine possible medullary sites of action for the various serotonin receptor subtypes, receptor autoradiography techniques were applied to sections of rat medulla, and the densities of the various serotonin receptor subtypes were examined 1) in areas of visceral afferent termination (Astrom, 19531, i.e., the NTS, the promontorium, the paratrigeminal islands, the area postrema, and the dorsomedial portion of the trigeminal nucleus and 2) in areas containing vagal efferent neurons, i.e., the DNIV and nucleus ambiguus.
unilateral cervical vagotomy or nodose ganglionectomy on serotonergic binding. Rats were anesthetized with chloral hydrate and the vagus nerve was isolated. In five rats, the vagus nerve was cut 10 mm from its entrance into the tenth cranial nerve foramen (i.e., distal to the nodose ganglion). In five rats, the vagus nerve was retracted to expose the nodose ganglion in the foramen and the ganglion was excised. In five shamoperated rats, the vagus nerve was exposed but not cut. After 10 days, the animals were sacrificed, and the medullae were processed as described above. Nuclear subdivisions of the NTS were based upon the descriptions of Kalia and Sullivan (1982).
RESULTS 3H-5HTbinding In general, 3H-5HTbinding reflected the combination of binding seen with 3H-8-OH-DPAT and 1251-CYP, which are described below in detail. The relative distribution of 3H-5HTbinding among the various autonomic areas of the dorsomedial medulla are listed in Table I.
3H-8-OH-DPATbinding
METHODS
The highest density of 3H-8-OH-DPATbinding in the NTS was in the interstitial (Fig. 1B to 1D) and central Detailed methods can be found in Thor et al. (1992). subnuclei (Fig. 1D to 1F). Binding in the commissural Briefly, adult male Sprague-Dawley rats were decapitated, and the brains were rapidly removed and frozen subnucleus of the NTS (Kalia and Sullivan, 1982) was on powdered dry ice. Frozen sections (20 pm thick) were intermediate in density, while other portions of the NTS cut on a cryostat, placed on subbed slides, desiccated in exhibited lower levels of binding (Fig. 1, Table I). At a vacuum overnight at 4"C, and stored at -70°C until rostral levels of the medulla, 3H-8-OH-DPATbinding in incubation. 5HT, sites were indiscriminately labelled the NTS was moderately dense, with an increase in using 31H1-5HT,5HT,, sites were labelled using 3[Hl-8- density medially to laterally (Fig. 1,Table I). The dense OH-dipropyl aminotetraline (3H]80H-DPAT), and binding in the lateral NTS at rostral levels was contig5HTIBsites were labelled using 125[I]-iodocyanopindolol uous with dense binding in the dorsomedial subnucleus (l"I-CYP) with 30 pM isoproterenol to block beta- of the trigeminal complex. Cervical vagotomy and nodose ganglionectomy each adrenergic binding sites. Nonspecific binding of ligands was assessed using 1 pM 5HT. Radioactive standards caused a decrease (49 8%and 57 5 6%, respectively; and correction for lipid quenching of the 3H signal have n = 5 for each) in 5HTlAbinding sites in the interstitial subnucleus (Fig. 2) ipsilateral t o the lesion, indicating been described in Thor et al. (1992). Fifteen rats were studied to determine the effects of an association of these sites with visceral afferent pathways. No other changes were detected in these lesioned animals. 3H-8-OH-DPAT binding was sparse in the DMV (Fig. 1) and NA (Table I; cf. Fig. 4, Thor et al., 1992). Abbreviations However, the lateral portion of the nucleus intercalatus 10 dorsal motor nucleus of the vagus nerve (NU,which lies between the DMV and the hypoglossal 12 hypoglossal nucleus Ap area postrema nucleus, exhibited substantial binding (Fig. 1A-B, TaCEN central subnucleus of the NTS ble I). Binding in the area postrema was very low DM5 dorsomedial portion of the trigeminal nucleus IST interstitial nucleus of the NTS (Fig. lC, Table I). NA nucleus ambiguus Areas of the medulla that are not in the NTS proper NC nucleus cuneatus NG nucleus gracilis but also receive visceral primary afferent input (AsNI nucleus intercalatus trom, 1953) also exhibited substantial 3H-8-OH-DPAT NTS nucleus tractus solitarius binding. The promontorium, which caps the dorsomePr promontorium PrH prehypoglossal nucleus dial aspect of the trigeminal nucleus at caudal levels, PTI paratrigeminal islands the paratrigeminal islands, and the dorsomedial subnupyramidal tract, decussation PY>PYX SG substantia gelatinosa cleus of the trigeminal nucleus oralis each exhibited so superior olives substantial binding (Table I; cf. Figs. 4 and 5 , Thor spinal tract of the trigeminal nerve SP5 ts tractus solitarius et al., 1992).
*
~~
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MEDULLARY AUTONOMIC SEROTONERGIC BINDING SITES TABLE I. Specific binding (expressed as femtomoles/mg wet weight) of 3H-5HT,3H-8-OH-DPAT,and 1251-CYPin subnuclei of the NTS, areas of the medulla that are closely associated with the NTS, and in uagal efferent nuclei Ligand Concentration
3H-5HT (5 nM)
Caudal/intermediate NTS subnuclei Commissural 124.7 f 11.2 Dorsal 85.1 f 6.0 Dorsolateral 118.2 f 7.6 Medial 98.5 f 6.9 Interstitial 147.2 f 7.3 Central 150.1 f 9.9 Intermediate 126.0 f 9.1 Substantia gelatinosa 137.8 f 8.7 Ventrolateral 121.6 =k 8.2 Ventral 129.7 f 6.5 Rostra1 NTS 165.2 f 8.7 Medial Lateral 174.9 f 7.6 Associated areas Promontorium 164.3 f 9.7 Paratrigeminal 108.7 f 8.6 DM5 82.4 f 7.6 Area postrema 38.5 f 5.7 Vagal motor nuclei and nucleus intercalatus 119.8 f 12.2 DMV, DMV, 115.4 10.1 Nucleus intercalatus 185.1 18.3 Nucleus ambiguus 79.3 f 8.2
*
*
3H-8-OH-DPAT (2 nM)
'25I-CYP (50 PM)
34.5 f 1.9 21.4 f 1.7 24.5 f 2.3 28.0 f 1.8 58.7 f 3.0 55.6 f 2.2 33.9 f 1.9 25.8 f 2.0 27.0 f 2.3 27.9 f 2.9
6.98 f 0.46 4.84 f 0.41 5.63 k 0.39 6.34 f 0.42 6.01 f 0.21 5.11 f 0.32 5.30 f 0.28 8.18 f 0.69 7.41 f 0.64 7.23 f 0.57
42.3 f 3.1 49.2 f 2.8
8.63 f 0.49 8.93 f 0.32
59.7 31.8 33.8 9.7
f 5.6 f 4.9 f 2.2 f 1.3
5.89 f 0.47 5.31 f 0.51 4.91 f 0.42 2.08 f 0.11
18.9 f 1.1 19.7 f 1.4 38.0 f 2.6 18.6 f 1.4
6.89 f 0.43 5.32 f 0.51 9.93 f 0.58 3.20 f 0.27
DM5 = dorsomedial portion of the trigeminal nucleus; DMV = dorsal motor nucleus of the vagus nerve.
1251-CYPbinding
binding in the dorsal portion of nucleus reticularis parvocellularis. At caudal levels, binding in the DMV proper was moderately dense, being homogeneous with that seen dorsally in the commissural subnucleus of the NTS (Fig. 3A-C, Table I). However, at intermediate levels, the DMV became distinguished by its sparse binding, compared to the denser binding seen in the nucleus intercalatus ventrally and the substantia gelatinosa dorsally (Fig. 3D and E). Similar to the sparse binding that distinguished the DMV at intermediate levels, the nucleus ambiguus (NA) was also distinguished by exhibiting less binding than the surrounding neuropil (Fig. 3G1, Fig. 4, Table I). The sparse binding in NA was conspicuous (Fig. 4C-F) from the caudal border of the seventh motor nucleus to the level of the nucleus reticularis lateralis. At this more caudal level (Fig. 4A and B), the pattern of sparse binding in NA became less distinct, presumably because adjacent structures, such as the nucleus reticularis lateralis, also exhibit a paucity of '251-CYPbinding and because neurons in NA at this level are distributed more diffusely than they are at rostral levels (Bieger and Hopkins, 1987). No changes were observed in 1251-CYPbinding following cervical vagotomy or nodose ganglionectomy.
The highest levels of 1251-CYPbinding were found in the rostral third of the NTS (Fig. 3 H 4 , Table I), with greater density in the lateral portion, which was contiguous with dense binding in the dorsomedial portion of the trigeminal nucleus. At intermediate levels, the NTS binding diminished medial to the tractus solitarius, except for a circular area in the dorsomedial quadrant of the NTS adjacent to the ventricle, where binding was denser (Fig. 3E-F; Table I).This area of denser binding in the NTS corresponds to the substantia gelatinosa subnucleus. Further caudally, this area of denser binding in the dorsomedial portion of the NTS became less distinct as binding in the commissural and medial subnuclei increased (Fig. 3A-D). Ventrolateral portions of the NTS exhibited a moderately dense level of 1251CYP binding at all levels. The relationship between 1251-CYP binding and the dorsal motor nucleus of the vagus nerve ( D W ) was complex (Fig. 3) due to the heterogeneous pattern of binding in this area. On the one hand, there was a highly distinguishable, thin (50 x 150 pm), horizontal strip of extremely dense binding that appeared to be localized along the boundary of the DMV and hypoglossal nucleus, i.e., in the nucleus intercalatus (Fig. 3B-E). This strip of dense binding, seen at both the intermediate and DISCUSSION caudal levels of the NTS, extended laterally from the The NTS receives a dense innervation from the medfloor of the IV ventricle and from the central canal, respectively (Fig. 3B-E), and was contiguous with dense ullary raphe and associated groups of serotonergic neu-
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Figure 1.
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Fig. 2. Loss of high-density 3H-80H-DPATbinding sites in the IST followingunilateral vagotomy (right side). Calibration bar = 250 pm.
rons (Thor and Helke, 1987). The purpose of the present study was t o anatomically describe the relative distributions of certain 5HT binding sites among the various portions of the medulla oblongata that are directly involved in autonomic control. The anatomical distributions of the serotonin binding sites will be discussed in the context of the viscerotopic organization of the NTS, as revealed by horseradish peroxidase (HRP) tracing studies of visceral primary afferent fibers, and with regards to the various physiological roles that serotonin has been proposed to play in autonomic control. The results indicate that both 5HTlA and 5HTlB sites are very specifically and differentially distributed among
Fig. 1. 'H-SOH-DPAT binding in the dorsomedial medulla (caudalrostral, A-H, respectively, all sections from a single animal). A-D: Binding is moderately dense in the commissural region of the NTS. At the level of the AP,dense binding can be seen in the IST. At levels rostral to the AP, dense binding occurs in the CEN as well. Binding in the AP is sparse. E-H: 3H-80H-DPAT binding remains dense in the CEN. At rostral levels, binding within the NTS is homogeneous and contiguous with binding in DM5. The calibration bar in the lower right of panel A = 200 km.
the various subnuclei of the NTS and the autonomic motor nuclei. The 5HTlA sites were most dense in the interstitial, intermediate, and central subnuclei of the NTS, with moderate binding in the commissural subnucleus. The interstitial and commissural subnuclei have been shown to receive primary afferent terminals from fibers contained in the aortic depressor and carotid sinus nerves of rats (Housley et al., 1987; Seiders and Steusse, 1984; Ciriello, 1983). Thus, 5HTIAreceptors in the NTS may have a role in regulation of baroreceptor and chemoreceptor inputs. In fact, direct application of 5HT into the NTS has been shown to alter blood pressure (Laguzzi et al., 1984). In addition, the interstitial and the central subnuclei of the NTS receive inputs from primary afferent fibers that innervate the larynx, pharynx, and esophagus (Hamilton and Norgren, 1984; Hisa et al., 1985; Altschuler et al., 19891, structures responsible for coordinating the act of swallowing. In fact, the distribution of 5HTIAbinding sites in this area of the NTS is remarkably similar to the distribution ofHRP labelling of upper
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Figure 3A-H.
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Fig. 3. '251-CYPbinding in the dorsomedial medulla (caudal-rostral, A-I, respectively, all sections from a single animal). A-D: Dense homogeneous binding occurs in the commissural subnucleus of the NTS. Moderately dense binding is seen in the dorsal column nuclei, NG and NC. A very dense band of binding occurs in NI (white arrows in panels B and C). Note that 10 contains less binding (see also Fig. 11). AF' contains sparse binding. In panel D, a circular area of denser
binding in the SG (white arrows) appears, which becomes more distinct rostrally (see panels E and F). E, F: 'z51-CYPbinding remains dense in the SG. G, H: At rostral levels ofthe medulla, where NAis compact and distinct, the sparse binding differentiates it from the surrounding neuropil. I, J: '"'I-CYP binding in the NTS remains dense at rostral levels. Calibration bar at bottom right of panel A = 1 mm.
alimentary tract afferent fibers (cf. Figs. 7-11, Altschuler et al., 1989). The anatomical association between 5HTlA sites and swallowing is strengthened by pharmacological studies that have shown that swallowing can be modulated by serotonergic drugs or electrical stimulation of raphe nuclei that contain serotonergic projections to the NTS (Bieger, 1981; Hashim and Bieger, 1987; Kessler and Jean, 1985,1986). A component of the serotonergic modulation of swallowing may occur via a direct presynaptic action on primary afferent terminals, since electrical stimulation of nucleus raphe magnus, which contains a large population of serotonergic neurons that project to the NTS (Thor and Helke, 1987), produces a depolarization of laryngeal primary afferent terminals (Lucier and Sessle, 1981).The possibility of a direct action of serotonin on primary afferent terminals in the NTS is supported by the present findings of a decrease in 5HTIA binding sites in the interstitial subnucleus following cervical vagotomy or nodose ganglionectomy. Since cervical vagotomy (which presumably does not destroy the central terminals of vagal afferent neurons) reduced 5HTlA binding to a level comparable to the reduction seen following nodose ganglionectomy (which does destroy the central terminals of vagal afferent neurons), one must assume that cervical vagotomy causes a "chromatolytic-like" decrease in the expression of 5HTlA receptors if these receptors are on primary afferent terminals. Otherwise, the receptors could be on interneurons, whose expression of the receptor site is dependent upon normal levels of vagal primary afferent activity. In conjunction with the localization of 5HTlA sites on vagal primary afferent terminals, it is interesting to
note that this receptor subtype has also been postulated to be on spinal-cord primary afferent terminals (Daval et al., 1987). Since 5,7-DHT treatment did not reduce 5HTlA sites in the dorsomedial medulla (Thor et al., 1992), it is unlikely that these receptors are located presynaptically on serotonergic terminals, which is consistent with pharmacological studies of serotonergic terminal autoreceptors being of the 5HTIB subtype (Maura et al., 1986; Palacios and Dietl, 1988). 5HTlB sites were fairly dense and uniformly distributed amongst the caudal NTS subnuclei. At intermediate levels of the NTS, however, dense binding was distinctively associated with the substantia gelatinosa area. The binding in this area correlates with the distribution of HRP-labeled primary afferent fibers that innervate the stomach (cf. Fig. 12,Altschuler et al., 1989; cf. Fig. 2B, Leslie et al., 1982; Gwyn et al., 1979; cf. Fig. 7B, Gwyn et al., 1985; cf. Fig. 1, Shapiro and Miselis, 1985; Kahlia and Mesulam, 1980), pancreas (Norgren and Smith, 19881, and liver (Rogers and Hermann, 1983; Norgren and Smith, 1988). However, it is doubtful that the 5HTl, binding sites are on primary afferent terminals, since this binding was not affected by cervical vagotomy or nodose ganglionectomy. Similarly, it is unlikely that a significant portion of the 5HTlB sites were located on serotonergic terminals, since 5,7-DHT pretreatment did not affect binding density (Thor et al., 1992). This latter finding is similar to previous studies that have been unable to show a loss of 5HT1, binding in most brain regions following 5,7-DHT lesions (Palacios and Dietl, 1988). 5HTlB binding in the commissural subnucleus may
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Fig. 4. Sparse lZ6I-CYPbinding in vagal motor nuclei (caudal-rostral, A-F, respectively, all sections from a single animal). At rostral levels of the medulla (C-F), where NA (white arrows) is compact, sparse binding makes it distinct. At caudal levels (A-B), where the motor neurons are more diffusely distributed, the binding is less distinct. 10 (white arrowheads) also shows a paucity of binding that is contrasted with the dense binding just ventral to it in NI and 12. Calibration bar at lower right of panel A = 1mm.
also suggest a role for this receptor subtype in cardiovascular regulation, since the commissural subnucleus also receives an input from primary afferent fibers in the aortic depressor and carotid sinus nerves (Ciriello, 1983; Housley et al., 1987). Areas that contained high densities of both 5HTIA and 5HT,, binding sites were the paratrigeminal islands, the promontorium, and the dorsomedial portion of the rostral trigeminal nucleus. These regions of the trigeminal system are unique in receiving convergent inputs from visceral and somatic afferent terminals
(Astrom, 1953; Contreras et al., 1982; Hamilton and Norgren, 1984; Menetrey and Basbaum, 1987; Altschuler et al., 1989). In addition, the promontorium and paratrigeminal islands contain neurons that project to the NTS (Menetrey and Basbaum, 1987; Thor, unpublished observations) and the parabrachial nucleus (Cechetto et al., 1985; Standaert et al., 19861, which is also important in central autonomic control. This association of 5HTlA and 5HTlB binding sites with areas of somatovisceral primary afferent overlap is maintained along the spinal cord, where 5HTlA and 5HTIBbinding
MEDULLARY AUTONOMIC SEROTONERGIC BINDING SITES
is exceptionally dense in lamina I of the dorsal horn at the thoracolumbar and sacral levels (Thor and Helke, in progress), i.e., areas of overlap of visceral and somatic primary afferent fibers (Morgan et al., 1981; Thor et al., 1989). The anatomical association of serotonin binding sites with areas of overlap of terminals of somatic and visceral afferent neurons is strengthened by a recent pharmacological study that has implicated serotonin in the regulation of somatovisceral interactions in the spinal cord (Thor et al., 1990). A final area that contained high densities of both ~ H T and ~ A5HTlB sites was the rostra1 NTS, an area that receives primary afferent input from gustatory receptors (Hamilton and Norgren, 1984). Thus, it is tempting to speculate that serotonergic drugs are involved in the regulation of taste perception. Surprisingly, there was not a remarkable correlation between 5HT binding sites and vagal motor nuclei. The only portion that displayed moderate levels of binding was the DMV caudal to the area postrema where moderate 5HTIB binding was observed. Binding around other vagal efferent neurons was low. The lack of 5HT binding sites in the vagal nuclei proper might be interpreted to reflect a lack of involvement of 5HT in the control of visceral efferent activity. However, this interpretation may be erroneous in light of 5HTlAand 5HTlB binding in the nucleus intercalatus, which lies along the ventral border of the DMV and receives dendritic projections from vagal preganglionic neurons (Shapiro and Miselis, 1985; Norgren and Smith, 1988). In addition to containing dendrites of vagal preganglionic neurons, the nucleus intercalatus also contains neurons that project to the cerebellum (Kotchabhakdi et al., 1978)and the superior colliculus (Stechison et al., 1985). Furthermore, the nucleus intercalatus receives 1)central autonomic inputs from the prefrontal cortex, amygdala, and hypothalamus (van der Kooy et al., 1984; Schwaber et al., 1982; Hopkins and Holstege, 1978), 2) primary afferent inputs from the cervical dorsal root ganglion neurons (Stechison and Saint-Cyr, 1986), 3) proprioceptive inputs from the vestibular nuclei (Mergner et al., 1977), and 4) cerebellar inputs from the fastigial nucleus (Thomas et a]., 1957).Neurons in the fastigial nucleus can be activated by vagal (Perrin and Crousillat, 1985) and splanchnic (Perrin and Crousillat, 1979) visceral afferent stimulation, and electrical or chemical stimulation of the fastigial nucleus produces marked changes in cardiovascular function (Chida et al., 1986; Dormer et al., 1986). Thus, this area of the cerebellum is considered to be involved in autonomic control. In light of the other above-mentioned autonomic connections, it is tempting to speculate that the nucleus intercalatus may be involved in cerebellarautonomic integration and that 5HTIA and 5HTlB receptors may be involved in regulating this integration. Again, an area of visceral-somatic integration exhibits both 5HTlA and 5HTlB receptors. The restriction of 5HTIAsites to the lateral portion of the nucleus sug-
225
gests that these sites may regulate primary afferent input from cervical dorsal-root ganglion neurons, since the terminal distribution from these neurons is also restricted to the lateral portion of the nucleus (Stechison and Saint-Cyr, 1986). A recent paper (Manaker and Verderame, 1990) also examined 5HTIAand 5HTlBbinding sites in the NTS in a highly quantitative manner. The results of the present study confirmed many of their findings but also revealed additional anatomical relationships, e.g., the dense 5HTlA binding in the interstitial nucleus and dense 5HTIB binding in the substantia gelatinosa, which can be vaguely discerned in their Figs. 4 and 13, respectively, but were not outstanding, as they appear in the present autoradiographs. In addition, the Manaker and Verderame (1990) paper concludes that the dorsal motor nucleus of the vagus nerve had dense 5HTlB binding, while the present paper concludes that this nucleus has sparse binding. This difference in conclusions is not due to differences in data, only to differences in interpretation of the autoradiograms. For example, from their own figures (cf. Figs. 11 and 12, Manaker and Verderame, 19901, it appears that the area of dense 5HTIB binding is ventral t o the dorsal motor nucleus of the vagus nerve (i.e., in the nucleus intercalatus, as described in the present paper), while the dorsal motor nucleus proper has sparse binding. Finally, the present paper additionally provides evidence l) against 5HTIB sites in the NTS being on serotonergic terminals, since the 5HT neurotoxin 5,7DHT did not significantly modify binding, and 2) for an association of 5HTIA binding sites with primary afferent inputs from the vagus nerve. The present study has shown a distinctive distribution of 5HTIAand 5HTIB sites among the various subnuclei of the NTS and related areas of the dorsomedial medulla oblongata. Recent studies have also shown that medullary 5HT3 receptor sites are localized in the NTS, with half of the binding associated with primary afferent fibers (Pratt and Bowery, 1989; Waeber et al., 1988; Barnes et al., 1990). Hopefully, future pharmacological studies will examine the specific role of each 5HT receptor subtype in determining the complex role of serotonin in the central medullary control of autonomic function. ACKNOWLEDGMENTS This work was supported by NIH grants NS20991 and NS24876 to CJH. REFERENCES Altschuler, S.M., Bao, X., Bieger, D., Hopkins, D.A., and Miselis, R.R. (1989) Viscerotopic representation of the upper alimentary tract in the rat: Sensory ganglia and nuclei of the solitary and spinal trigeminal tracts. J. Comp. Neurol., 283:24&268. Astrom, K.E. (1953) On the central course of afferent fibres in the trigeminal, facial, glossopharyngeal and vagal nerves and their nuclei in the mouse. Acta Physiol. Scand., 29(Suppl. 106):209-320. Barnes, J.M., Barnes, N.M., Costall, B., Naylor, I.L., Naylor, R.J., and Rudd, J.A. (1990) Topographical distribution of 5-HT, receptor
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