Ascending Projections from the Area Postrema and The

Okajimas Folia Anat. Jpn., 75(1): 9-32, May, 1998

Ascending Projections from the Area Postrema and the Nucleus of the Solitary Tract of Suncus Murinus: Anterograde Tracing Study Using Phaseolus Vulgaris Leucoagglutinin

Hisao ITO and Makoto SEKI

First Department of Anatomy, Hyogo College of Medicine, Mukogawa-cho 1-1, Nishinomiya, Hyogo 663-8501, Japan

-Received for Publication, March 13, 1998-

Key Words: Area postrema, Solitary nucleus, Suncus murinus, Vomiting, Phaseolus vulgaris leucoagglutinin

Summary: Suncus murinus (suncus) is a new experimental animal model for research on the mechanisms underlying emesis. In the present study, we examined the ascending projections from the area postrema (AP) and the nucleus of the solitary tract (NTS) in suncus based on anterograde transport of phaseolus vulgaris leucoagglutinin. The AP projected heavily to the dorsal vagal complex, especially in the commissural and medial subnuclei of the NTS, and the dorsal motor nucleus of the vagus. Some ascending fibers from the AP projected bilaterally to the parabrachial nucleus (Pb), but no labeling was observed rostral to this area. In contrast, the NTS had extensive projections as far as the basal forebrain. The NTS projections were observed in the AP, ventrolateral reticular formation including the nucleus ambiguus, A5 noradrenergic area, locus coeruleus, Pb, and central gray matter of the midbrain. At the level of the diencephalon, the NTS projections were seen in the dorsomedial, lateral, paraventricular, periventricular, supraoptic, retrochiasmatic and arcuate nuclei of the hypothalamus, in addition to the paraventricular nucleus of the thalamus. Terminal fields within the basal forebrain were also shown to include the medial preoptic area, the bed nucleus of the stria terminalis, the substantia innominata and the ventral pallidum. The results indicated that the neurological relationship between the chemo- and/or barosensitive systems including the trigger of the emetic response and the general viscerosensory and/or -motor systems may exist also in the suncus.

Suncus murinus (suncus), a species of Insec- lie the emetic reflex via the AP. Some studies have tivora, is being used as a relatively new animal reported on the nature of the connectivity of the model for experimental research in Japan. Suncus AP, but most have used the rat as their model, are easily induced to vomit by exposing them to which, unlike the suncus, is not prone to vomit- various stimuli, e.g., the emetic agents nicotine and ing (van der Kooy and Koda, 1983; Shapiro and veratrine (Ueno et al., 1987), or reciprocal shaking Miselis, 1985a; Herbert et aL, 1990; Cunningham et (Ueno et aL, 1988), and this makes the suncus a aL, 1994). In the ferret, a species with a high inci- valuable experimental model for emetic research. dence of vomiting, Strominger et aL (1994) demon- The area postrema (AP) in the suncus, as in the strated that the AP has reciprocal connections only rat and rabbit, is an unpaired midline structure that with the nucleus of the solitary tract (NTS), which ocupies the wall of the obex of the fourth ventricle. is generally accepted as the primary recipient of It is a highly vascular structure with a very weak gustatory, cardiovascular, pulmonary, and gastro- blood-brain diffusion barrier, allowing most sub- intestinal sensory afferents. stances to penetrate into the interstitial space be- Studies on the innervation of the NTS have tween the neurons. Based on physiological studies, suggested that functionally classified columns are the AP has generally been recognized to act as a arrayed rostrocaudally in this nucleus; for example, "chemoreceptor trigger zone" for the induction of gustatory afferents occupy the rostral third portion emesis (Borison et al., 1984; Carpenter, 1989), how- of the column (Whitehead and Frank, 1983; Hamil- ever, there have been few morphological studies ton and Norgren, 1984), while visceral afferents concerning the neuronal mechanisms which under- occupy a more caudal region (Kalia and Mesulam ,

Correspondence to Dr. Hisao Ito, First Department of Anatomy, Hyogo College of Medicine , Mukogawa-cho 1-1, Nishinomiya, Hyogo 663-8501, Japan

9 10 H. Ito and M. Seki

1980; Panneton and Loewy, 1980; Ciriello, 1983; ture, postfixed with 4% paraformaldehyde in the Rogers and Hermann, 1983; Seiders and Stuesse, phosphate buffer overnight, and then stored in 20% 1984; Gwyn et aL, 1985; Shapiro and Miselis, 1985b; sucrose solution for 2 days. Frozen frontal or Sweazey and Bradley, 1986; Housley et al., 1987; sagittal sections were cut serially at a thickness of Norgren and Smith, 1988; Altschuler et al. , 1989, 40 gm with a freezing microtome, and every third 1991). Based on neurological studies showing that section was collected and treated for immunohisto- the NTS controls the gastrointestinal tract via the chemical PHA-L reaction. The procedures for dorsal motor nucleus of the vagus (DMV) (Morest, PHA-L were carried out according to Gerfen and 1967; Cottle and Calaresu, 1975; Norgren, 1978; Sawchenko (1984). In brief, the sections were Beckstead et aL, 1980; Rogers et aL, 1980; Arends treated with 1% albumin solution for 1 hour, and et al. , 1988), and controls the pharynx, larynx, and then incubated in rabbit anti-PHA-L serum (dilu- esophagus via the nucleus ambiguus (Amb) tion 1 : 2000, EY Lab.) for 2-3 days. The primary (Morest, 1967; Cottle and Calaresu, 1975; Loewy antibodies were localized by incubation in biotiny- and Burton, 1978; Norgren, 1978; Ricardo and Koh, lated goat anti-rabbit IgG serum (1 : 5000, Vector) 1978; Beckstead et al., 1980; ter Horst et aL, 1984; overnight, and then in an ABC kit (Vector) over- Ross et al., 1985; Arends et aL, 1988; Cunningham night at 4 °C. Between each incubation, the sections and Sawchenko, 1989; Cunningham et al., 1991), were rinsed thoroughly with a Tris-buffer solution the NTS appears to play an important role as the containing Triton-X100 at pH 7.4. After incubation, afferent system of all visceromotor reflexes, includ- peroxidase was visualized by the cobalt-glucose oxi- ing the emetic response. Nevertheless, to define the dase method with diaminobenzidine (DAB) (Itoh neural pathways involved in the vomiting reflex, et aL, 1979). After mounting on gelatin-coated further comparative studies are necessary, espe- slides, the sections were dehydrated in alcohol, cially in species having vomiting peculiarities. cleared in xylene, coverslipped, and observed The purpose of the present study was to examine under a light microscope. In order to clarify the in detail the ascending pathways and projection normal cytoarchitecture, adjacent sections on one targets of the AP as well as those of the viscero- side were stained with thionin. sensory region of the NTS of the suncus by using In order to investigate the relationship between the anterograde tracing method with Phaseolus anterograde labelings in the specific catechol- vulgaris leucoagglutinin (PHA-L). aminergic and peptidergic neurons, adjacent sections on the opposite side were immunostained by the peroxidase-antiperoxidase (PAP) method Materials and Methods (Sternberger, 1979) using mouse anti-tyrosine hydroxylase (TH) (Chemicon, dilution 1 : 1000), The experiments were carried out on 68 adult rabbit anti-phenylethanolamine-N-methyltrans- suncus of both sexes. The males weighed 50-90 g, ferase (PNMT) (Chemicon, dilution 1 : 1000), and the females weighed 35-55 g; 57 animals were rabbit anti-arginine vasopressin (AVP) (Chemicon, used for the PHA-L anterograde tracing study and dilution 1 : 2000) and rabbit anti-oxytocin (OT) 11 for the fluoro-gold (FG) retrograde tracing study. (Funakoshi, dilution 1 : 500) sera. Each section was All surgical procedures were carried out under so- incubated with the respective primary antiserum dium pentobarbital anesthesia (40-50 mg/kg, i.p.). for 2 days at 4 °C, and subsequently treated with a The cytoarchitectonic identifications and the brain secondary antibody (goat anti-mouse IgG; Cappel, nomenculture are according to the descriptions of dilution 1 : 100 or anti-rabbit IgG; Cappel, dilution Paxinos and Watson (1986) in the rat. 1 : 100) and a final third layer (mouse PAP; Dako, In the anterograde tracing experiments, injec- dilution 1 : 100 or rabbit PAP; Dako, dilution tions of 2.5% PHA-L (Hohnen Inc., in 0.1 M Tris- 1 : 400) overnight at 4 °C. The immunoreaction was buffer solution containing 0.3 M KC1 at pH 8.2) visualized by DAB reaction. were made into either the AP or the NTS. The To confirm the results of the anterograde tracing tracer was injected iontophoretically with a current study by retrograde transport, 0.02-0.04 gl of of 10-20 gA applied in an alternating mode (5 s 1% FG (Fluorochrome Inc.) was injected into the on/5 s off). Seven to ten days after the injection, the parabrachial nucleus (Pb) under pressure with a 1 animals were perfused with 50 ml saline, followed gl Hamilton syringe. Two days after the microinjec- by 100 ml of a solution containing 4% paraformal- tion, the animals were anesthetized and perfused dehyde and 0.1% glutaraldehyde in 0.1 M phos- transcardially with 50 ml saline, followed by 300 ml phate buffer at pH 7.2, and then with 250 ml of 4% of a solution containing 4% paraformaldehyde in paraformaldehyde. The brains were immediately phosphate buffer. The brains were removed and removed and embedded in an agarose-gelatin mix- embedded in an agarose-gelatin mixture, postfixed Area Postrema and Solitary Nucleus Projections in the Suncus 11 with the same fixative overnight, then stored in extent of the injection site in animal No. 47, in- 20% sucrose solution for 2 days. Serial frontal fro- cluding the entire DVC with slight overlap into zen sections were cut at a thickness of 40 pm with regions immediately lateral and ventral to it (i.e., a freezing microtome, mounted on slides, and air- the gracile and the hypoglossal (XII) nuclei, respec- dried at room temperature. The preparations were tively) but without any overlap into the AP. observed under a fluorescent microscope equipped In the DVC itself, PHA-L labeled fibers were with a UV filter system (Olympus, New Vanox). observed in the entire NTS ipsilateral to the injection (Fig. 3C, D). In addition, numerous fibers crossed the midline through the commissural sub- Results division and extended to the contralateral NTS region (Fig. 3A). In the AP, a small number of Projectionsfrom the AP fibers were observed immediately medial to the Thirty-seven animals were used for PHA-L in- injection site (Fig. 3A, B). jection into the AP. In the representative animal The majority of labeled fibers leaving the DVC No. 12, the injection deposit was centered in the coursed ventrolaterally, forming dense terminals in AP and spread completely throughout the nucleus. the Amb, the caudoventrolateral (CVL), and ros- However, no diffusion was observed in the nearby toventrolateral (RVL) reticular nuclei (Figs. 3B—D regions of the dorsal vagal complex (DVC) (Fig. and 4A). In a supplementary immunohistochemical 1A). Although Fig. 1 shows only one side of the study using catecholaminergic markers, TH- and brainstem, the labelings were generally observed PNMT-immunoreactive neurons could be found in bilaterally. areas of the CVL and RVL, respectively (Fig. 4B). Labeled fibers left the AP in the direction of In a more rostra! region of the ventrolateral the adjacent DVC, where the majority terminated pons, the labeled fibers coursed through the lateral immediately (Figs. 1B and 2A). Caudal to the AP, region of the facial nucleus, where some terminal terminal labeling was observed in the medial and labeling was also seen in the region surrounding the commissural subdivisions of the NTS. Slightly ros- facial nerve roots (Fig. 3E—G). tral to the AP, abundant labeling was observed in At the level of the rostral pole of the principal the medial and periventricular subdivisions of the trigeminal nucleus, labeled fibers turned sharply NTS and the DMV (Figs. 1C and 2B), but labeling dorsally along the lateral lemniscus toward the was rare in the lateral subdivisions around the soli- dorsolateral pontine tegmentum (Fig. 3G, H). A tary tract. There was no labelling in the more ros- moderate number of labeled fibers and terminal tral regions of the NTS (Fig. 1D). A portion of boutons were also observed in the locus coeruleus the labeled fibers passing through the DVC then (LC), where numerous TH-immunoreactive neu- entered the ventrolateral medullary area (Fig. 1B, rons were detected (Figs. 3F, G and 4C, D). In the C), but no terminal labeling could be observed LPb, dense plexuses of labeled fibers and terminals in the ventrolateral reticular nucleus (VL) or the were observed in the external, central and dorsal Amb. The fibers then passed rostrally through the subdivisions (Figs. 3H and 4E, F). Heavy terminal pons immediately medial to the spinal trigeminal labeling was also seen in the KF (Fig. 3H). In the nucleus (Sp5) (Fig. 1D—G).Rostral to the superior MPb, fibers formed a dense terminal field sur- olivary nucleus, the labeled fibers turned dorsally rounding a limited area immediately ventromedial and ended in a pattern of dense terminal arboriza- to the superior cerebellar peduncle (scp), whereas tions in the central and external subdivisions of the only a few labeled fibers were seen in the remaining lateral parabrachial nucleus (LPb) (Figs. 1H and region of the MPb and scp (Figs. 3H and 4E). 2C). Few labeled fibers were seen in the medial Additionally, some labeled fibers and terminals parabrachial (MPb) and K011iker-Fuse(KF) nuclei. were found in the lateral region of the central gray In a more rostral portion of the Pb, no antero- matter (CG) at the rostra! end of the pons (Fig. gradely-labeled fibers arising from the AP were 3H). found (not illustrated in Fig. 1). Rostral to the pons, the ascending fibers divided into two primary pathways: the dorsal pathway Projections from the NTS and the ventral pathway. The fibers of the former Twenty animals were used for the PHA-L injec- coursed dorsomedially from the rostral end of the tion into the NTS. In the present study, we were LPb, and then entered the dorsal longitudinal able to confine the injections to almost entirely fasciculus (d1f) (Fig. 31). Many terminal boutons within the NTS and the DMV in eight animals, and were observed in the ventromedial region of the similar patterns of anterograde labeling were ob- CO at the caudal midbrain level, but few were de- tained in all of these animals. Figure 3A shows the tected at the middle or rostral levels (Fig. 3J, K). At 12 H. Ito and M. Seki

the level of the caudal diencephalon, the ascending lateral preoptic area. Further rostral to this, no fibers in the dlf again divided into two pathways: anterograde labeling was observed anywhere, ex- the secondary dorsal pathway and the secondary cept occasionally in the lateral septum, the diagonal ventral pathway (Fig. 3L—N).The fibers of the sec- band, and the nucleus accumbens (Fig. 3T). All of ondary dorsal pathway entered the medial region of the labelings in the present study were generally the dorsal thalamus, sending abundant collaterals observed bilaterally, with dominant distribution on to the thalamic paraventricular nucleus (PVT) (Fig. the ipsilateral side. 3N—P), whereas those of the secondary ventral pathway entered the hypothalamus (Fig. 3N). Retrograde tracing experiment In contrast, the less abundant fibers of the pri- The anterograde tracing studies suggested an mary ventral pathway coursed rostrally from the extensive projection system from both the AP and ventral region of the Pb to the ventral tegmental the NTS to the LPb. Retrogradely labeled neurons area (Fig. 31—M).This pathway was occasionally were consistently found throughout both regions observed in and around the medial lemniscus, but after a large injection of FG into the Pb (Fig. 8A, no terminal labeling was observed in any region B). Labeled cells were randomly distributed in the of the midbrain. In the diencephalon, this pathway AP, with a majority found ipsilateral to the injec- continued along the medial forebrain bundle (mfb) tion site (Fig. 8C). These cells were round or oval in (Fig. 3N). shape. In the NTS, a very large number of labeled The labeled fibers reaching the hypothalamus via cells was found ipsilaterally (Fig. 8C), whereas only the secondary ventral pathway and/or the primary a few were found contralaterally. These cells tended ventral pathway showed characteristic distribu- to be elliptical or polygonal in shape and to have tional patterns in their target nuclei compared with their long axis in a mediolateral orientation. those of the rat. The large number of labeled fibers and terminal boutons were observed in the dorsomedial hypothalamic nucleus (DMH) (Figs. 3N and Discussion 5), as well as in the lateral hypothalamic nucleus (LH) (Fig. 3N, 0). Fewer were observed in the In the present study, we examined the ascending paraventricular hypothalamic nucleus (PVH) pathways and the projection targets from the AP where immunohistochemistry revealed a smaller and the caudal portion of the NTS (Fig. 9), and number of AVP- and OT-immunoreactive neurons confirmed some reciprocal connections between the (Figs. 30 and 6). Such labeling was sometimes seen AP and the NTS at the level of the dorsal medulla. in the ventral narrow region of the periventricular The target of the AP projection beyond the level of hypothalamic nucleus (Figs. 3P and 6A). Few, but the dorsal medulla is the Pb. In contrast, the NTS distinctly labeled, fibers and terminals were also has a long projection system extending as far as the found in the retrochiasmatic area and in the supra- basal forebrain, the targets including the Amb and optic nucleus (Fig. 30—Q). Occasionally, a small VL in the medulla, the Pb and LC in the pons, the number of labeled fibers and terminals were seen in CG in the mesencephalon, the DMH, LH and PVT the arcuate nucleus (Fig. 3M, N). A small number in the diencephalon, and the BST, SI, VP and MPA of labeled fibers were seen in the anterior hypo- in the telencephalon. thalamic and suprachiasmatic nuclei, but no terminal labeling was identified. Projections from the AP In areas further rostral, fibers projecting along In all species examined to date, the AP has been the mfb gave off many collaterals to the substantia shown to have abundant reciprocal connections innominata (SI), but relatively few to the amygda- with the adjacent nuclei of the DVC. In the sixties, loid nucleus (Amy) (Fig. 30). The projection fibers Morest (1960) reported on the existence of a local to the basal forebrain structures were distributed connection between the AP and the adjacent NTS via the mfb and/or the secondary dorsal pathway. in a study using the Golgi staining technique. More The heaviest terminal labeling was found in the bed recently, as advances have been made in axonal nucleus of the stria terminalis (BST) immediately transport technology, a number of studies on the ventral to the lateral ventricle (Figs. 3R and 7A, B). connectivity of the AP have been carried out using Labeled fibers and terminals in this area extended rats as the model. Efferent fibers of the AP of the laterally to the ventral pallidum (VP) (Figs. 3R, S rat were found most heavily in the dorsal, medial, and 7A). It is notable that a considerable amount of and commissural subnuclei of the NTS (Vigier and terminal labeling was found in the medial preoptic Portalier, 1979; Vigier and Rouviere, 1979; van der area (MPA) (Figs. 3Q—S,7C), but that no more Kooy and Koda, 1983; Shapiro and Miselis, 1985a; than a few labeled fibers could be found in the Kachidian and Pickel, 1993; Cunningham et aL, Area Postrema and Solitary Nucleus Projections in the Suncus 13

1994). These subnuclei have been reported to re- the rat. According to their findings, the core of the ceive dense vagal inputs from the cardiovascular AP projected to the central, external, and dorsal and gastrointestinal organs (Shapiro and Miselis, subdivisions of the LPb. Our findings in the present 1985b; Norgren and Smith, 1988; Altschuler et al., study are entirely consistent with their results. In 1989, 1991;Miselis et al., 1989). In a recent study in the rat and golden hamster, the external subdivi- the ferret, the AP was found to have reciprocal sion of the LPb was reported to project extensively connections with the medial and gelatinous sub- to the basal forebrain region including to the Amy nuclei of the NTS (Strominger et aL, 1994), and the and the hypothalamus (Fulwiler and Saper, 1984; present study in the suncus revealed dense inputs Moga et aL, 1990;Halsell, 1992). Based on a physio- from the AP to the medial and commissural sub- logical study, electrical or chemical stimulation of divisions of the NTS, in agreement with the findings this region should produce profound pressor, tachy- in previous reports on the rat and ferret. As de- cardic, and tachypneic responses in the rat (Herbert scribed later in detail, the caudal portion of the et al., 1990). It seems likely that the AP-Pb pro- NTS has been reported to project to the DMV and jection system contributes to the transmission of the Amb in the rat, cat, and monkey (Morest, 1967; cardiovascular impulses, but the exact nature of this Cottle and Calaresu, 1975; Loewy and Burton, connection is not known. 1978; Norgren, 1978; Ricardo and Koh, 1978; In contrast to the findings in the present study in Beckstead et al., 1980; Rogers et al., 1980; ter Horst the suncus, King (1980) reported the absence of et al., 1984; Ross et al., 1985; Cunningham and any projection from the AP to the Pb in the cat. Sawchenko, 1989; Cunningham et al., 1991). Thus, Vigier and co-workers studied the AP projections there appears to be a connection for an autonomic in the rat in detail, but they did not describe any reflex arch conveying the visceral information of parabrachial connection (Vigier and Portalier, 1979; the AP as well as the NTS to the visceromotor Vigier and Rouviere, 1979). Strominger et al. preganglionic nuclei. The local pathway from the (1994) reported no ascending fibers arising from AP to the NTS may play a role in modifying the the AP in the ferret, except for a local connec- autonomic reflex arch with the chemo- and baro- tion to the NTS. The ferret, like the suncus, vomits receptive information. frequently, and thus the difference between the It was interesting that the local projection targets findings in this study and theirs intrigues us. As of the AP noted in the present study directly discussed above, there may be a species-related involved not only the nearby NTS neurons but difference in AP-Pb connectivity. Further studies the parasympathetic preganglionic neurons of the are needed to clarify the significance of the AP-Pb DMV. Few such projections have been reported connection. in the rat (Vigier and Portalier, 1979; Vigier and In addition to projections to the DVC and the Rouviere, 1979; van der Kooy and Koda, 1983; Pb, other targets of the AP have been proposed Cunningham et aL, 1994). Quoting a passage from in tracing studies on the rat. Vigier and Rouviere Cunningham et al. (1994), however, 'the possibility (1979) described AP efferent projections to the XII, of AP inputs to the distal dendrites of the DMV the mesencephalic trigeminal nucleus, the LC, and neurons could not be excluded.' The results of the the inferior and superior colliculi, and van der Kooy present study suggests that the AP-DMV projection and Koda (1983) reported on the existence of a few might form a bisynaptic modifying pathway through AP projections to the dorsal and dorsolateral teg- which chemo- and barosensory information could mental nuclei. Shapiro and Miselis (1985a) de- be transferred directly to the visceromotor system scribed the system of direct projections to the Amb, of the vagus. the Sp5, the paratrigeminal nucleus, the ventro- The ascending projection arising from the AP lateral medullary catecholaminergic region, the neurons of the suncus was shown to innervate the mesencephalic CG, and the cerebellar vermis. In LPb, especially in the areas of its external and cen- contrast to the findings in these reports, Cunning- tral subdivisions. In other species, the existence of ham et aL (1994) reported that their PHA-L tracing a connection between the AP and the Pb was first study did not reveal any labeled fibers or any ter- confirmed in the rat (Cedarbaum and Aghajanian, minal boutons in these regions; a finding consistent 1978) and cat (Loewy and Burton, 1978). It was with those of the present study. The reason for such suggested later that the major neural target of the a discrepancy is unclear, but it may be due to the AP might be a specific region within the LPb, the different techniques used in each study. external subdivision (van der Kooy and Koda, 1983; Cunningham et aL, 1994). Herbert et al. (1990) used Projections from the NTS an anterogarde tracing technique to examine the In studies in some mammals and birds, the DMV h subnuclear organization of the AP-Pb projection in as been found to be a major target of projections 14 H. Ito and M. Seki

from the NTS (Morest, 1967; Cottle and Calaresu, generally been regarded as a relay of visceral and 1975; Norgren, 1978; Beckstead et aL, 1980; Rogers gustatory afferents to higher levels (Norgren and et aL, 1980;Arends et al. , 1988). Rogers et aL (1980) Leonard, 1973; Sakai et al., 1977; Norgren, 1978; suggested that this pathway was responsible for Ricardo and Koh, 1978; Beckstead et al., 1980; the elaboration of a variety of the so-called "vago- King, 1980; Herbert et al., 1990). Physiological vagal" gastrointestinal reflexes. Due to certain studies have suggested that taste-responsive neu- technical limitations, we could not confirm whether rons are primarily located in the MPb and that vis- such projections exist in the suncus. We assume, cerosensory-responsive neurons are located in the however, that they exist in this species, the same as LPb (Norgren and Pfaffmann, 1975; Hermann and in other species, because of the necessity of a direct Rogers, 1985; Ward, 1989; Halsell and Frank, autonomic reflex arch via the NTS as well as the AP 1991). Furthermore, anatomical studies on the as- in the suncus. cending NTS-Pb projection suggested that there Projections from the NTS to the Amb have fre- was a subnuclear organization in their connection quently been reported (Morest, 1967; Cottle and (Norgren, 1978;Ricardo and Koh, 1978;Herbert et Calaresu, 1975; Loewy and Burton, 1978;Norgren, al., 1990). According to Herbert et al. (1990), the 1978; Ricardo and Koh, 1978; Beckstead et al., medial NTS, which they refer to as "the general 1980;ter Horst et aL, 1984;Ross et aL, 1985; Arends visceral part", projects to restricted terminal re- et al., 1988; Cunningham and Sawchenko, 1989; gions in the external, central and dorsal subdivi- Cunningham et al., 1991). Lawn (1966a, b) first sions of the LPb and to the waist area of the scp, proposed that the Amb is the origin of the motor while the ventrolateral NTS, or "the respiratory nerves that innervate the special visceral striated area", projects to the KF and to the restricted areas musculature involved in breathing, swallowing, in the LPb and MPb. As described above, heavy and phonation. More recently, Bieger and Hopkins terminal labeling was observed in the suncus almost (1987) studied the cytoarchitecture and viscerotopic entirely within the LPb, a limited area of the MPb, organization of the Amb in the rat. They noted that and in the KF. The pattern of labeling observed in the Amb was composed of compact, semicompact, our present experiment could thus be considered loose and external formations that contained the as representing a combination of the patterns of esophagomotor, pharyngolaryngomotor, laryngo- medial NTS projection and ventrolateral NTS pro- motor, and preganglionic neurons innervating the jection reported by Herbert et aL (1990). Our re- heart and supradiaphragmatic structures, respec- sults may be attributable to the extent of the injec- tively. Given such a diversity of visceral innervation tion territory including the entire medial and the from the Amb, projections from the NTS to the ventrolateral regions of the NTS (see Fig. 3A). Amb most likely to mediate a variety of vagal vis- However, it seems certain that a few differences in ceral informations including vomiting, swallowing, organization of the NTS-Pb projection exist be- respiratory, and cardiac responses. However, no tween the suncus and the rat. For example, heavy such viscerotopic representation of the Amb has terminal labeling in the waist area of the scp was yet been shown in the suncus. seen in the rat, but not seen in the suncus. In contrast, Ross et aL (1985) suggested that ter- Direct projections from the NTS to the hypo- minals arising from the NTS were confined largely thalamus were described by Ricardo and Koh to areas outside the Amb itself. According to their (1978) and ter Horst et aL (1989) in the rat and study, the NTS innervates a region of the RVL by Arend et aL (1988) in the pigeon. In general, containing Cl adrenergic neurons and the contig- the hypothalamic targets of ascending fibers from uous region of the CVL containing Al noradre- the caudal NTS in the suncus are similar to those nergic neurons. In our investigation in the suncus, in the rat. However, some differences between the the distribution pattern of TH- and PNMT-im- two species in areas receiving the NTS inputs were munoreactive neurons in the ventrolateral medulla also seen. The most striking difference was in the was shown to be similar to that of the rat. The RVL PVH, where ter Horst et al. (1989) observed a and the CVL may therefore contain adrenergic and dense network of efferent fibers from the NTS, noradrenergic neurons, respectively. Therefore, the whereas in the present study we observed only a NTS-medullary projection may form an anatomical few labeled fibers and terminals. This may be due substrate for the catecholaminergic system respon- to the PVH of the suncus being underdeveloped; sible for baroreceptive, chemoreceptive, and other for example, the small number of AVP- and OT- visceral information in the suncus as well as in the immunoreactive cells distributed in the dorsal re- rat. gion of the nucleus (Fig. 6B), which may corre- In the suncus, as in other mammals, the Pb is the spond to the magnocellular subnucleus of the PVH major target for projections from the NTS, and has in the rat. In general, the distributional pattern of Area Postrema and Solitary Nucleus Projections in the Suncus 15 the neuropeptide-containing cells in the PVH of the upper alimentary tract in the medulla oblongata in the the suncus is to some degree different from that of rat: The nucleus ambiguus. J Comp Neurol 1987; 262:546- 562. other mammalian species. 6) Borison HL, Borison R and McCarthy LE. Role of the area It was interesting that only a few labeled fibers postrema in vomiting and related functions. Federation could be found in the Amy. In the rat, a strong Proc 1984; 43:2955-2958. amygdaloid projection from the NTS has been 7) Carpenter DO. Central nervous system mechanisms in demonstrated in studies using an anterograde deglutition and emesis. In: Handbook of physiology, sec- transport technique (Ricardo and Koh, 1978; ter tion 6, The gastrointestinal system (Schultz SG ed.), vol. I (Wood JD ed.), chapter 18, 685-714, American Physio- Horst et al. , 1989) and a retrograde transport tech- logical Society, Bethesda, 1989. nique (Ottersen, 1981; Zardetto-Smith and Gray, 8) Cedarbaum JM and Aghajanian GK. Afferent projections 1990; Petrov et aL , 1993). In the rabbit, Kapp et al. to the rat locus coeruleus as determined by a retrograde tracing technique. J Comp Neurol 1978; 178:1-16. (1989) confirmed that the NTS projects to the central subdivision of the Amy both directly and in- 9) Ciriello J. Brainstem projections of aortic baroreceptor afferent fibers in the rat. Neurosci Lett 1983; 36:37-42. directly, primarily by way of the LPb. They sug- 10) Cottle MKW and Calaresu FR. Projections from the nu- gested that these projections form an anatomical cleus and tractus solitarius in the cat. J Comp Neurol 1975; substrate by which visceral afferent impulses may 161:143-158. influence the limbic forebrain. In contrast to the 11) Cunningham ET Jr and Sawchenko PE. A circumscribed results reported in the rat and rabbit, the afferent projection from the nucleus of the solitary tract to the nucleus ambiguus in the rat: Anatomical evidence for connection leading from the NTS to the Amy in somatostatin-28-immunoreactive interneurons subserving the cat was found to be weak (Volz et al., 1990) or reflex control of esophageal motility. J Neurosci 1989; completely absent (Ottersen, 1981; Russchen, 1982). 9:1668-1682. These findings in the cat agree with those obtained 12) Cunningham ET Jr, SimmonsDM, Swanson LW and Saw- in our present study in the suncus. In conclusion, a chenko PE. Enkephalin immunoreactivity and messenger RNA in a discrete projection from the nucleus of the soli- species-related distinction certainly exists in regard tary tract to the nucleus ambiguus in the rat. J Comp to the direct NTS-Amy projection. The reason for Neurol 1991; 307:1-16. such an underdeveloped projection in the cat and 13) Cunningham ET Jr, Miselis RR and Sawchenko PE. The suncus is unclear. Further detailed studies including relationship of efferent projections from the area postrema the possibility of an indirect connection by way of to vagal motor and brain stem catecholamine-containing cell groups: An axonal transport and immunohistochemical the Pb are needed. study in the rat. Neurosci 1994; 58:635-648. 14) Fulwiler CE and Saper CB. Subnuclear organization of the efferent connections of the parabrachial nucleus in the rat. Acknowledgements Brain Res Rev 1984; 7:229-259. 15) Gerfen CR and Sawchenko PE. An anterograde neuro- anatomical tracing method that shows the detailed mor- We thank Dr. K. Doi for his helpful comments phology of neurons, their axons and terminals: Immuno- and suggestions in regard to this manuscript. We histochemical localization of an axonally transported plant are also grateful to Mrs. M. Hatta and Mr. N. Oka- lectin, phaseolus vulgaris leucoagglutinin (PHA-L). Brain mura for their secretarial assistance. Res 1984; 290:219-238. 16) Gwyn DG, Leslie RA and Hopkins DA. Observations on the afferent and efferent organization of the vagus nerve and the innervation of the stomach in the squirrel monkey. References J Comp Neurol 1985;239:163-175. 17) Halsell CB. Organization of parabrachial nucleus efferents 1) AltschulerSM, BaoX, BiegerD, HopkinsDA and Miselis to the thalamus and amygdala in the golden hamster. J RR. Viscerotopicrepresentation of the upper alimentary Comp Neurol 1992;317:57-78. tract in the rat: Sensoryganglia and nucleiof the solitary 18) Halsell CB and Frank ME. Mapping study of the para- and spinaltrigeminal tracts. J CompNeurol 1989; 283:248- brachial taste-responsive area for the anterior tongue in the 268. golden hamster. J Comp Neurol 1991;306:708-722. 2) AltschulerSM, Ferenci DA, Lynn RB and MiselisRR. 19) Hamilton RB and Norgren R. Central projections of gus- Representationof the cecumin the lateral dorsal motor tatory nerves in the rat. J Comp Neurol 1984;222:560-577. nucleusof the vagusnerve and commissuralsubnucleus of 20) Herbert H, Moga MM and Saper CB. Connections of the the nucleustractus solitariiin rat. J Comp Neurol 1991; parabrachial nucleus with the nucleus of the solitary tract 304:261-274. and the medullary reticular formation in the rat. J Comp 3) Arends JJA, Wild JM and Zeigler HP. Projectionsof Neurol 1990;293:540-580. the nucleusof the tractussolitarius in the pigeon(Columba 21) Hermann GE and Rogers RC. Convergence of vagal and livia).J CompNeurol 1988; 278:405-429. gustatory afferent input within the parabrachial nucleus of 4) BecksteadRM, Morse JR and NorgrenR. The nucleusof the rat. J Autonom Nervous Syst 1985; 13:1-17. the solitarytract in the monkey:Projections to the thala- 22) Housley GD, Martin-Body RL, Dawson NJ and Sinclair mus and brain stemnuclei. J CompNeurol 1980; 190:259- JD. Brain stem projections of the glossopharyngeal nerve 282. and its carotid sinus branch in the rat. Neurosci 1987; 5) BiegerD and HopkinsDA. Viscerotopicrepresentation of 22:237-250. 16 H. Ito and M. Seki

23) Itoh K, Konishi A, Nomura S, Mizuno N, Nakamura Y and 43) Ricardo JA and Koh ET. Anatomical evidence of direct Sugimoto T. Application of coupled oxidation reaction to projections from the nucleus of the solitary tract to the electron microscopic demonstration of horseradish per- hypothalamus, amygdala, and other forebrain structures in oxidase: cobalt-glucose oxidase method. Brain Res 1979; the rat. Brain Res 1978; 153:1-26. 175:341-346. 44) Rogers RC and Hermann GE. Central connections of the 24) Kachidian P and Pickel VM. Localization of tyrosine hepatic branch of the vagus nerve: A horseradish peroxi- hydroxylase in neuronal targets and efferents of the area dase histochemical study. J Autonom Nervous Syst 1983; postrema in the nucleus tractus solitarii of the rat. J Comp 7:165-174. Neurol 1993;329:337-353. 45) Rogers RC, Kita H, Butcher LL and Novin D. Afferent 25) Kalia M and Mesulam MM. Brain stem projections of sen- projections to the dorsal motor nucleus of the vagus. Brain sory and motor components of the vagus complex in the Res Bull 1980;5:365-373. cat: II. Laryngeal, tracheobronchial, pulmonary, cardiac, 46) Ross CA, Ruggiero DA and Reis DJ. Projections from the and gastrointestinal branches. J Comp Neurol 1980; nucleus tractus solitarii to the rostral ventrolateral medulla. 193:467-508. J Comp Neural 1985; 242:511-534. 26) Kapp BS, Markgraf CG, Schwaber JS and Bilyk-Spafford 47) Russchen FT. Amygdalopetal projections in the cat. II. T. The organization of dorsal medullary projections to the Subcortical afferent connections. A study with retrograde central amygdaloid nucleus and parabrachial nuclei in the tracing techniques. J Comp Neural 1982;207:157-176. rabbit. Neurosci 1989;30:717-732. 48) Sakai K, Touret M, Salvert D, Leger L and Jouvet M. 27) King GW. Topology of ascending brainstem projections Afferent projections to the cat locus coeruleus as visualized to nucleus parabrachialis in the cat. J Comp Neurol 1980; by the horseradish peroxidase technique. Brain Res 1977; 191:615-638. 119:21-41. 28) Lawn AM. The localization, in the nucleus ambiguus of the 49) Seiders EP and Stuesse SL. A horseradish peroxidase in- rabbit, of the cells of origin of motor nerve fibers in the vestigation of carotid sinus nerve components in the rat. glossopharyngeal nerve and various branches of the vagus Neurosci Lett 1984; 46:13-18. nerve by means of retrograde degeneration. J Comp Neu- 50) Shapiro RE and Miselis RR. The central neural connec- ral 1966a; 127:293-306. tions of the area postrema of the rat. J Comp Neurol 1985a; 29) Lawn AM. The nucleus ambiguus in the rabbit. J Comp 234:344-364. Neurol 1966b; 127:307-320. 51) Shapiro RE and Miselis RR. The central organization of 30) Loewy AD and Burton H. Nuclei of the solitary tract: the vagus nerve innervating the stomach of the rat. J Comp Efferent projections to the lower brain stem and spinal cord Neurol 1985b;238:473-488. of the cat. J Comp Neurol 1978; 181:421-450. 52) Sternberger LA. Immunocytochemistry. 2nd ed. John Wiley 31) Miselis RR, Rogers WT, Schwaber JS and Spyer KM. & Sons, New York, 1979. Localization of cardiomotor neurons in the anaesthetized 53) Strominger NL, Knox AP and Carpenter DO. The connec- rat; cholera toxin HRP conjugate and pseudorabies label- tivity of the area postrema in the ferret. Brain Res Bull ling. J Physiol, Lond. 1989;416:63P. 1994;33:33-47. 32) Moga MM, Herbert H, Hurley KM, Yasui Y, Gray TS and 54) Sweazey RD and Bradley RM. Central connections of the Saper CB. Organization of cortical, basal forebrain, and lingual-tonsillarbranch of the glossopharyngeal nerve and hypothalamic afferents to the parabrachial nucleus in the the superior laryngeal nerve in lamb. J Comp Neurol 1986; rat. J Comp Neurol 1990; 295:624-661. 245:471-482. 33) Morest DK. A study of the structure of the area postrema 55) ter Horst GJ, De Boer P, Luiten PGM and Van Willigen with Golgi methods. Am J Anat 1960; 107:291-303. M. Ascending projections from the solitary tract nucleus to 34) Morest DK. Experimental study of the projections of the the hypothalamus. A phaseolus vulgaris lectin tracing study nucleus of the tractus solitarius and the area postrema in in the rat. Neurosci 1989;31:785-797. the cat. J Comp Neural 1967; 130:277-300. 56) ter Horst GJ, Luiten PGM and Kuipers F. Descending 35) Norgren R. Projections from the nucleus of the solitary pathways from hypothalamus to dorsal motor vagus and tract in the rat. Neurosci 1978;3:207-218. ambiguus nuclei in the rat. J Autonom Nervous Syst 1984; 36) Norgren R and Leonard CM. Ascending central gustatory 11:59-75. pathways. J Comp Neural 1973; 150:217-238. 57) Ueno S, Matsuki N and Saito H. Suncus murinus: A new 37) Norgren R and Pfaffmann C. The pontine taste area in the experimental model in emesis research. Life Sci 1987; rat. Brain Res 1975; 91:99-117. 41:513-518. 38) Norgren R and Smith GP. Central distribution of sub- 58) Ueno S, Matsuki N and Saito H. Suncus murinus as a new diaphragmatic vagal branches in the rat. J Comp Neurol experimental model for motion sickness. Life Sci 1988; 1988;273:207-223. 43:413-420. 39) Ottersen OP. Afferent connections to the amygdaloid 59) van der Kooy D and Koda LY. Organization of the pro- complex of the rat with some observations in the cat. III. jections of a circumventricularorgan: The area postrema in Afferents from the lower brain stem. J Comp Neurol 1981; the rat. J Comp Neural 1983;219:328-338. 202:335-356. 60) Vigier D and Portalier P. Efferent projections of the area 40) Panneton WM and Loewy AD. Projections of the carotid postrema demonstrated by autoradiography. Arch Ital Biol sinus nerve to the nucleus of the solitary tract in the cat. 1979; 117:308-324. Brain Res 1980; 191:239-244. 61) Vigier D and Rouviere A. Afferent and efferent connec- 41) Paxinos G and Watson C. The Rat Brain in Stereotaxic tions of the area postrema demonstrated by the horseradish Coordinates. 2nd ed. Academic Press, Sydney, 1986. peroxidase method. Arch Ital Biol 1979; 117:325-339. 42) Petrov T, Krukoff TL and Jhamandas JH. Branching 62) Volz HP, Rehbein G, Triepel J, Knuepfer MM, Stumpf H projections of catecholaminergic brainstem neurons to the and Stock G. Afferent connections of the nucleus centralis paraventricular hypothalamic nucleus and the central nu- amygdalae. A horseradish peroxidase study and literature cleus of the amygdala in the rat. Brain Res 1993; 609:81- survey. Anat Embryol 1990; 181:177-194. 92. 63) Ward DG. Neurons in the parabrachial nuclei respond to Area Postrema and Solitary Nucleus Projections in the Suncus 17

hemorrhage. Brain Res 1989; 491:80-92. 65) Zardetto-Smith AM and Gray TS. Organization of pepti- 64) Whitehead MC and Frank ME. Anatomy of the gustatory dergic and catecholaminergic efferents from the nucleus of system in the hamster: Central projections of the chorda the solitary tract to the rat amygdala. Brain Res Bull 1990; tympani and the lingual nerve. J Comp Neurol 1983; 25:875-887. 220:378-395.

Abbreviations

4V 4th ventricle Mo5 motor trigeminal nucleus 7n facial nerve, root MPA medial preoptic area A5 AS noradrenaline cells MPb medial parabrachial nucleus ac anterior commissure MVe medial vestibular nucleus Acb accumbens nucleus NTS nucleus solitary tract Amb ambiguus nucleus opt optic tract Amy amygdaloid nucleus ox optic chiasm AP area postrema Pb parabrachial nucleus Arc arcuate hypothalamic nucleus pc posterior commissure BST bed nucleus of the stria terminalis PDTg posterodorsal tegmental nucleus CG central gray Pe periventricular hypothalamic nucleus cp cerebral peduncle Pn pontine nuclei CPu caudate putamen Pr5 principal sensory trigeminal nucleus Cu cuneate nucleus PVH paraventricular hypothalamic nucleus CVL caudoventrolateral reticular nuclei PVT paraventricular thalamic nucleus DB diagonal band RCh retrochiasmatic area DMH dorsomedial hypothalamic nucleui Red red nucleus DMV dorsal motor nucleus vagus RRF retrorubral field f fornix RVL rostroventrolateral reticular nucleus Fl flocculus s5 trigeminal nerve, sensory root fr fasciculus retroflexus SC superior colliculus Gr gracile nucleus scp superior cerebellar peduncle ic internal capsule SI substantia innominata IC inferior colliculus sm stria medullaris thalami IP interpeduncular nucleus SN substantia nigra KF K011iker-Fuse nucleus SO supraoptic nucleus LC locus coeruleus Sp5 spinal trigeminal nucleus lfp longitudinal fasciculus pons SpVe spinal vestibular nucleus LH lateral hypothalamic area st stria terminalis LL lateral lemniscus nucleus SuO superior olive LPA lateral preoptic area SuVe superior vestibular nucleus LPb lateral parabrachial nucleus tz trapezoid body LS lateral septal nucleus VC ventral cochlear nucleus LV lateral ventricle VII facial nucleus m5 trigeminal nerve, motor root VL ventrolateral reticular nucleus Mam mammillary nucleus VP ventral pallidum ml medial lemniscus XII hypoglossal nucleus 18 H. Ito and M. Seki

Explanation of Figures

Plate I

Fig. 1. A. Photomicrograph of the PHA-L injection site in the AP in animal No. 12. Scale bar is 200 gm. B-H. Schematic repre- sentations of the distribution of anterogradely PHA-L labeled fibers (dashed lines) and terminal boutons (dots) after injection into the AP in the same animal. Although these drawings show only one side of the brainstem, labeling was generally observed bilaterally. Area Postrema and Solitary Nucleus Projections in the Suncus 19

Plate I 20 H. Ito and M. Seki

Plate II

Fig. 2. Photomicrographs of PHA-L labeled fibers and terminal boutons after injection into the AP. A. Labeled fibers in the NTS and DMV. B-C. High-power photomicrographs of labeled fibers and terminal boutons in the NTS and the LPb, respectively. Scale bars are 100 tun in A, and 20 gm in B and C. Area Postrema and Solitary Nucleus Projections in the Suncus 21 Plate Il 22 H. Ito and M. Seki

Plate III and IV

Fig. 3. A. Photomicrograph of the PHA-L injection site in the NTS in animal No. 47. Scale bar is 200 gm. B-T. Schematic repre- sentations of the distribution of anterogradely PHA-L labeled fibers (dashed lines) and terminal boutons (dots) after injection into the NTS in the same animal. Although these drawings show the fibers seen on the same side as the injection, labeling was generally observed bilaterally. Area Postrema and Solitary Nucleus Projections in the Suncus 23 Plate III 24 H. Ito and M. Seki

Plate IV Area Postrema and Solitary Nucleus Projections in the Suncus 25 Plate V

Plate V

Fig. 4. A. Photomicrograph of PHA-L labeled fibers and terminal boutons in the ventrolateral medulla after injection into the NTS. Extremely dense labeling is seen in the Amb, and extensive labeling in the RVL. B. Photomicrograph of PNMT-immunoreactive neurons in the RVL in the serial section rostral to the section shown in A (arrows). Note the absence of immunoreactivity in the Amb. C. Photomicrograph of PHA-L labeled fibers and terminal boutons in the LC after injection into the NTS. D. Photomicrograph of TH-immunoreactive neurons in the LC in the serial section rostral to that in C. E. Low-power photomicrograph of PHA-L labeled fibers and terminal boutons in the Pb after injection into the NTS. Note the dense labeling seen in the LPb. A few labeled fibers are seen in the MPb (arrow). F. High-power photomicrograph of PHA-L labeled fibers and terminal boutons in the LPb after injection into the NTS. Scale bars are 50 inn in A, B, C and D, 100 pm in E, and 20 pm in F. 26 H. Ito and M. Seki

Plate VI

Fig. 5. High-power photomicrograph of PI-IA-L labeled fibers and terminal boutons in the DMH after injection into the NTS. Scale bar is 20 pm.

Fig. 6. A. Photomicrograph of PHA-L labeled fibers and terminal boutons in the PVH and Pe after injection into the NTS. B. Photomicrograph of AVP-immunoreactive neurons in the PVH and Pe in the serial section rostral to the section in A. Most of immunoreactive neurons are seen in the dorsal region of the PVH (arrows), where few labeled fibers are observed in A. Scale bars are 50 pm. Area Postrema and Solitary Nucleus Projections in the Suncus 27 Plate VI

Fig. 5

Fig. 6 28 H. Ito and M. Seki

Plate VII

Fig. 7. Photomicrographs of PHA-L labeled fibers and terminal boutons after injection into the NTS. A. Labeled fibers and terminal boutons in the BST and VP. B-C, High-power photomicrographs of labeled fibers and terminal boutons in the BST and the MPA, respectively. Scale bars are 100 gm in A, and 20 gm in B and C. Area Postrema and Solitary Nucleus Projections in the Suncus 29 Plate VII 30 H. Ito and M. Seki

Plate VIII

Fig. 8. A. Fluorescent photomicrograph of the large injection site of FG in the dorsolateral pontine tegmentum including the LPb. B. Bright-field photomicrograph of the thionin-stained control section adjacent to the section in A. C. Fluorescent photomicrograph of FG labeled neurons in the AP and NTS. Scale bars are 500 pm in A and B, and 100 pm in C. Area Postrema and Solitary Nucleus Projections in the Suncus 31 Plate IX

Plate IX,

Fig. 9. Summary of ascending pathways from the AP (broken line) and the NTS (solid line) in the brain of the suncus.