Connections of the parabrachial nucleus with the nucleus of the solitary tract and the medullary reticular formation in the rat.

THE .JOURNAL OF COMPARATIVE NEIJROLOGY 293~540-580 (1990)

Connections of the Parabrachial Nucleus With the Nucleus of the Solitary Tract and the Medullary Reticular Formation in the Rat HORST HERBERT,MARGARET M. MOGA, AND CLIFFORD B. SAPER Departments of Pharmacological and Physiological Sciences and Neurology, Committee on Neurobiology, and the Brain Research Institute, University of Chicago, Chicago, Illinois 60637

A4BSTRACT We examined the subnuclear organization of projections to the parabrachid nucleus (PB) from the nucleus of the solitary tract (NTS), area pastrema, and medullary reticular formation in the rat by using the anterograde and retrograde transport, of wheat germ agglutinin-horseradish peroxidase conjugate and anterograde tracing with Phascolus uulgaris-leiicosgglutinin. Different functional regions of the NTS/area postrema complex and medullary reticular formation were found to innervate largely nonoverlapping zones in the PR. The general uisceral part of the N T S , including the medial, parvicellular, intermediate, and commissural NTS subnuclei and the core of the area postrema, projects t o restricted terminal zones in the inner portion of the external lateral PB, the central and dorsal lateral PR suhnuclei, and the “waist” area. The dorsomedial NTS subnucleus and the rim of the area postrema specifically innervate the outer portion of the external lateral P B subnucleus. In addition, the medial N‘I’S innervates the caudal lateral part of the external medial PB subnucleus. The respiratory part of the N T S , comprising the ventrolat.era1, intermediate, and caudal commissural subnuclei, is reciprocally connected with the Kolliker-Fuse nucleus, and with the far lateral parts of the dorsal and central lateral PR subnuclei. There is also a patchy projection to the caudal lateral part of the external medial P B subnucleus from the ventrolateral NTS. The rostral, gustatory part of the N T S projects mainly to the caudal medial parts of the P B complex, including the “waist” area, as well as more rostrally to parts of the medial, external medial, ventral, and central lateral PB subnuclei. The connections of different portions of the medullary reticular formation with the PB complex reflect the same patterns of organization, but are reciprocal. The periamhiguus region is reciprocally connected with the same PR subnuclei as the ventrolateral NTS; the rostral uentrolaterat reticular nucleus with t,he same P B suhnuclei as both the ventrolateral (respiratory) and medial (general visceral) NTS; and the p a r i k l l u l a r relicular area, adjacent to the rostral NTS, with parts of the central and ventral lateral and the medial P B subnuclei that also receive rostral (gustatory) NTS input. In addition, the rostral ventrolateral reticular nucleus and Ihe parvicellular reticular

Accepted Oct.ober4,1989. Address reprint requests to Dr. C.B. Saper, Dept. of Pharm. B Physiol. Sci., IJniversity of Chicago. 947 E.58th St., Chicago, 11.60637. Dr. Horst Herbert is now at the Dept. of Animal Physiology, University of Tubingen, Morgenstelle 28, D-7400 Tubingen, FRG.

0 1990 WILEY-LISS, INC.

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MEDULLO-PARABRACHIAL CONNECTIONS formation have more extensive connections with parts of the rostral P B and the subjacent reticular formation that receive little if any NTS input. The P B contains a series of topographically complex terminal domains reflecting the functional organization of its afferent sources in the NTS and medullary reticular formation. Key words: area postrema, nucleus ambiguus, cardiovascular, respiratory, gustatory, gastrointestinal

The parabrachial nucleus (PB) is the main relay for ascending visceral sensory information from the nucleus of the solitary tract (NTS) to the forebrain (Norgren, '76, '78; Loewy and Burton, '78; Ricardo and Koh, '78; Saper and Loewy, '80; Fulwiler and Saper, '84; Cechetto and Calaresu, '85). Early studies on the ascendiilg projections from the NTS suggested that, the rostral, gustatory part of the NTS primarily innervates the medial PB, whereas the caudal medial NTS, including the cardiovascular relay area, projects to the lateral P B (Ricardo and Koh, '78; Norgren, '78). However, the exact topographic relationships of the two projections have never been clarified. More recently, it has become clear that both the N T S and the P B can be further subdivided into subnuclei with distinct connections and physiological roles. In the NTS, t,he rostral subnucleus, which receives gustatory and oral sensory afferents (Whitehead and Frank, '83; Hamilton and Norgren. '84; Travers et al., '87; Whitehead and Altschuler et al.. '89) has been distinguished from the intermediate and ventrolateral subnuolei, which are dominated by respiratory and upper airway afferents (Kalia and Mesulam, 'Sob; Kalia and Richter, '85, '88; Altschuler e t al., '89). The caudal medial and commissural subnuclei, which receive cardiovascular afferents (Panneton and Loewy, '80; Kalia and Mesulam. '80a; Ciriello et al., '81; Davies and Kalia, '81: Ciriello, '83; Seiders and Stuesse, '84; Housely et al., '87),have been differentiated from the rostral part of the medial NTS, the site of termination of gastrointestinal afferents (Leslie et al., '82; Rogers and Hermann, '83; Shapiro and Miselis, '85b; Norgren and Smith, '88; Altschuler e t al., '89). Finally, the area postrema has traditionally been considered a neurohema1 chemoreceptor zone for control of the gastrointestinal and cardiovascular systems (Borison and Wang, '53; Hyde and Miselis, '83; Gatti e t al., '85; Shapiro and Miselis, '85a,b; Carpenter and Rriggs, '86; Edwards and Ritter, '86; Fink et al., '87;Skoog and Mangiapane, '88). However, the area postrema also receives some vagal afferents (Kalia and M e s u ~ lam, '80a; Ciriello et al., '81; Contreras et al., '82; Kalia and Sullivan, '82) and is sometimes classified with the NTS complex. The P B contains a t least 10 cytoarchitectonically distinct subnuclei, each of which has a different pattern of forebrain projection (Fulwiler and Saper, -84; Lind and Swanson, '84; Cechetto and Saper, '87). Recent studies of afferents to the PR from the spinal cord (Cechetto e t al., '85),the A P (Shapiro and Miselis, %a), and the forebrain (Moga e t al., '90) demonstrate that afferents to the PB also respect these subnuclear boundaries. Comparably detailed information is lacking, however, on the projection to the PB from the NTS o r related areas of the medullary reticular formation. We therefore set out to examine systematically the subnuclear organization of the medullary afferent and efferent connections of the PB.

MATERIALS AND METHODS Experiments were performed in male Sprague-Dawley rats ranging in weight from 200 to 350 g. The animals were anesthetized with 7?C chloral hydrate (0.8 m1/100 g) and fixed in a stereotaxic device (David Kopf Instruments). For t.he PB and the rostral medullary injections, the skull was exposed and a hole was drilled in the parietal and interpaietal bones. The injections were guided by stereotaxic coordinates from Paxinos and Watson ('86). For NTS and caudal medullary injections, the dorsal surface of the lower medulla was exposed through the foramen magnum. For rostral N T S injections, part of the occipital bone was removed and the caudal cerebellum aspirated. Injections were then placed under direct visual guidance using dorsal medullary structures as landmarks. Retrograde and anterograde tracing experiments with wheat germ agglutinin-horseradish peroxidase conjugate (WGA-HRP). Three to six nl injections of a 1''i solution of WGA-HRP (Sigma) were made into the P B (n = 18) or medullary nuclei (n = 23) by using calibrated glass micropipettes and an air pressure injection system. Five additional animals each received two or three injections of approximately 6 nl each into the PB, t o label as many of its afferents as possible. After 30-60 hours survival, the animals were reanesthetized with 7 "0 chloral hydrat,e and perfused through the aorta with 0.9 9; saline followed by 500 ml of fixative containing 0.5% or 1", paraformaldehyde and 1.251:O glutaraldehyde in 0.1 M phosphate buffer pH 7.4 (PR), and followed by 10% sucrose in PB. The brains were removed and soaked overnight in a 30% sucrose solution in P B at 4°C; 50-pm-thick sections were cut on a freezing microtome and processed according to the tetramethylbend i n e (TMB) method of de Olmos et al. ('78). Sections were then mounted on gelatin-coated slides, air dried, rapidly counterstained with buffered 0.125 '% thionin, dehydrated in graded alcohols, cleared in xylenes, and coverslipped with Histoclad mounting medium. The staining and dehydration in alcohols was done on ice at about 8°C to prevent loss of T M W H R P reaction product. Sections were viewed with both brightfield and polarization optics and the localion of labeled neurons and anterogradely labeled neuropil was mapped by using a camera lucida drawing tube. The nuclear and subnuclear boundaries were drawn from the same counterstained section under brightfield illumination. The border of an injection site was defined as the area of uniformly dense reaction product surrounding the pipette track, as previously described (Saper and Levisohn, '83). Fine punctate laheling over the neuropil was taken to represent axonal labeling. Anterograde tracing experiments with phaseolus oulga.ris-leucoagglutinin(PHA-L). The injections into medullary nuclei (n 21) were made iontophoretically from ~

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glass micropipettes with tip diameters of 25-50 pm, filled with a 2.5"0 PHA-L solution (Vector) in 10 mM phosphate buffer pH 7.5. A 7 second on-7 second off pulsed positive current of 5-8 pA was applied for 15-30 minutes. After 7-9 days survival time, the animals were reanesthetized with 7", chloral hydrate and perfused through the aorta with 0.9"" saline followed by 500 rnl of 450 paraformaldehyde in 0.1 M P B and postfixed for 1.5 hour; or perfused by 0.5cc glutaraldehyde-2.500 paraformaldehyde in 0.1 M PI3 pH 7.4 followed by 250 ml cold 10% sucrose in P B and stored in the same solution a t 4°C. Sections were cut on a freezing microtome at 50 pm and processed for immunohistochemistry. Briefly, sections were rinsed in PB-0.9% saline (PBS) and preincubated for 1 hour at room temperature in a diluent containing 3 cc normal swine serum and 0.25 70Triton-X 100 in PBS. The sections were then incubated in primary antiserum (goat anti-PHA-(E + L), Vector), diluted 1:1,000 with the diluent, and incubated for 12-48 hours at 4OC on a shaker. After this incubation the sections were washed three

times in PBS and the staining was visualized by either an ininiunoprroxidase or an immunofluorescence method. For immunoperoxidase staining, the sections were incubated for 1 hour at room temperature in a horseradish-peroxidase-laheled swine anti-goat IgG (Tago) diluted 1:50 in diluent. After three rinses in PBS the sections were incubated for 4-5 minutes in a solution containing 0.05$> 3,3diaminobenzidine (Sigma) and 0.01 5% hydrogen peroxide in PBS, then washed again in PBS, mounted on gelatin-coated slides, dehydrated in a series of graded ethanols, cleared in xylrnes, arid coverslipped with Histoclad mounting medium. In some cases sections were counterstained with 0.125 Cr, thionin. Immrinofluorescent staining was performed by using a double bridging technique with two FITC-labeled secondary antibodies. First, the sections were incubated for 1hour at room temperature with a rabbit anti-goat IgG-FITC (Sigma), then, after three rinses in PBS for another hour with a goat anti-rabbit IgG-FITC (Sigma). Both antibodies

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Amb AP Bar

nucleus ambiguus area postrema Barrington's nucleus CC central canal CG central gray matter CGPn central gray pons CnF cuneiform nucleus CU cuneate nucleus cuneate fasciculus cu DC dorsal rochlear nucleus n u dorsal nucleus of the lateral lemniscus DM. DMSpR dorsomedial spinal trigeminal nucleus external cuneate nucleus ECu gigantocellular reticular nucleus GI gigantocellular reticular nucleus, alpha G1.4 gracile nucleus Gr inferior colliculus IC inferior cerebellar peduncle LfP intercalated nucleus In inferior olive 10 intermediate reticular nucleus IRt locus cueruleus LC laterudorsal tegmental nucleus LDTg linear nucleus of t h e medulla 1,1 lateral reticular nucleus LRt lateral lemniscus I1 lateral paragigantocellular reticular nucleus LPGl ventral medullary reticular area MdV mesencephalic trigeminal nucleus Me.; mesencephalic trigeminal tract me6 medial longitudinal fascirulus mlf motor trigeminal nucleus Mo5 motor root of the trigeminal nerve mo5 medullary reticular formation mRF medial vestibular nucleus MVP medial vestibular nucleus, ventral part MVPV nucleus of the solitary tract NTS periambiguns region of the ventrolateral medulla pAmb parabrachial nucleus PU parviccllular reticular nucleus PCRt posterodorsal tegmental nucleus PDTg paragigantocellular reticular nucleus PGI Phaseolus vulgaris-leucoagglutinin PH4-1, prepasitus hypoglossal nucleus PrH trigeminal promontorium Pro principal sensory trigeminal nucleus Pr5 pyramidal tract PY pyramidal decussatian PYX retroambiguus nucleus RAmb reticular formation RE

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rctrofacial segment of t h e nucleus ambiguus (compact formation) rostral ventrolateral reticular nucleus superior cerebellar peduncle subpostrema zone spinal vestibular nucleus spinal trigeminal nucleus. caudal division spinal trigeminal nucleus. interpolar division spinal trigeminal nucleus. oral division spinal trigeminal tract supratrigeminal nucleus solitary tract ventral cochlear nucleus vestibular nucleus ventral spinocerebellar tract wheat germ agglutinin-horseradish peroxidase conjugate trochlear nerve facial nucleus facial nucleus, intermediate part facial nucleus, lateral part facial nucleus, medial part vestibulocochlear nerve dorsal motor vagal nucleus hypoglossal nncleus hypoglossal nerve NTS subnuclei: central subnncleus commissural subnucleus dorsomedial subnucleus gelatinous subnucleus intermediate subnucleus nredial subnucleus parvicellular subnucleus rostral subnucleus ventrolateral subnucleus PU subnuclei: central lateral subnucleus dorsal lateral subnuclcus external lateral subnucleus extreme lateral subnucleus external medial subnucleus internal lateral subnucleus Kolliker-Fuse nucleus medial subnucleus superior lateral subnucleus ventral lateral subnucleus "waist" area

MEDULLO-PARABRACHIAL CONNECTIONS wcre diluted 1:100 with diluent. Following the last incubation the sections were rinsed thoroughly in PBS and mounted on gelatin-coated slides, air dried, cleared in xylenes, and coverslipped with Histoclad mounting medium containing 5 v/v of 6-mercaptoethanol (Sigma) to retard fading (Franklin and Filion, '85; Renfroe et al., '84). The immunoperoxidase t,reated sections were viewed with both brightfield and darkfield optics. Anterograde fiber labeling in the P B was plotted with a camera lucida drawing tube; nuclear and subnuclear boundaries were drawn from t,he same counterstained section or from sections of the adjacent series, which were only stained with thionin. The fluorescent material was used to take high power photomicrographs of the anterograde fiber labeling in the P B subnuclei to demonstrate the course of fibers, the density of the fiber plexus, and the presence of' fiber varicosities and terminal boutons. Cgtoarchiteeture of the nucleus of the solitarg tract. T o examine the subnuclear organization of the NTS, a series of rats was perfused with 4 % paraformaldehyde and the brains embedded in paraffin or celloidin. Sections were cut in transverse or horizontal planes at 15 pm (paraffin) or 35 pm (celloidin), then stained for Nissl-substance with thionin or for fibers with hematoxylin and viewed with brightfield microscopy. Subdivisions of the NTS were determined on the basis of the size, shape, and orientation of the neurons. Neuronal diameters were measured across the longest axis of the perikaryon in the plane of the nucleolus, in 15-pm paraffin sections. In addition to the Nissl-and fiber-stained material, 50-pm-thick frozen sections stained histochemically for acetylcholinesterase (hlesulam, '82) cyt,ochrome oxidase (Wong-Riley, '79), and NADPH-diaphorase (Scherer-Singler et al., '83) provided furt.her evidence of the subnuclear organization of the NTS. %)

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RESULTS Cytoarchitecture of the NTS in the rat Previous investigators have described the subnuclear cytoarchitecture of the caudal two-thirds (general visceral portion) of the NTS in the rat (Kalia and Sullivan, '82; Kalia et al., '84) and the cat (Loewy and Burton, '78) in some detail. These descriptions tend to use the same terms somewhat differently, and therefore disagree in certain details with one another, and with our own observations. The following description is an attempt to clarify the terminology for the caudal NTS subnuclei that we have adopted. In addi tion, we add some observations on the rostral (gustatory) NTS (see Fig. 1). The NTS can be divided somewhat arbitrarily into three rostro-caudal levels: The rostral level extends from the point a t which the NTS first buds off from the dorsomedial edge of the spinal trigeminal nucleus, back to the level a t which the NTS touches the edge of the fourth ventricle; the intermediate level extends farther caudally to the obex; and the caudal level extends back into the medulla to the junction with the spinal cord. The N l S can further be subdivided into a medial subdivision, which includes a number of subnuclei located medial to the solitary tract, and a lateral subdivision, which becomes progressively less prominent at the far rostral and caudal extremes ofthe NTS. Within the lateral dioision of the NI'S, we identify only one subnucleus. The ucntrolatwal N T S consists of relatively large (15-20 pm), darkly staining neurons, intermixed

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with smaller (12-15 pm) less intensely staining cells. The ventrolateral NTS extends laterally almost to the medial edge of the spinal trigeminal nucleus, being separated from it by a prominent band of fibers. Cells similar to those of the ventrolateral NTS are intercalated among the fascicles of the solitary tract. Some investigators have separated this as an interstitial nucleus, and have further identified ventral, ventrolateral, and dorsolateral subnuclei in the lateral NTS of the rat (Kalia and Sullivan, '82; Kalia et al., '84; Altschuler et al., '89). We have not been able to distinguish these subnuclei based on cytoarchitectonic criteria in our preparations, nor do our connectional studies provide evidence for or against such subdivisions. Hence, for the purpose of this study we have not subdivided the ventrolateral su bnucleus. Within the medial subdiuisiori of h e N T S the various subnuclei are more prominent at different rostrocaudal levels. Several are best defined a t the intermediate level of the NTS. Just dorsal to the dorsal motor vagal nucleus is a population of medium-size (12-15 pm) neurons that are more darkly staining than those in the remainder of the medial division of the NTS. Many of these cells stain immunohistochemically for a variety of neuropeptides, including galanin and corticotropin-releasing hormone (Herbert and Saper, '90), and others helong to the A2 noradrenergic cell group (see Armstrong et al., '82; Milner et al., '84, '86). We have termed this the m.edial subnucleus. At the level of the obex and for several hundred microns rostrally, a separate cell population is found in the ventrolateral part of the medial subdivision, adjacent, to the medial subnucleus. This cluster of relatively small (10-12 pm), round, darkly staining neurons can be easily distinguished in Nissl-stained material due to the dense packing of its cell bodies. In NADPH-diaphorase stained material, this cell group stands out clearly because of the dense staining of its neuropil (Fig. lA,B'). This group was named by Ross and colleagues ('85) the central subnucleus, and we have also used this term. Just medial to the medial subnucleus, a t the same levels as the central NTS, is a collection of relatively small (10-12 pm), more lightly staining neurons that tend to be oriented parallel to the face of the fourth ventricle. Many of these neurons have projections similar to the medial NTS subnucleus (see below), but, fewer of them stain immunohistochemically for galanin or corticotropin releasing hormone (Herbert and Saper, '90). l n NADPH-diaphorase stained mat,erial, many neurons in this cell group are clearly stained (Fig. lA',B'). Loewy and Burton ('78) described a similar appearing cell group in the cat as t.he parvicellular NTS. Although it is not clear that their cell group has the same connections as the one we have identified in the rat, we have chosen lo use their term. ,Just lateral to the medial NTS subnucleus is a collection of larger (K-I8 pm), moderately darkly staining neurons that tend to be elongated in an orientation parallel to the medial face of the solitary tract. In agreement with Kalia and Sullivan ('82) and Loewy and Burton ('781, we have named this the intermediate subnucleus. Dorsal to the medial NTS subnucleus is a distinct collection of smaller (I 0-12 pm), relatively pale, teardrop-shaped neurons that tend to be oriented in a medio-lateral direction. The neurons in this region project to a distinct terminal field in the P B (see below), and stain immunohistochemically for a different set of putative neurotransmitters (e.g., cholecytokinin, neurotensin, and epinephrine; see Ruggiero

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Fig. 1. Series of photomicrographs through four different levels of the caudal two-thirds of the NTS illustrating the cytoarchitecture in Nissl-stained sections (left column: A-D, rostral to caudal) and the che-

H. HERBERT ET AL.

moarchitecture in NADPH-diaphorase stained sections (right column: A'-D', rostral t u caudal). See descriptions of the NTS subnuclei in the text,. Scale = 0.5 mm.

MEDULLO-PARABRACHIAL CONNECTIONS

Fig. 2. Line drawings of coronal sections through the pons and the medulla in experiment R40i illustrating the distribution of retrograde neuronal labeling in the brainstem (B-K, caudal to rostral) following

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multiple WGA-HRP injections into the parabrachial nucleus (shaded area in A). Dots depict individual retrogradely labeled cells.

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Fig. 3. Brightfield photomicrographs of WGA-HRP injection sites into lateral and medial parabrachial nuclei in experiment R407 (A) and the Kollik-r-Fuse nucleus in experiment R609 (B). Arrows indicate the

border of the dense uniform label that demarcates the actual injection sites. Scale = 250 fim.

et al., '85; Kawai et al., '88; Herbert and Saper, '90) than those in the medial subnucleus (e.g., galanin, corticotropin releasing factor, norepinephrine, Armstrong et al., '82; Milner et al., '84, '86; Herbert and Saper, '90). In NADPHdiaphorase stained material, the neuropil in this dorsumedial subnucleus stains somewhat more darkly than that in the medial subnucleus (Fig. 1B). The lateral portion of the dorsomedial NTS, a t the level just rostral to the obex, is relatively cell-poor. Leslie et al. ('82) and Shapiro and Miselis ('85),noting that this fibrous area contains the terminations of' many gastric afferents, called this region the gelatinous nucleus (Fig. lA,A'). At the level of the obex and caudally, a distinct group of relatively small (8-12 pm) medium staining elongated neurons appears ventral to the area postrema and rapidly replaces the parvicellular NTS. By the caudal end of the area postrema, this commissural subnucleus has expanded to replace the dorsomedial NTS as well. These neurons tend to be oriented in a medial-to-lateral direction, except at the level of the obex, where they are situated with their long axes parallel to the surface of the area postrema. The commissural NTS, first distinguished by Cajal ( ' 5 2 ) , was included by Torvik ('56) in the medial NTS. Our connectional studies (below) indicate that the commissural subnucleus caudal to the area postrema has connections (e.g., with the Kiilliker-Fuse nucleus in the parabrachial complex) that are

distinct from those of most of the remainder of the medial division of the NTS. On the other hand, the rostral portion of the commissural NTS may be more closely related to the parvocellular subnucleus. The area postrema is separated from the commissural NTS ventrally by a narrow cell-poor hand, which we have distinguished as the subpostrema zone. In NADPH-diaphorase stained material, the neuropil in this region is distinctly more densely stained than the remainder of the commissiiral subnucleus (Fig. 1"). Injections of PHA-L into the area postrema (below) often label cell bodies in the subpostrema zone whose dendrites can be followed into the area postrema (see also hlorest, '60). The nrea postrema itself is not usually considered a portion of the NTS. This collection of small (6-10 pm), densely packed, darkly staining neurons sits astride the obex and forms an inverted triangular profile in the midline along the dorsal surface of the medulla in coronal sections. The most prominent distinguishing feature of the area postrema is the large number of blood vessels it contains, and the virtual absence of NADPH-diaphorase staining within its borders (Fig. lB',C'). It is generally accepted that the neurons in this circumventricular organ primarily respond to substances circulating in the blood, which gain access to the area postrema neurons via fenestrated capillaries (see Broadwell and Brightman, '76). Nevertheless, recent axonal tracer


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studies demonstrate that the area postrema receives primary afferent fibers from the vagus nerve (Ciriello et al., '81; Contreras et al., '82; Kalia and Sullivan, '82). As demonstrated below, the projections from the area postrema to the parabrachial nucleus are virtually identical to those from the medial subdivision of the NTS. Consequently, for the purposes of this study we have chosen to treat the area postrema as a part of the NTS complex. At the junction of the rostral and intermediate levels of the NTS, at about the point a t which the cuneate nucleus and the central NTS subnucleus begin to disappear, two additional subnuclei are seen (not illustrated). Just dorsolateral and dorsal to the solitary tract is a distinct collection of small (8-10 pm), oval, medium-staining cells. This group of neurons, which has been termed the parasolitary nucleus, is thought to be part of the olivo-cerebellar system (see Loewy and Burton, '78). It was not involved by the labeling in any of our experimental material and is not considered further here. At the same level that the parasolitary nucleus appears, a collection of relatively small (10-12 pm) round and oval neurons appears between the medial and the parvicellular NTS subnuclei, just dorsal to the dorsal motor vagal nucleus. More rostrally, this rostral subnucleus expands in size, eventually displacing the other subnuclei of' the medial division. As the NTS draws away from the fourth ventricle, the rostral NTS shifts farther laterally, displacing the lateral subdivision of the NTS as well. The cytoarchitecture of the rostral NTS in the rat is similar to that described in detail in the hamster by Whitehead ('88) and by Davis and ,Tang ('88).

Retrograde tracing experiments: large injections Iqjections into the parabrachial nucleus. Two or three injections of about 6 nl each of WGA-HHP were placed into the PB complex in a series of five rats to provide a comprehensive map of PB afferent neurons in the medulla and forebrain (see Moga et al., '90). Similar patterns of retrograde labeling were obtained in the medulla in each of these experiments. A representative case (R407) is shown in Figure 2 (the forebrain labeling pattern in this case is illustrated in Moga et al., '90). The injection site (Fig. 3A) included most of the PB complex, although the superior lateral, extreme lateral, and Kiilliker-Fuse subnuclei were only slightly involved. The injection spread to some extent into the supratrigemirial nucleus, between the medial P B and the trigeminal motor nucleus. Retrogradely labeled neurons were found in numerous cell groups throughout the medulla including the NTS, the AP, the periambiguus region, the rostral ventrolateral reticular nucleus, the parvicellular reticular area, the spinal trigeminal nucleus, and the trigeminal promontorium. N T S Retrogradely labeled cells were found throughout the rostrocaudal rxtent of the NTS. Caudal to the calamus scriptorius many neurons were present in the commissural and medial subdivisions of the NTS with a slight ipsilateral predominance (Fig. 2B,C). Some retrogradely labeled neurons were found intermixed with the cells of the dorsal motor vagal nucleus, hut their size and morphology resembled other labeled cells in the NTS more than the larger vagal motor neurons. By the level of the A P most of the ipsilateral medial subdivision of the NTS was filled with retrogradely labeled neurons, except for the subpostrema zone, which was free of labeling (Fig. 2E,F); the contralateral

Fig. 4. Polarization and brightfield photomicrographs of coronal sedions through the nucleus of the solitary tract. A. Labeling in the NTS following a large WGA-HRP injection into the parabrachial nucleus in experiment R663. The central subnucleus is completely free of labeling; i t apparently provides nu input to the PB. B. Labeling in the NTS lollowing an injection into the region of the nucleus amhiguus in experiment R668. Note that the neurons in the central NTS subnucleus are now heavily labeled. C. Brightfield photomicrograph of the same section as shown in (B). Th e small closely packed cells of the central NTS stand out. Scales = 100 pm.

medial and ventrolateral NTS subdivisions contained only a few scattered retrogradely labeled cells (Fig. 2D). Rostra1 to the obex a densely packed cluster of labeled cells appeared just medial to the solitary tract (Fig. 2G); most of these cells

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Fig. 5. Photomicrographs of coronal sections through the ventrolateral medulla following a WGA-HRP injection into the central subnuclew of the NTS just rostral to th e obex in experiment R518. A. Polarization photomicrograph illustrating the dense labeling in the rostral part of the nucleus ambiguus. Scale 100 Fm. B. High power photomi-

crograph with Nomarski optics of the same section shown in (A) illustrating that the fine punctate labeling in the neuropil closely surrounds the ambiguus motor neurons. but is not seen within their cytoplasm. Scale 20 wn.

were located in the intermediate NTS subnucleus. The dorsomedial NTS contained only a moderate number of labeled cells, whereas the central subnucleus was completely free of labeling (Figs. 2G,H, 4A). This last subnucleus was the only part of the NTS other than the subpostrema zone providing no input to the PB. The absence of labeling in the central NTS subnucleus was not due to an inability of these cells to transport WGAHRP. After WGA-HRP injections into the ventrolateral medulla including the nucleus ambiguus, a dense cluster of retrogradely labeled cells was found in the central subnucleus (Fig. 4B,C: see also Ross e t al., ’85).In turn, injections into the central subnucleus just rostral to the obex produced anterograde iabeling closely surrounding the motor neurons of the rostral compact formation of the nucleus ambiguus, just dorsal to the rostral ventrolateral reticular nucleus of the medulla (Fig. 5A,B). Large PB injections also consistently resulted in extensive retrograde labeling in the rostral NTS (Fig. 21,K). At the very rostral pole of the NTS the labeled neurons were continuous with labeled cells in the dorsal aspect o f parvocellular reticular area and the dorsomedial part of the spinal trigeminal nucleus. Area postrema. Labeled cells were most numerous in the caudal two-thirds of the area postrema, whereas the rostralmost portion was almost free of labeling (Fig. 2E,F). In addition, we distinguished two populations of retrogradely labeled cells in the area postrema: a “shell” group along the

lateral margin of the area postrema, with mainly ipsilateral projections, and a “core” group without lateral dominance (Fig. 2E). Medullary reticular formation. Labeled neurons were mainly found in the dorsal parvicellular reticular area. Rostrally. the density of labeled cells in this region increased as they merged with the labeled neurons in the overlying rostral NTS and the dorsomedial part of the spinal trigeminal nucleus (Fig. 2K). Medial parts of the medullary reticular formation contained only a few randomly scattered cells. In addition, small numbers of retrogradely labeled neurons were seen in the periambiguus region of the reticular formation and in the rostral ventrolateral reticular nucleus. Spinal trigeminal nucleus. The pars caudalis of the spinal trigeminal nucleus contained many labeled neurons, which were mainly confined to its lamina I (Fig. 2B-D). At the transition zone to pars interpolaris, labeled neurons filled out the dorsal portion of the pars caudalis. In contrast, the pars interpolaris as well as the pars oralis contained only a few scattered neurons (Fig. 2G-I; for spinal trigeminal afferents to the P B see Cechetto et al., ’85).Rostrally, where the rostral NTS emerges from the spinal trigeminal nucleus, numerous labeled cells were seen in the dorsomedial part of the spinal trigeminal nucleus (Fig. 2K). Finally, a dense cluster of retrogradely labeled neurons was found in the trigeminal promontorium starting at the obex level and extending rostrally for about one mm (Fig. 2F-H; see also Cechetto et al., ’85).

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Fig. 6. Line drawings of coronal sections through the pons and the medulla in experiment R609 illustrating an injection site in the KiillikerFuse nucleus (shaded area in A) and the resulting labeling in the

medulla (B-H, caudal to rostral). Large dots depict retrogradely labeled neurons. fine dots depict axonal labeling.

Injections into the Kolliker-Fuse nucleus. Several parabrachial injections that involved the Kolliker-Fuse nucleus to a greater extent than case R407 showed a somewhat different and more extensive pattern of afferent and effererit connections within the medulla. The results are illus-

trated by reference to experiment R609 (Fig. 6) in which the injection site was centered in the Kdliker-Fuse nucleus (Fig. 3B), with only moderate spread of WGA-HRP into the principal sensory trigeminal nucleus and the ventral edge of the external lateral and medial PR subnuclei. The pattern of'

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Fig. 7. Fluorescence photomicrographs of two coronal sections through the dorsal vagal complex. A illustrates a PHA-L injection site in the medial NTS in experiment R571. Note that many intracellularly labeled neurons are present in the center of the injection site. B illus-

trates a PHA-1, injection site in the area postrema in experiment R607. Arrows indicate anterogradely labeled fibers in the dorsomedial NTS. Scales = 250 pm.

retrograde and anterograde labeling in the N T S and ventrolateral medulla in this case was unlike that seen after injections into the adjacent cell groups. NTS. Caudally, there was heavy cellular and terminal labeling in the commissural NTS (Fig. 6R). The retrogradely labeled cells were almost evenly distributed over the ipsi- and contralateral commissural subnucleus, with their long axes oriented mediolaterally. At the level of the caudal area postrema (Fig. 6C) the number of labeled cells in the commissural and the medial NTS decreased, whereas the ventrolateral NTS contained numerous heavily labeled neurons as well as anterograde terminal labeling in the neuropil. From a level just caudal to the obex to about 0.8 mm rostral to the obex (Fig. 6D-E), retrogradely labeled cells and axonal labeling were most prominent in the ventrolateral NTS; some labeled cells were also found medially just adjacent to the solitary tract in the intermediate subnucleus. On the contralateral side, only a few labeled neurons were found in the ventrolateral NTS, but there was substantial terminal labeling. Nucleus ambiguus and medullary reticular formation. A column of retrograde and terminal labeling was found extending the entire length of the ventrolateral medulla (Fig. 6B-G), from the level of the pyramidal decussation up to the facial nucleus. In the caudal two-thirds of the medulla, up to about 0.5 mm rostral to the obex, the labeling was concentrated in and around the nucleus ambiguus. In addition, a band of mainly axonal labeling extended dorsomedially from the periambiguus region to the ventrolateral NTS (Fig. 6R-I)). Rostrally, retrogradely labeled

cells and terminal labeling were mainly found ventral to the nucleus ambiguus in the rostral ventrolateral reticular nucleus (Fig. 6F-G). Heavy terminal labeling continued rostrally capping the lateral and intermediate subdivisions of the facial motornucleus. The same distribution of labeling was found contralaterally, but it was much less intense. Labeled cells were also aligned parallel to the spinal trigeminal tract in layer I throughout the length of the pars caudalis (Fig. 6B-H; see also Cechetto et al., '85). A layer of terminal labeling was present in the outer lamina ofthe dorsal cochlear nucleus (Fig. 6H).

Anterograde tracing experiments To demonstrate the terminal distribution of medullary afferents within parabrachial subnuclei in detail, discrete injections of WGA-HRP or PHA-L were made into different NTS subnuclei, the area postrema, the parvicellular reticular area, the periambiguus region, the rostral ventrolateral reticular nucleus, and the facial nucleus. Nucleus of the solitarg tract and area postrema. Medial NTS. In eight animals, WGA-HRP (n 3) or PHA-L (n = 5) was injected into the medial division of the NTS. Injections that were centered at different rostro-caudal and dorso-ventral levels in the medial part of the NTS resulted in slightly different terminal patterns of labeling in the parabrachial subnuclei. The basic pattern is illustrated by the PHA-L labeling in experiment R571. The injection site (Fig. 7A) was located dorsal to the vagal motor nucleus a t the midlevel of the area postrema, involving the medial, parvicellular, and dorsomedial N'I'S subnuclei. PHA-L ~

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Fig. 9. Fluorescence photomicrographs of four coronal sections through the parabrachial nucleus (A-D, rostra1 to caudal) illustrating PHA-L labeled fibers in the dorsal lateral PB (A; cf. Fig. 8C), the external lateral PH (B; cf. Fig. 8D), the central lateral PI3 ( C ;cf. Fig. BE), and the “waist area” (D; cf. Fig. 8G) following an injection into the medial NTS in experiment R571. Scales = 100 pm.


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Fig. 10. Photomicrographs of two coronal sections through the parabrachial nucleus illustrating the lack of labeling in the outer portion of the external lateral P B following NTS injections that did not include the dorsomedial subnucleus. A. PHA-L labeling in the inner portion of the external lateral PB in experiment R606. Scale = 50 prn. B. Anterograde WGA-HRP labeling in lateral P B subnuclei in experiment R518. Arrow indicates Ihe weaker labeling in the outer portion of the external lateral PB. Scale = 100 pm.

labeled fibers ran rostrally through the lateral medullary reticular formation, turning dorsally a t the caudalmost portion of the PB to cut through the superior cerebellar peduncle and form a small terminal field in the “waist” of the peduncle (Figs. 8G, 9D). Some labeled fibers were also found in the lateral part of the pontine central gray matter, but these avoided the locus coeruleus and Barrington’s nucleus (Fig. 8F,G). Farther rostrally, a dense plexus of laheled fibers and terminals extended into the central lateral PB and completely filled the external lateral P B (Figs. 8DF, 9B,C). At the level of the internal lateral P B the PHA-I, labeled fibers ran dorsally to form a dense terminal plexus in the medial portion of the dorsal lateral subnucleus (Figs. 8C,I), 9A). In the rostralmost part of the PB, the labeled fibers were found dorsally in the central lateral PB from which they ran medially through the fascicles of the trochlear nerve and the mesencephalic trigeminal tract to enter the midbrain central gray matter (Fig. 8A,B). The densiiy of the anterogradely labeled fiber plexus indicates that the medial subdivision of the NTS is a major source of afferents to the PB. The photomicrographs in Figure 9 demonstrate the PHA-L labeling in the “waist” area and several lateral subnuclei of the PB. The fibers show numerous varicosities, presumably representing sites of synaptic contact. Our results show further that there i s a specific projection from the dorsomedial NTS to the outer portion of the external lateral PB. Injections into the medial subdivision of the NTS at the rostro-caudal level that did not involve the dor-

somedial NTS subniicleiis labeled few terminals in the outer part of the external lateral PR (experiments 12606 and R518 illustrated in Fig. 10, and experiment R510; see Fig. 18). The labeling in the remainder of the PR was otherwise as described above. In one experiment (R487), a PHA-L injection was centered in the dorsomedial NTS, with little involvement of the underlying medial subnucleus. Here, the fiber labeling was densest in the outer part of the external lateral PB, whereas the inner portion was nearly free of labeling (not illustrated). Again, the labeling in the rest of the lateral P B was the same as illustrated in experiment R571. In summary. the medial sirbdivision of the NTS provides a heavy input to several P B subnuclei, including the “waist” area, the central lateral PB, the medial aspect of the dorsal lateral PB, and the external lateral PB (Figs. 8,9). Furthermore. t,he external lateral P B may be subdivided into an outer portion innervated by the dorsomedial NTS, and an inner portion innervated by the medial and perhaps the parvicellular NTS subnuclci. Area postrpma. In six animals that. received injections either of WGA-HRP (n = 2) or PHA-L (n = 4),the injection sites were clearly confined to the area postrema. The absence of labeling in the central nucleus of the amygdala and the bed nucleus of the stria terminalis confirmed that, there was little or no spread of tracers into the subjacent NTS (see also Shapiro and Miselis, ’85a). A distinct pattern of labeling, consistently found bilaterally in the lateral PB subnuclei, is demonstrakd by reference to case R607, a rep-

554 resentative PHA-L injection into the area postrema (Fig. 7B). Caudally in the PB. labeled fibers entered the central and external lateral subnuclei. In contrast to medial NTS injections, the “waibt” area was free of labeling. At the midlekel of the P B a dense fiber plexus was centered almost exclusively in the external lateral P B (Figs. IlB, 12A). Some fibers then ran dorsomedially through the central lateral P B into the medial portion of the dorsal lateral PB where they formed a circular terminal field (Fig. I 1B). Occasional labeled fibers were also found in the superior lateral PB. The PHA-L labeled fibers in these lateral P B subnuclei demonstrated numerous varicosities, resembling terminal boutons (Fig. 12B). In experiment R521, two PHA-L injections were placed into the ventral rim of the area postrema. In this case, there was a dense plexus of‘ labeled fibers in the outer portion of the external lateral PR, but few labeled fibers were seen in the inner part of this subnucleus (not illustrated). The labeling in this experiment was similar to the labeling after the dorsomedial N’I’S injection (R487) described above. In addition, a unique belt of cell and fiber labeling was present in the NTS/subpostrema region just adjacent to the injection site in the area postrema (Fig. 13). Dendrites from many of these neurons could be traced into the adjacent area postrema (cf. Fig. 13; Fig. 3 in Morest, ’60). The results demonstrate that the distribution of afferents to the parabrachial subnuclei from the “core” of the area postrema is virtually identical with that from the medial NTS subnucleus, whereas the projection from the “shell” of the area postrema is more similar to that from the dorsmedial NTS. Vmtrolnteral N T S Eleven animals received injections into lateral parts of the NTS (WGA-HRP: n = 4, PHA-L: n = 7). In some cases the injection sites involved the solitary tract as well as the intermediate subnucleus; some of the injections reached slightly into the medial NTS or the underlying reticular formation. In each case, a characteristic pattern of labeling, attribulable to the ventrolateral NTS, was seen in the PB. When injections involved the medial subdivision of the NTS, additional labeling was seen in the distribution described above. A control injection (R544) into the reticular formation just ventral to the ventrolateral NTS did not result in any labeling in the PB. The typical pattern of ventrolateral NTS projection to the PB was demonstrated in experiment R543, in which the WGA-HRP injection was clearly centered in the ventrolateral subnucleus (Fig. 14A), with only minor involvement of the adjacent cuneate nucleus (which does not project to the PB; see Figs. 2 , 6). A patchy pattern of axonal labeling was found caudally in several PB subnuclei including the external medial, external lateral, and central lateral P R (Fig. 14E). Farther rostrally, heavy retrograde perikaryal and terminal labeling was seen in the Kolliker-Fuse nucleus (Figs. 14C,D, 15C). At the level of the internal lateral PB, a few retrogradely labeled cells were seen scattered around the ventral tip of the superior cerebellar peduncle, among the lateral PB subnuclei (see Fig. 14B), but these seemed continuous with the population of labeled cell bodies in the Kiilliker-Fuse nucleus. Patchy anterograde labeling was present in the lateral PB, in a crescent including the far lateral part of the central lateral suhnucleus, but avoiding the external lateral group. Additional axonal labeling was seen in the medial P B just adjacent to the superior cerebellar peduncle (Fig. 14B). At far rostral levels, no labeling was seen in the P B complex.

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Fig. 11. Series of camera lucida drawings through coronal sections of the parahrachial nucleus (A-C, rostral to caudal) illustrating the distri bution of PHA-I, labeled fibers following a n injection into the area postrema in experiment R607. T h e corresponding injection site is illustrated in Figure 7B.

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Fig. 12. Darkfield photomicrographs of a coronal section through the parabrachial nucleus illustrating labeled fibers in the external latera1 PB following a PHA-L injection into the area postrema in experi-

ment R607. A. Scale = 50 pm. B. Higher magnification of area in box in A illustrating the numerous fiber varicosities and presumed terminal boutons. Scale = 20 pm.

Fig. 13. Photomicrograph with Nomarski optics illustrating the unique PHA-L labeling in the NTS just ventral to the area postrema following an injection into the ventral rim of the AP in experiment H521.

Labeled dendrites can be followed from some neurons into the AP. Scale = 50 pm.

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C Fig. 14. Line drawings of coronal sections showing (A) the center of a WGA-HRP injection site in the ventrolateral NTS (v1NTS) (shaded area) in experiment R543, and (B-E, rostra1 t u caudal) the distribution

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of retrograde and anterograde labeling in the Kolliker-Fuse nucleus and

the parabrachial subnuclei. Large dots depict retrogradely labeled cells, fine dots depict axonal labeling.

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Fig. 15. Fluorescence and polarization photomicrographs of coronal sections through the Kolliker-Fuse nucleus follnwing tracer injections into the ventrolateral NTS. In A the PHA-L fiber labeling in the midlevel of the KP is illustrated in experiment R606; in B the PHA-L fiber labeling in a more rostra1 part of the KF is shown capping the ventrolateral edge of the superior cerebellar peduncle in experiment R619. Note the numerous fiber varicosities. Scales = 50 pm. In C retrograde and anterograde WGA-HRP labeling is seen in the midlevel of the Kdliker-Fuse nucleus (arrows) in experiment R543. Scale 100 pm. ~

A similar pattern of axonal labeling was demonstrated in the experiments employing PHA-L. Fiber and terminal labeling filled the Kolliker-Fuse nucleus (Fig. 15A) and capped the ventrolateral edge of the superior cerebellar peduncle (Fig. 15B). The fibers entered these areas from the ventral direction, apparently undergoing a tremendous terminal arhorization and demonstrating numerous varicosities (Fig. lGA,B). To summarize, the ventrolateral NTS is reciprocally connected with the midlevel of the Kolliker-Fuse nucleus and, to a lesser extent, with adjacent areas in the lateral and medial PB. Caudal comrnissural NTS. In four rats WGA-HRP (n - 3) or PHA-L (n 1) was injected into the caudal cominissural NTS. A representative case, experiment R510, is described here in detail. The WGA-HRP injection site (Fig. 17A) was centered in the commissural NTS caudal to the AP with minimal spread into the gracile, dorsal motor vagal, or hypoglossal nuclei (none of which projects to the PB, Fig. 2). ~

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Fig. 16. Fluorescence photomicrographs demonstrating the course of individual fibers running through the Kiilliker Fuse nucleus (A, midlevel; B, rostra1 third) following injections of PHA-L into the ventrolateral NTS in experiment R619.In A ascending fibers are seen coursing dorsa!ly into the Kolliker-Fuse nucleus from the lateral pontine reticular formation. In B a single labeled fiber is shown coursing ventrodor

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sally through the Kolliker-Fuse nucleus into the external lateral PB. Note the varicose appearance of this fiber ventrally in the KF. T h e lower two arrows indicate the origins of long axon collaterals; the upper arrows indicate two short axonal collaterals that appear to end abruptly in terminal houtons. Scales = 50 pm.

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C Fig. 17. Line drawings of coronal sections through the medulla and the pons illustrating the center of a WGA-HRP injection site in the caudal rornrnissiiral NTS (cNTS) in experiment R510 (shaded area in A), and the distribution of retrograde and anterograde labeling in the

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Kiilliker-Fuse nucleus and lateral parabrachial subnuclei (B-E, rostra1 to caudalj. Large dots depict retrogradely labeled neurons, fine dots depict axonnl laheling. Note that there is little laheling in the outer part of the external lateral PB subnucleus.

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Axonal labeling was found in the PB complex caudally in the central lateral subnucleus and extending into the inner portion of the external lateral subnucleus (Fig. 1D) and farther rostrally into the dorsal lateral subnucleus (Fig. 17C-E). At the level of the internal lateral PB (Fig. 17B), anterograde labeling was confined to t,he medial part of the dorsal lateral subnucleus. In the rostral PB only sparse labeling was found dorsally in the central lateral subnucleus. In addition, retrogradely labeled cells were restricted to the mid-rostro-caudal level of the Kolliker-Fuse nucleus (Fig. 17R-D). In general, the pattern of labeling in the PB (Fig. 18) was a conibination of that seen following mediai NTS/core area postrema injections and ventrolateral NTS injections. Rostra1 N T S Five rats received WCA-HRP injections into different portions of the rostral NTS. Some of these injections involved parts of the vestibular nuclei, the parvicellular reticular formation, or the dorsomedial part of the spinal trigeminal nucleus. The latter two sites do have connections with the PB complex that need to be distinguished from those of the rostral NTS (see below). Experiment R523, in which the injection site was centered in the rostral NTS, largely avoiding these other areas, is presented here in detail (see Fig. H A ) . A unique pattern of axonal labeling was seen involving the ventromedial part of the “waist” area caudally and extending rostrally into the ventral and central lateral PB closely surrounding the medial part of the superior cerebellar peduncle (Fig. 19D,E). Farther rostrally, patchy anterograde axonal labeling was found in the medial part of the external medial subnucleus (Fig. 19C,D), and extending medially in a distinct band of labeling across the ventral part of the medial subnucleus (Fig. 19B,C). Even farther rostrally the labeling in the medial and ventral lateral subnuclei became gradually less intense, until the most rostral part of the PB where labeled fibers could be followed from the ventral lateral nucleus, extending medially through the mesencephalic trigeminal tract into the pontine central gray matter (not illustrated). Two other experiments (R524, R547) resulted in a very similar labeling in the P B (see Fig. 20A,R). Injections into the most anterior part of the rostral NTS (e.g., experiment R669) showed a pattern similar to the one described above, except that the band of labeling in the medial PB (Fig. 19B) was missing, as was the projection to the most caudal part of the “waist” area. In summary, the afferents from the rostral NTS mainly terminate in the ventral lateral, medial, and external medial PB subnuclei. In addition, the caudal part of the rostral NTS, near the transition to the medial subdivision of the N‘I’S, joins the medial subnucleus in innervating the caudal “waist” area. Medullary reticular formation. Periambiguus region. In five animals injections (WGAHRP: n = 2; PHA-L: n = 3) were placed just dorsal to, or into the nucleus ambiguus a t different anteroposterior levels. As demonstrated with WGA-HRP experiment R546, extensive axonal and retrograde labeling was found in the P B complex (Fig. 21). Caudally, both axonal and cellular labeling was seen in the Kolliker Fuse nucleus and the central lateral PB. A few labeled neurons also were seen along the border of the principal sensory trigeminal nucleus (Fig. 21F). At the midlevel of the PB, retrograde and axonal labeling was most prominent in the Kolliker-Fuse (Fig. 21D; see also Fig. %A), though a band of less intense terminal labeling was present in the dorsal portion of the central lateral subnucleus. At this level and farther rostrally, the retro-

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Fig. 18. Polarization photomicrograph of a coronal section through the midlevel of the parabrachial nucleus illustrating the labeling in the PB following a WGA-HRP injection into the caudal commissural NTS in experiment R510. Anterograde labeling was most prominent in the lateral PH subnuclei. Note the zone of sparse labeling in the outer portion of the external lateral PB (indicated by two arrows). T h e KiillikerFuse nucleus contained predominantly retrogradely labeled neurons (arrowhead). Scale - 100 rm.

grade cell labeling extended beyond the boundary of the Kiilliker-Fuse nucleus into the lateral parts of the adjacent medial and central lateral P B subnuclei (Fig. 21C,D; see also Fig. 23A). A few labeled cells were also seen along the lateral margins of the dorsal and external lateral P B and within the lateral half of the superior cerebellar peduncle. At the level of the internal lateral PB (Fig. 21A), only a few labeled fibers and cells were seen, mainly within the superior cerebellar peduncle and the central lateral PB subnucleus; the Kiilliker-Fuse nucleus was notahly bare of labeling at this level. The PHA-L injections confirmed that the pattern of axonal labeling seen in the WGA-HRP experiments represented a dense terminal plexus containing numerous varicosities (Fig. 22). T o summarize, the periambiguus region of the ventrolateral medulla is reciprocally connected with the KollikerFuse nucleus and some rostrally located parts of‘the medial and lateral P R subnuclei. The pattern generally resembles the connections of the ventrolateral NTS with the PB complex (compare Figs. 14 and 21).

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C Fig. 19. Line drawings of coronal sections through the medulla and the pons illustrating a WGA-HRP injection site in the rostral NTS (rNTS) in experiment R523 (shaded area in A). and the subnuclear dis-

tribution of anterogradely labeled fibers and terminals in the parabrachial nucleus (B-E, rostral to caudal).

Rostra1 uentrolateral reticular nucleus. Two animals received WGA-HRP injections into the rostral ventrolateral reticular nucleus (as defined by Ross et al., '84a.b). Both experiments resulted in the same distinctive pattern of cellular and axonal labeling in the PB complex. The results are demonstrated with one of these experiments, case R676 (Fig. 24). Caudally the first labeled cells and fibers were

found in the dorsal lateral PB (Fig. 24F) and principal sensory trigeminal nucleus (Fig. 24E). A few sections farther rostrally, numerous retrogradely labeled neurons as well as profuse axonal labeling appeared in the Kiilliker-Fuse nucleus (Figs. 238,24C E). Some labeling was also present in the central and dorsal lateral PB, but the external lateral and external medial subnuclei were almost completely

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spared. Furthermore, the labeling extended beyond the Kolliker-Fuse nucleus into the ventrolateral portion of the medial subnucleus just rostral to the external medial P B (Figs. 23B, 24C,D). The labeling in the Kolliker-Fuse nucleus and the medial PR was heaviest rostrally. The labeled cells and fibers were oriented dorsoventrally, extending from the Kolliker-Fuse nucleus through the medial PB and the superior cerebellar peduncle into the rostral portion of the central lateral PB (Fig. 24C). In the most rostral part of the PR some axonal labeling was still present around the ventrolateral edge of the superior cerebellar peduncle (Fig. 24B). Following a control injection just lateral to the rostral ventrolateral reticular nucleus (R666), labeling was primarily seen in the principal sensory trigeminal nucleus with only occasional cells labeled in the Kiilliker-Fuse nucleus. Thus the labeled neurons in and around the principal sensory trigeminal nucleus in R676 (Fig. 24E) may have been due to tracer spread into the spinal trigeminal nucleus lateral t.o the rostral ventrolateral reticular nucleus. In summary, neurons in the rostral ventrolateral reticular nucleus are reciprocally connected with the Kolliker-Fuse nucleus and with adjacent far lateral parts of the medial, central lateral, and dorsal lateral subnuclei. This pattern of connectivity with the PB is similar to that of the periambiguus region and the ventrolateral NTS. However, the rostral ventrolateral reticular nucleus receives afferents from, and projects to, more rostral parts of the PB complex (particularly in the Kiilliker-Fuse nucleus) than do the other two medullary sit.es. Parvicellular reticular area. Three animals received injections of WGA-HRP (n = 2) or PHA-L (n I) into the parvicellular reticular area. The pattern of labeling was similar in each case; the group is represented by experiment R688. The injection of WGA-HRP was placed in the rostral part of the parvicellular reticular area, just rostral to the appearance of the NTS (Fig. 25A). There was no involvement of the adjacent spinal trigeminal nucleus and vestibular nuclei. Both retrograde and axonal labeling were found both in the PB complex and in adjacent areas of the dorsal pons. Caudally, many labeled cells and dense axonal labeling were present in the supratrigeminal nucleus, capping the trigeminal motor nucleus dorsally (Fig. 25E). In the PB, terminal labeling filled the medial PB and extended through the superior cerebellar peduncle into the most medial part of the lateral P B subdivision. A dense field of axonal labeling and retrogradely labeled cells was present in the ventral lateral PR, located along the dorsomedial edge of the superior cerebellar peduncle. The “waist area,” however, contained a zone that was almost free of labeling (Fig. 25E). Farther rostrally, heavy axonal labeling was found just adjacent to the superior cerebellar peduncle in the ventral lateral and medial PB; at this level, labeled fibers avoided the ventralmost, strip of the medial PB, which is a terminal field for rostral NTS fibers (Figs. 25C,D, 26A). Less intense terminal labeling was found in the external medial subnucleus. In the most rostral part of the P B prominent axonal labeling still was present in the ventral lateral PB extending into the pontine central gray matter. Less intense fiber labeling was widely distributed through the lateral and medial PB subnuclei at this level, and some retrogradely labeled cells were present ventrolateral to the superior cerebellar peduncle (Fig. %R). In addition, many cells of the mesencephalic trigeminal nucleus were retrogradely labeled. In an experiment in which PHA-L was injected into the more caudal portion of the parvicellular reticular area (R573), hasically ~

Fig. 20. Polarization photomicrographs of two coronal sections through caudal levels of the parabrachial nucleus (A, caudal third of PB; B, most caudal part of PB) following an injection of WGA-HRP into the rostral NTS in experiment H.547. Note the anterograde axonal labeling in the “waist” area, and in the ventral lateral and central lateral PR just adjacent to the superior cerebellar peduncle, as well as in the external medial PB (A). Arrows indicate a band of labeling in the medial PB subnucleus. Scales = 100 pm.

the same pattern of axonal labeling was seen. The anterogradely labeled fibers showed many varicosities, presumably indicating that there are numerous synaptic contacts within the P R complex (not illustrated). Briefly, the parvicellular reticular area is reciprocally connected with parts of the PB just adjacent to the superior cerebellar peduncle, including the medial and ventral lateral PB subnuclei throughout their rostro-caudal extent. Facial nucleus. In two rats WGA-HRP was injected into the facial motor nucleus. The injection site in experimerit R661 was centered in the lateral subdivision of the facial motor nucleus, clearly extending into the perifacial zone. Many retrogradely labeled cells were present in the Kiilliker-Fuse nucleus (Fig. 26B), and extending beyond the boundaries of the Kolliker-Fuse nucleus into the lateral portion of the medial PR subnucleus (Fig. 26B). Little if any axonal labeling was seen in the Kiilliker Fuse nucleus. A small injection that did not exceed the bounds of the facial motor nucleus did not result in any labeling in the KollikerFuse nucleus.

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Fig. 21. Camera lucida drawings of coronal sections through the medulla and the pons illustrating a WGA-HRP injection site into the periambiguus region of the ventrolateral medulla (shaded area in A) in experiment R546 and the subnuclear distribution of retrograde and

anlerograde labeling in the parabrachial complex (B-F, rostra1 to caudal). Large dots depict retrograde neuronal labeling, fine dots depict anterugradely laheled fibers and terminals.

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Fig. 22. Fluorescence photomicrograph montage of a coronal section through the parabrachial complex illustrating the dense plexus of PHAL labeled fibers in the Kolliker-Fuse nucleus and around the ventro-

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lateral tip of the superior cerebellar peduncle in experiment R621. The injection was placed into the periambiguus region of the ventrolateral medulla. Scale = 50 pm.

MEDULLO-PARABRACHIALCONNECTIONS

Fig. 23. Polarization photomicrographs of two coronal sections through the parabrachial complex illustrating the retrograde and axonal labeling in the Kolliker-Fuse nucleus, following WGA-HRP injection (A) into the periamhiguus region of the ventrolateral medulla (experiment R.546) and (B) into the rostral ventrolateral reticular nucleus (ex-

Retrograde transport experiments:small injections T o confirm the results of the anterograde tracing experiments and to identify more precisely the cells of origin of each projection, an additional series of experiments was done in which discrete WGA-HRP injections were placed int.o various PB subnuclei (Fig. 27). Although the injection sites in most experiments involved more than one PB subnucleus, the analysis of numerous small, overlapping injections allowed a number of useful distinctions to be made (see Table 1). Injections into the lateral PI3 subnuclei (e.g., R614, R622, R645, R683; see Fig. 27) resulted in retrogradely labeled neurons in the medial NTS subdivision and the area postrema. With these experiments we were able to verify the specific projection from the dorsomedial NTS and the rim of the area postrema to the outer part of the external lateral PB. The injections in experiment R622 and R683 both were centered in the external lateral subnucleus (Fig. 27). However, in R683 t,he outer part of external lateral PB was only slightly involved, and no retrogradely labeled cells were present in the dorsomedial NTS (compare the labeling in Fig. 28A arid B). Similarly, in experiments R614 and R645

565

perimenl R676). Note the prominent retrograde neuronal and the axonal labeling in the ventrolateral part of the medial parabrachial nucleus (arrows) and extending into the superior cerebellar peduncle (curved arrow) in B (cf. Fig. 24C,D). Scales = 100 wrn.

the injections sites involved the central and dorsal lateral PB subnuclei, but missed the outer part of the external lateral group. Labeled cells in these cases were present in the medial and parvicellular NTS suhnuclei and the core of the area postrema, but not in the dorsomedial NTS or the “shell” of the area postrema (not illustrated). Following injections including the Kolliker-FusP nucleus (R609) or the far lateral parts of the central lateral and dorsal lateral P R subnuclei (R610, R622, R677, R683; see Fig. 27), both retrograde and terminal labeling were present in the caudal cornmissural and ventrolateral NTS suhnuclei, the periambiguus region, and the rostral ventrolateral reticular nucleus (see Fig. 29). In contrast, injections into rost.ral parts of the PB (R613, R614, 11639, R640; see Fig. 27) result,ed in prominent labeling in the rostral ventrolateral reticular nucleus, but very little labeling in the ventrolateral NTS or periambiguus region (see Table 1).These observations confirm that the afferent and efferent connect.ions of the rostral ventrolateral reticular nucleus with the P R extend farther rostrally than do those from the periambig u m region or the ventrolateral NTS. After WGA-HRP injections into caudal portions of the PR, including the “waist area,” the lateral PB and the external medial subnucleus (R665, R645, and R610, respectively),

H. HERBERT ET AL.

566 TABLE 1. Summary of the Retrograde Labeling of Medullary Cell Groups by Selected Injection Sites in the Parabrachial Nucleus

AP R 407 R 609 R 610 R 613 R 614 R 622 R 639 R 640 R 645 R 646 R662 R 663 R 664 R 665 R 677 R 680 R683 R 689 R 690

a

rnNTS’ dmNTS

a

INTS

vl NTS

pAm b

a

0

0

0

a

0

0

0

a

a

0

0

0

a a

RVL

rNTS - PCRt

DMSp 5

0

0

0

0

a

0

0

0

0

0

SP 5

a a a

0

a

0

0 0

0 0

a

a

a

a

a a

0

0

a

0

a a a

0

a 0

0

0

a

0

0

0 0

0

0

a

a

0

a

a

0

0

0

0

0

0

--

a

The sizes of the dots indicate relative intensity of retrograde neuronal labeling in different medullary nuclei following injections of WGA-HRP into different PB Yubnuclei. The injection sites for these experiments are illustrated diagramatically in Figure 27. h’ote that

the mNTS in (his table includes other closely related NTS subnuclei that are located medial to the solitary tract, i.e., the comNTS at the AP level and the pcNTS rostral to the obex. c. cumNTS caudal commissural NTS, posterior tu the area postrema level.

retrogradely labeled neurons were seen mainly in the rostral NTS, with few if any labeled neurons in the parvicellular reticular area (Fig. 30A; Table 1). More rostrally and medially placed injection sites (experiments R662, R663, R680 and R689) resulted in retrogradely labeled cells both in the parvicellular reticular area as well as the rostral NTS (Fig. 30B,C),confirming the differential distribution of projections from these to cell groups observed in the anterograde experiments. Retrograde labeling was found in the dorsomedial part of the spinal trigeminal nucleus mainly in experiments R610, R664, and R680, consistent with the earlier report from this laboratory (Cechetto et al., ’85) that the medial and external medial PB receive afferents from this part of the trigeminal complex (see Fig. 30D).

(Norgren, ’78;Ricardo and Koh, ’78). Our findings indicate that the projections to the P R from the NTS and associated regions of the medullary reticular formation are considerably more complicated (Fig. 31). Three basic patterns of eonnectivity have emerged.

DISCUSSION Previous studies of the projection from the nucleus of the solitary tract to the parabrachial nucleus have emphasized that the rostral (gustatory) NI’S innervates the medial subdivision of the PB (including the “waist area”) and that the caudal medial NTS projects to the lateral PB subdivision

-

1. G e n w a l riisceral pattern: The caudal two-thirds of the medial division of t h e N T S , including the medial, parvicellular, dorsomedial, intermediate and commissural subnuclei, and the adjacent area postrema, innervates a terminal field that includes the external lateral subnucleus and lateral part of the external medial subnucleus, as well as medial parts of the central and dorsal lateral PB subnuclei, and the dorsal part of the “waist area” at caudal levels. 2. R a p i r a t o r y pattern: The uentrolateral NTS is joined by the intermediate and commissural NTS subnuclei, the periamhiguus region, and the rostral ventrolateral reticular nucleus in having reciprocal connections with the KollikerFuse nucleus and the adjacent lateral parts of the medial. external medial, central lateral, and dorsal lateral PB subnuclei. 3. Gustatoryloral somatosensory p a t t e r n . The rostral N T S and the adjacent parvicellular reticular area innervate

I \

\

\ 0.5mm

Fig. 24. Camera lucida drawings of coronal sections through the medulla and the pons illustrating a WGA-HRP injection site in the rostral ventrolateral reticular nucleus in experiment R676 (shaded area in A), and the subnuclear distribution of retrograde and anterograde labeling in the PB complex. Large dots depict retrograde neuronal labeling,

fine dots depict axonal and terminal labeling. Note t h a t the connections of the rostral ventrolateral reticular nucleus extend much further rostrally in the PB complex (B,C) than those of the periambiguus region (most rostral labeling, shown in Figure ZlB, is a t the level of D in this figure).

B

C

0.5 mm

Fig. 25. Line drawings of coronal sections through the medulla and the pons illustrating a WGA-HRP injection site in the parvicellular reticular area in experiment R688 (shaded area in A), and the suhnuclear distrihution of retrograde and axonal labeling in the parabrachial

E nucleus and the supratrigeminal nucleus (B-E, rostral to caudal). Large dots depict the retrograde neuronal labeling, fine dots the axonal and terminal labeling. Note the bare spot in the "waist" area in (E) and along the ventral margin of the medial PB subnucleus in (C,D).


569

Fig. 2fi. Polarization photomicrographs of two coronal sections through the parabrachial complex. A. Retrograde neuronal and anterograde terminal labeling in the medial and ventral lateral parabrachial nucleus following a WGA-HRP injection into the parvicellular reticular

area in experiment H68H (cf. Pig. 25C). B. Retrogradely labeled neurons in the Kiilliker-Fuse nucleus following an injection of WGA-HRP into the facial motor nucleus and adjacent reticular formation in experiment R66l. Scales = 100 Mm.

a zone that includes most of the medial and ventrolateral subnuclei, the medial part of the external medial subnucleus, and the "waist" area.

tion of the neurons responding to different functional classes of axuns has not been identified in detail. Moreover, the tendency fur neurons in the NTS to have wide-ranging dendritic trees (Cajal, '52; Mifflin et al., '88) that may cross through the domains of several different afferent nerves makes it difficult to identify individual subnuclei with specific physiological roles. Subdivision of the medial part of the NTS and area postrema. Nevertheless, it is possible on the basis of cytoarchitecture and chemaarchitecture to identify a number of subdivisions within the medial part of the NTS. The siibnuclei that we have defined are largely in agreement with those of previous investigators and are further supported by our own connectional data. The central N T S subnucleus. for example, is the same group that Ross et al. ('85) and Cunningham and Sawchenko ('89) identified on the basis of its massive projection to the compact portion of the nucleus ambiguus. As the central subnucleus is the main terminal field for esophageal afferents (Altschuler et al., '89), and the compact portion of the nucleus ambiguus contains the esophageal motor neurons (Bieger and Hopkins, '87), this pathway may participate in the generation of esophageal reflex movement (Altschuler et al., '89). Our observations. confirming the massive projection from the central NTS to the nucleus ambiguus, support this suggestion. However, it has also been proposed that the central NTS may participate in esophageal control of the crural diaphragm, via projections to the respiratory portion of the PB (Altschuler et al., '89). We find no projection from the cen-

Taken together, these three projections subtend nearly all of the PB. (The extreme lateral and superior lateral PB subnuclei. located a t far rostra1 levels, receive relatively few afferents from the NTS. The internal lateral subnucleus, which we previously showed is a major site of termination for spinal and trigeminal afferents to the PB complex (Cechetto et al., '85), is unique in having no apparent input from the NTS.) These observations underscore the impression gained from previous work that the P B largely serves as a relay for visceral afferent information arising from the NTS (see Fulwiler and Saper, '84 for discussion of the efferent projections from PB subnuclei). In addition, the data suggest that the organization of this relay system is substantially more complex than had previously been realized.

General visceral pattern Most of the general visceral afferent fibers carried in the glossopharyngeal and vagal nerves terminate in the medial division of the NTS (Torvik, '56; Kerr, '62: Berger, '80; Kalia and Mesulam, '80a,b; Panneton and Loewy, '80: Ciriello et al., '81; Contreras et al., '82; Kalia and Sullivan, '82; Seiders and Steusse, '84; Ciriello, '83; Shapiro and Miselis, '85b; Norgren and Smith, '88; Altschuler et al., '89). Although cach peripheral branch of these nerves projects to a different terminal field in the NTS. the topographic organiza-

570

H. HERBERT ET AL.

tral NTS subnucleus to the PB complex at all, so that any role in respiration must be accomplished via connections with the ventrolateral medulla. The intermediute N T S subnucleus has been identified by previous investigators because of its relatively large, distinctively oriented cells, and its input from pulmonary stretch receptor aff'erents (Kalia and Richter, '85, '88), all of which properties are more similar to the ventrolateral NTS than to other medial NTS subnuclei. The intermediate NTS, like the other medial NTS subnuclei, was retrogradely labeled by large PB injections with WGA-HRP that spared the KBlliker-Fuse nucleus (Fig. 2G,H). On the other hand, after injections into the Kolliker-Fuse nucleus, the intermediate subnucleus was the only part of the medial division of the NTS rostral to the obex that was retrogradely labeled. These observations support the view that the intermediate NTS may share some properties both of the ventrolateral and the medial N'rS subnuclei. The medial NZ'S subnucleus has been differentiated from the parvicellular N T S in this and prior studies on the hasis of cell size, shape, and orientation. Immunohistochemical studies support this distinction, showing numerous galanin, corticotropin releasing hormone, and noradrenergic neurons in the medial, hut. not the parvicellular NTS (Milner e t al., '86; Herbert and Saper, '90). On the other hand, we have been unable to distinguish any major differences in the projections of these two subnuclei. The dorsompdial NTS subnucleus has not previously been distinguished from the medial and the parvicellular subnuclei on cytoarchitectonic grounds. Some investigators have designated a cell-poor region in the lateral part of this subnucleus, which receives gastric afferents as the gelatinous subnucleus (Leslie et al., '82; Shapiro and Miselis, '85b; Altschuler et al., '89). Neither our connectional nor our histochemical data indicate a distinction between the gelatinous subnucleus and the remainder of the dorsomedial NTS. On the other hand, immunohistochemical studies indicate that neurons in the dorsomedial NTS, as we define it, have quite different chemical properties from those in the remainder of the medial subdivision. A population of neurons in the dorsomedial NTS stains for the adrenaline synthetic enzyme, phenylethanolamine N-methyltransferase (Armstrong et al., '82; Ruggiero et a]., '85). Many of the same cells also stain with antisera against neurotensin or cholecystokinin (Milner and Pickel, '86b; Kawai et al., '88; Herbert and Saper, '90). The dorsomedial NTS also has a characteristic terminal distribution in the outer zone of the external lateral PB subnucleus that further distinguishes it from the medial and parvocellular NTS subnuclei (see discussion of this terminal field below). The area postrema, although often not considered a part of the NTS, clearly shares general visceral afferents with the adjacent medial division of the NTS (Ciriello et al., '81; Contreras et al., '82; Kalia and Sullivan, '82). Our studies confirm the earlier observations by Shapiro and Miselis ('85a) on the origin and distribution of the area postrema projection to the PB complex. In particular, our findings support their view that the caudal two-thirds, but not rostral third o f t h e area postrema projects to the PR. We have fur-

Fig. 27. Semischematic illustration of WGA-HRP injection sites into different parabrachial subnuclei. Shaded areas indicate the sizes and locations of the injection sites.

M

D

R645

1

R677


571

Fig. 28. Polarization photomicrographs of two coronal sections through the dorsal vagal complex illustrating the retrograde neuronal labeling following WGA-HRP injections into the parabrachial nucleus. A. Retrogradely labeled cells are present in the area postrema, the medial NTS, and the dorsomedial NTS following an injection into both

divisions of the external lateral P B (experiment R622). B. Retrogradely labeled cells in the dorsal vagal romplex after an injection into the external lateral PH where the outer part of this subnucleus is only slightly involved (experiment R683). Arrows indicate the dorsomedial NTS, which is nearly free of label in the latter case. Scales = 100 p m .

ther been able to subdivide the area postrema terminal field in the PI3 into a projection from the “core” of the area postrema that closely resembles the projection from the medial and parvicellular NTS and one from the “shell” of the area postrema that is similar to the projection from the dorsomedial NTS. This organization is similar to the subfornical organ, another circumventricular organ: the “shell” of the subfornical organ projects t o the median preoptic nucleus, whereas its “core” innervates the organum vasculosum of the lamina terminalis (Saper and Levisohn, ’83). Neurons in the subpostrema zone were not retrogradely labeled by any of our PI3 injections (see Figs. 2E,F, %A). However, following injections of PHA-L into the “shell” of the area postrema, neuronal perikarya in the subpostrema zone were labeled. Although dendrites from some of these cells could be followed into the injection site in the area pos trema (see also Morest, ’60), it is conceivable that some may have been retrogradely labeled (Gerfen and Sawchenko, ’84; Shu and Peterson, ’88). The subpostrema zone receives afferents from the hepatic branch of the vagus nerve (Rogers and Hermann, ’83: Norgren and Smith, ’88), which includes hepatic chemoreceptor information (Kahrilas and Rogers, ’84). Hence, neurons in the subpostrema zone potentially have access both to chemoreceptor information from the vagus nerve as well as direct chemoreception of blood-borne substances entering the brain a t the area postrema. However, their efferent connections are likely to be mainly local. The commissural NTS subnucleus is usually classified as part of the medial division of the NTS. Its projection pattern to the PB is similar to the medial N T S subnucleus and the core of the area postrema, supporting this view. Howevrr, like the intermediate NTS, the commissural subnucleus also has projections that overlap those of the ventrolateral NTS, and may more properly be considered to share the general visceral and respiratory patterns of PJ? projection.

Uiflerential projections to PB terminal fields. As noted above, one distinguishing characteristic of the various suhnuclei of the medial division of the NTS is that they have diffcrent patterns of terminal distribution in the PB complex. The most distinctive pattern is that seen in the externu1 luterol PB subnucleus. Whereas large injections of PHA-L or WGA-HRP into the medial NTS subdivision fill t.he entire external lateral PB with terminal labeling (Fig. 8), smaller injections into the medial, parvicellular, or commiss u r d NTS or the “core” of the area postrema show innervation mainly of the inner half of the subnucleus, adjacent to the superior cerebellar peduncle (Figs. 9, 17, 18). Conversely, injections into the dorsomedial NTS or the shell of the area postrema demonstrate projections primarily to the outer half of the external lateral subnucleus. These observations are consistent with immunohistochemical data showing that the projection from the dorsomedial NTS to the PB arises in part from cells that are irnmunoreactive for phenylethanolamine N-methyltransferase, neurotensin, and cholecystokinin (Milner et al., ’84, ’86; Milner and Pickel, ’86b: Kawai e t al., ’88; Herbert and Saper, ’90). All three of these markers are found in terminals concentrated in the outer half of the external lateral subnucleus. By contrast, many neurons in the medial NTS suhnucleus that project to the PB are immunoreactive for galanin, substance P or corticotropin releasing hormone (Milner and Pickel, ’86a; Herbert and Saper, ’90). Terminals that are immunoreactive with antisera against each of these substances are concentrated in the inner half of the external lateral nucleus. The significance of this dichotomy is not yet clear. Fulwiler and Saper (’84) and Moga e t al. (’90) found that the inner part of the external lateral PR projects mainly to the zona incerta and the suhstantia innominata, whereas the outer zone projects to the hypothalamus (paraventricular and median preoptic nuclei and lateral hypothalamic area) and amygdala (central nucleus). Presumably these chemi-

572

Fig. 29. Series of polarization photomicrographs through four levels of the rat medulla (A-D, caudal to rostral) in experiment R609 illustrating retrograde neuronal labeling and anterograde terminal labeling in the ventrolateral NT S (A),the periambiguus region of the ventrolateral

cally and anatomically distinct parallel pathways from the medial part of the NTS to the basal forebrain indicate some differentiation of function. However, it may be very difficult to distinguish the difference using standard chemical or electrical stimulation methods. For example, we have found that electrical stimulation of the external lateral PB with less than 10 y A of current or chemical stimulation with 100 pmoles of L-glutamate can produce profound pressor, tachycardic, and tachypneic responses in rats (Holmes et al., ’87; unpublished observations). Even using threshold stimu-

H. HERBERT ET AL.

medulla (B),the rostral ventrolateral reticular nucleus (C), and axonal and terminal labeling in the perifacial region (D).Th e injection site was centered in the Kolliker-Fuse nucleus (see Fig. 27). Scales = 100 pm. Fig. 30. Polarization photomicrographs through the rat medulla illustrating labeling in the rostral NTS, the parvicellular reticular area, and the dorsomedial spinal trigeminal nucleus following injection of WGA-HRP into the PB complex. A. There was heavy retrograde labeling in the rostral NTS, but relatively little in the parvicellular reticular area, following a WGA-HRP injection caudally into the “waist” area in experiment R665 (see Fig. 27). B. Following a more rostral injection into the medial and ventral lateral subnuclei and the “waist” area in experiment R689 (Fig. 27), labeling was seen both in the rostral NTS and the adjacent parvicellular reticular area. C. Labeling was seen not only in the rostral NTS and the parvicellular reticular area but also (D) extending rostrally and laterally into the dorsomedial spinal trigeminal nucleus following an injection into the medial PI3 t h at extended into t he supraLrigeminal region (experiment R680, Fig. 27). Scales = 100 pm.

573

MEDULLO-PARARRACHIAL CONNECTIONS

Figure 30

574

lation dosages, though, it has been difficult to distinguish differences between responses from the inner and outer zones of the external lateral subnucleus. Further dissection of the cardiorespiratory responses obtained from the external lateral subnucleus may require the use of transmitterspecific agonists and antagonists. The projection to the caudal ventrolateral PB and “ruaLst” a r m appears to arise from the medial and parvicellular NTS as well as the adjacent caudal part of the rostral subnucleus. This part of the PB remained unlabeled after injections into the dorsomedial or commissural NTS or area postrema that did not include the medial, parvicellular, or rostral NTS subnuclei. Following injections into the far rostral NTS or the parvicellular reticular area, the zone occupied by terminals from the medial and parvicellular NTS stood out as an unlabeled patch surrounded by dense terminal labeling (see Fig. 25E). These observations suggest that the caudal “waist” area may receive either glossopharyngeal or vagal gustatory inputs (which synapse most caudally in the rostral NTS; see Kerr, ’62; Contreras et al., ’82; Travers et al., ‘87) or perhaps related non-gustatory inputs from the pharynx or esophagus (which terminate a t the junction of the rostral NTS with the medial and parvicellular subnuclei; see Altschuler et al., ’89). Extracellular recordings from the caudal “waist” area, demonstrating that individual neurons respond both to vagal and to gustatory stimuli, support this view (Hermann and Rogers, ’85). The neurons located caudally in the “waist” of the superior cerebellar peduncle project mainly to forebrain sites, including the substantia innominata, zona incerta, central nucleus of the amygdala, and the insular cortex, but the significance of this projection is not known (Fulwiler and Saper, ’84). Following injections into the medial division of the NTS (and the area postrema), a dense field of terminal labeling was identified snaking through the central lateral PB from the border of the external lateral PB into the medial part of the dorsal lateral PB. The significance of this projection, which contains virtually superimposable terminal fields from the area postrema as well as several medial NTS subnuclei, is not clear. However, the medial part of the dorsal lateral PB projects primarily to hypothalamic sites, including the paraventricular nucleus and the lateral hypothalamic area, and especially the median prenptic nucleus (Fulwiler and Saper, ’84). This last site, in the anteroventral third ventricular region, has extensive connections with nuclei that are important in the regulation of blood volume and pressure (Saper and Levisohn, ’83). Stimulation of the dorsal lateral PB with small doses of L-glutamate (100-500 pmoles) produces decreases in blood pressure, although stimulation of the same area with small electrical currents often has the opposite effect (Holmes et al., ’87; unpublished observations). As fihers from the NTS and the rostral ventrolateral reticular nucleus pass through the dorsal lateral PB on their way into the central gray matter (Fig. 8), it is likely that the pressor responses are due to stimulation of fibers of passage, whereas the depressor responses reflect the activity of cell bodies in the dorsal lateral nucleus. Injections into the medial, parvicellular, or dorsomedial NTS subnuclei, but not the commissural NTS or the area postrema, labeled a group of fibers terminating in the external media[ subnucleus, particularly in its caudal half. Recent studies from our own laboratory indicate that the external medial subnucleus is the major source of afferents from the PB to the parvicellular component of the ventro-

H. HERBERT ET AL. basal thalamic nuclei that relays visceral sensory information to the cerebral cortex (Cechetto and Saper, ’87a,b; Yasui et al., ’89;see section on gustatory pattern below). The distribution of fibers from the NTS indicates that the topographic organization of the ascending visceral sensory system is maintained in the external medial subnucleus. The (gustatory) rostral NTS innervates the rostromedial part of the external medial PB (which projects to the medial parvocellular thalamic taste relay nucleus), whereas the (general visceral) medial subdivision of the NTS innervates the caudolateral part of the external medial PB (which in turn projects to the lateral parvicellular relay area concerned with cardiovascular and gastrointestinal information; see Cechetto and Saper, ’87a,b).

Respiratory pattern Respiratory-related activity is so prominent in the lateral subdivision of the NTS in the cat that this region is often referred to as the “dorsal respiratory group” (see Feldman, ’86 for review). Neurons in this area frequently display activity related to pulmonary stretch, particularly during inspiration. In the cat, slowly adapting pulmonary stretch receptors terminate in the ventral, ventrolateral, interstitial, and intermediate NTS subnuclei, whereas rapidly adapting receptors terminate in the dorsal, dorsolateral, interstitial, and intermediate subnuclei (Kalia and Richter, ’85, ’88). The lateral subdivision of the NTS in the rat has not received such thorough physiological analysis, but appears to contain neurons with similar activities (Saether et al., ’87; Ezure et al., ’88). Injections of anterograde tracers into the lateral subdivision of the NTS in the rat produced a highly distinctive pattern of terminal labeling, involving parts of the PB (Kolliker-Fuse nucleus and adjacent parts of the medial and central lateral PB) and the ventral lateral medulla that also have been identified with respiratory function (the so-called pontine and ventral respiratory groups; see Feldman, ’86). We consequently refer to this as the respiratory pattern of labeling. Subdiaision of the lateral NTS and related portions of the medullary reticular formation. We have been unable, based upon either the cytoarchitecture or our connectional data, to further subdivide the ucntrolateral N T S into dorsolateral. dorsal, ventral, or interstitial nuclei in the rat. Such subnuclei are widely recognized in the cat (Loewy and Burton, ’78; Kalia and Richter, ‘85, ’88) and have been proposed in the rat (Kalia and Sullivan, ’82; Kalia et al., ’84; Altschuler et al., ’89). Where these subnuclei are based upon difference in peripheral afferents (e.g., the interstitial NTS receives distinctive superior laryngeal and pharyngeal afferents in the rat; Altschuler et al., ’89) or efferent connections (the NTS projection to the spinal cord in the cat originates mainly from the intermediate and ventrolateral subnuclei; Loewy and Burton, ’78), the distinctions may prove useful. However, we found it difficult to distinguish these cell groups reliably on cytoarchitectonic grounds. More importantly, in our experiments all parts of the ventrolateral NTS were retrogradely labeled by injections of WGA-HRP into the Kolliker-Fuse nucleus or the far lateral part of the central lateral PB. Hence, for the purpose of this discussion, we have elected to treat this subdivision as a single entity. In addition to the ventrolateral NTS, two parts of the medial NTS subdivision were found to contribute to the respiratory projection pattern in the PB. As already noted,


575

NTS

3

Fig. 31. Summary diagram illustrating the connection pattern ofthe nucleus of the solitary tract and the area postrema with the parabrachial nucleus. Left, schematic drawings of the NTS in the horizontal (top) and coronal planes (bottom). Right, a series of coronal sections of the PB at four levels arranged from rostra1 (top) to caudal (bottom). NTS cell

groups and their terminal fields in the PB are coded by different shad-

ing. Note that the functional-topographic specificity of visceral afferents in the NTS is maintained by their relay through the PB. Areas in the NTS and the PB marked by the horizontal shading are reciprocally connected.

676

neurons in the intermediate N T S are retrogradely labeled by injections of WGA-HRP into either the parts of the P B that receive input from the medial division of the NTS (see above) or the lateral (respiratory) division. The caudal commissurnl NTS, likewise, shares this dual pattern. Two regions of the medullary reticular formation associated with respiratory control also project to the PR in the respiratory pattern. In the caudal medulla (roughly coextensive in the rostro-caudal plane with the lateral reticular nucleus), the neurons in and around the nucleus ambiguus (prriambiguus region) were retrogradely labeled by injections of WGA-HRP into the Kolliker-Fuse nucleus. Injections into the periambiguus region demonstrated that this is a reciprocal projection and that the periambiguus neurons innervate the lateral parts of the medial and central lateral PI3 in a pattern that is virtually identical with that arising from the ventrolateral NTS (Figs. 14, 21). This region constitutes the caudal portion of the "ventral respiratory group," which contains neurons that fire mainly during expiration (Feldman, '86; Ezure et al., '88). Numerous retrogradely labeled neurons were also found in the rostral ventrolateral reticular nucleus following injections of WGA-HHP into the Kolliker-Fuse nucleus. Terminal labeling was found in the same area and extending rostrally to surround the lateral part of the facial motor nucleus. Injections of WGA-HRP into these latter fields confirmed that the descending projection originates from the same PR subnuclei that receive afferents from the rostral ventrolateral reticular nucleus: the Kiilliker-Fuse nucleus and the lateral portions of the medial and the central and dorsal lateral PB subnuclei. Many neurons in the rostral ventrolateral reticular nucleus have inspiratory-related activity, except for a small group located just ventral to the compact formation of the nucleus ambiguus (in the retrofacia1 area; see Bieger and Hopkins, '87, Altschuler et al., '89) that show mainly expiratory-related firing (the Botzinger complex, see Feldman, '86; Otake et al., '87, '88; Ezure et al., '88). The rostral ventrolateral reticular nucleus also contains the neurons that support tonic vasomotor tone (Ross et al., '84a,b; Brown and Guyenet, '85; Ciriello et al., '86). It is interesting in this regard t,hat the rostral ventrolateral reticular nucleus innervates, albeit less heavily, the P B terminal fields that receive their major input from the medial NTS subnuclei (see above). Dtrerential projections l o PB terrninalBe1d.s. The main P B terminal field for the lateral NTS and related projections is the Kiilliker-Fuse nucleus. It is interesting that projections from the ventrolateral and the commissural NTS subnuclei and the periambiguus region mainly terminate within the caudal two-thirds of the Kijlliker-Fuse nucleus. Only the rostral ventrolateral reticular nucleus projects strongly to the rostralmost part of the KollikerFuse nucleus. This result is consistent with our earlier retrograde transport study indicating that the projection from the Kiilliker-Fuse nucleus to the spinal cord originates in the caudal part of t,he nucleus; the projection to the NTS in the middle third; and the projection to the ventrolateral medulla in the rostral two-thirds (Fulwiler and Saper, '84). Our current findings refine these observations, demonstrating that the most rostral part of the Kiilliker-Fuse nucleus is specifically and reciprocally related to the rostral ventrolateral reticular nucleus. As pointed out, many neurons in the rostral ventrolateral reticular nucleus have inspiratory-related firing patterns,

H. HERBERT ET AL. although a specific cluster near the compact portion of the nucleus ambiguus (the Botzinger complex) is expiratoryrelated (Feldman, '86; Otake et al., '87, '88; Ezure et al., '88). It. is interesting in this regard that electrical stimulation in the region of the Kolliker-Fuse nucleus in the cat primarily facilitates expiratory activity (Cohen, '71). In our own microstimulation experiments in the rat, respiration is halted in the expiratory phase by stimulation sites specifically in the rostral part of the Kolliker-Fuse nucleus (Holmes et al., '87). These observations suggest that the projection from the rostral Kijlliker-Fuse nucleus to the rostral ventrolateral reticular nucleus may inhibit neurons that cause inspiration or excite neurons that cause expiration, or possibly have both effects. Further studies of the projections specifically from the expiratory-facilitating zone of the Kiilliker-Fuse nucleus (which does not give rise to cardiovascular responses in our own experiments) may help resolve this issue. Expiratory facilitation is also obtained by electrical stimulation in the lateral part of the medial PB adjacent to the Kolliker-Fuse nucleus (Younes et al., '87). Our studies show that the part of the medial P B subnucleus just, rostral to the external medial subnucleus is reciprocally related with the rostral ventrolateral reticular nucleus and the periambiguus region, and receives smaller numbers of afferents from the ventrolateral and comrnissural NTS. Projections from the rostral NTS and the parvicellular reticular area avoid this region. terminating in the more medial parts of the medial PB. These observations suggest that the medial P B contains functionally specific domains, some of which are probably related to respiratory rather than gustatory function (or perhaps their coordination-see below). The projection from the ventrolateral NTS to the m t e r rral m.edial PB is mainly concentrated in the ventrolateral and caudal parts of the cell group. The rostral ventrolateral reticular nucleus also projects to the external medial nucleus, but its projection pattern is more similar to that of the medial N'L'S, i.e., the dorsolateral and caudal portion of the suhnucleus. This topographic organization is consistent with the observation that the caudolateral part of the external medial nucleus is the main source of afferents to the contralateral thalamic relay nucleus, concerned with the relay of respiratory and cardiovascular information to the visceral sensory cortex (Cechetto and Saper, '87a,b). The distinction between the terminal zones from the ventrolateral and the medial NTS subnuclei further hints a t topographic separation of visceral sensory function in the external medial nucleus and its thalamic relay (see Cechetto and Saper, '87a,b). The lateral part of the central arid dorsal lateral PEI subnuclei is reciprocally connected wit,h the ventrolateral NTS, the periambiguus region, and the rostral ventrolateral reticular nucleus, and receives afferents from the commissural NTS as well. This "lateral crescent" of tissue along the extreme lateral edge of the P R complex also receives afferents from lamina I of all levels of the spinal and trigeminal dorsal horn (Cechetto et al., '85). It is interesting that electrical stimulation in this area has been reported in the cat to have the opposite effect of stimulation in the Kolliker-Fuse nucleus: there is facilitation of inspiratory activity, and tachypnea (Cohen, '71). It is not clear whether this response was due to spread of the rather large currents used (300 pAj into the external lateral nucleus (or its homologue in the cat). On the other hand, we have on occasion been able to

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stimulate tachypnea, without cardiovascular changes, with electrical stimulation along the dorsolateral border of the external lateral nucleus (Holmes et al., unpublished observations). It would be quite useful to determine the differences in terminal distribution of the descending projections from the Kolliker-Fuse nucleus versus the "lateral crescent."

Gustabry/oral somatosensory pattern The gustatory afferents carried in the facial, glossopharyngeal, and vagus nerves terminate in the rostral portion of the NTS, in a roughly topographic rostro-caudal order (i.e., in roughly the same order as their entry to the brainstem), although there is considerable overlap of terminal fields (Torvik, '56; Kerr, '62; Contreras et al., '82). Somatosensory fibers from the mandibular branch of the trigeminal nerve, conveying thermal, tactile, and nociceptive information from the tongue and oral cavity, also terminate in the rostral NTS, mainly in its lateral portion (Jacquin et al., '83; Hamilton and Norgren, '84; Marfurt and Turner, '84; Pfaller and Arvidsson, '88). Neurons throughout this territory respond to sapid stimuli (see Norgren, '84; Travers et al.. '87 for reviews), and often to thermal or mechanical stimuli that may be associated with food texture or temperature. The parvicellular reticular area adjacent to the rostral NTS also receives somatosensory input from the mandibular branch of the trigeminal nerve, as well as information from jaw muscle spindles and periodontal mechanoreceptors, via the mesencephalic trigeminal nucleus (Matesz, '81; Ruggiero et al., '82; Marfurt and Turner, '84). Neurons with sensory fields including intraoral structures form a continuum running from the dorsomedial spinal trigeminal nucleus through the parvicellular reticular area into the lateral part of the rostral NTS (see, e.g., Fig. 7 in Hamilton and Nnrgren, '84). The parvicellular reticular area projects to the cranial nerve oral motor nuclei (trigeminal, facial, ambiguus, and hypoglossal) and is thought to be involved in coordination of masticatory and swallowing behaviors (Holstege and Kuypers, '77; Holstege et al., '77; Huggiero et al., '82; Travers and Norgren, '83; Bieger and Hopkins, '87). Antidromic activation of neurons in the parvicellular reticular area from the P R complex confirms that, unlike neurons in the ventrolateral medulla, those in the dorsal part of the parvocellular region do not have respiratory related activity (King and Knox, '84). Our observations indicate that the parvicellular reticular area projects to some of the same PB terminal fields as the rostral NTS, as well as having extensive connections with adjacent parts of the PB complex and reticular formation that do not receive direct inputs from the NTS. Subdivision of the rostral NTS and related reticular formation. Although the terminations of different afferent nerves within the rostral N T S are topographically organized, there is too much overlap to allow the rostral NTS to be subdivided on this basis. Similarly, neither electrophysiological recording studies nor our own data on efferent connections indicate any functional subdivisions of the rostral NTS with respect to gustatory stimuli (Norgren. '84). The parucellular reticular area adjacent to the rostral NTS bears no cytoarchitectonic characteristics that would distinguish it from the remainder of the parvicellular medullary reticular formation (see Valverde, '61, '62). King ('80) has emphasized that the neurons in this region pro-

jecting to the PB complex in the cat are organized into sheets that run rostro-caudally in a dorsomedial-to-ventrolateral orientation, along the penetrating blood vessels. Our own retrograde transport data do not support this view in the rat. Digerential projections to PB terminalfields. The projection from the rostral NTS to the P B is considerably less extensive than previous studies would suggest, particularly in t,he nipdial PB subnucleus (Norgren, '78). Following injections int,o the caudal part of the rostral NTS, we saw a dense band of terminal labeling in the most ventral part of the medial PB rostrally. A large gap with no labeling separates this ventral band from the superior cerebellar peduncle. The projection from the parvicellular reticular area fills in this gap while scrupulously avoiding the most ventral part of the medial PB, where the rostral NTS input is most intense (cf. Figs. 19B, 25C). The projection from the parvicellular reticular area extends ventrally from the P B into the subjacent reticular region, including the supratrigeminal nucleus (see Rokx et al., '86). This cell group provides input to the motor trigeminal, facial, and ambiguus nuclei, and has inhibitory effects upon masseteric motor neurons, implicating it in chewing and swallowing (see Travers and Norgren. '83 for review). Both the medial PB and the supratrigeminal nucleus project back to the parvicellular reticular area, but not to the rostral NTS. l'he projections from the rostral NTS and the parvicellular reticular area overlap in the ventral lateral PB adjacent to the medial tip of the superior cerebellar peduncle, in the rostral two-thirds of the PR. At rostral levels, fibers from the parvicellular reticular area wrap around the medial edge of the superior cerebellar peduncle and enter the mesencephalic trigeminal nucleus and the lateral part of the central gray matter. The projection from the parvocellular reticular area leaves bare the most caudal part of the ventral lateral PB and the adjacent waist area, corresponding to the terminal field originating in the medial and parvicellular NTS subnuclei and the caudal part of the rostral NTS. The rostral NTS and the parvicellular reticular area also innervate the rostro-medial part of the external medial PB subnucleus, which projects to the contralateral gustatory t,halamic relay nucleus that is reciprocally related to the taste cortex (Cechetto and Saper, '87a,b; Yasui et al., '89). Our current results support. the view that the rostro-medial part of the external medial nucleus deals with gustatory information. We have remarked elsewhere on the close physical relationship of these neurons with the dorsomedial edge of the principal sensory trigeminal nucleus (see Fig. IIE, Fulwiler and Saper, '84). This relationship may promote the sharing of lingual tactile and thermal information between gust,atory and oral somatic sensory systems. In addition, the close relationships of the terminal fields in t.be rostral medial PB originating in the parvicellular reticular area and the rostral ventrolateral reticular nucleus may facilitate coordination of the use of the oral cavity for feeding and respiration.

CONCLUSIONS Taken together, our studies on the subnuclear distribution of the afferent connections of the P B complex (Fulwiler and Saper, '84; Cechetto et al., '85; Cechetto and Saper, '87; Moga et al., '90; this study) demonstrate that functionally

578

different afferent sources innervate largely separate spatial domains. whereas related afferent sources occupy overlapping terminal fields. This is true both for ascending afferent sources, from the medulla and spinal cord (which mark out general visceral, respiratory, and gustatory domains) and for descending projections from the forebrain (which innervate cortical, amygdaloid, and hypothalamic domains; see Moga et al., '90). It is remarkable that the ascending inputs to the P R are largely complementary, occupying nearly all of the complex, whereas the descending afferents are also complementary and fill most of the P B but in a different pattern (cf. Fig. 31 vs. Fig. 21 in Moga et al., '90). One problem with attempting to draw functional conclusions about the roles of neurons in different PB subnuclei based on their afferent innervation is that PB neurons may have widespread dendritic trees that extend beyond the borders of individual subnuclei. Of course, it is likely that neurons are more heavily influenced by afferents that terminate closer to their cell bodies. Perhaps of greater importance, however, is the extent to which a dendritic arborization remains within the domain of a specific functionally defined type of aff'erent. For example, neurons in the gustatory part of the "waist" area often have dendrites that extend through the superior cerebellar peduncle into the ventrolateral and medial subnuclei (Lasiter and Kachele, '88). All of these areas are within the gustalory domain of the PB, however, so that the spread of individual dendritic trees may not affect the functional specificity of the afferent termination patterns. On the other hand, the fact that ascending and descending inputs to the PI3 mark out terminal domains that are organized differently may allow individual neurons to attend to different aspects of the same visceral stimulus. For example, neurons throughout the length of the medial PB subnucleus receive gustatory afferents, but those in the far rostral part are under the influence of descending projections from the hypothalamus, whereas more caudally cortical descending projections predominate (see Moga, '90). Consequently, neurons within a single visceral sensory field in the P B may be modulated by quite different forebrain influences. In comparing the distribution of both the ascending and descending afferents to the PI3 with its output, it is clear that yet a third level of organization exists. In some cases, t,here is a clearcut topographic ordering that carries through the system: different visceral sensory modalities are topographically organized in the external medial nucleus much the same way they are in the NTS, and the projection from the external medial nucleus to the forebrain is equally topographically specific. Similarly, the dorsomedial NTS projects specifically to the outer segment of the external lateral PB, which in turn innervates hypothalamic sites and the central nucleus of the amygdala; in contrast, the medial, parvicellular, and commissural NTS subnuclei innervate the inner segment of the external lateral PB, which projects mainly to the substantia innominata and zona incerta. On the other hand, some populations of P B neurons with a common projection target receive quite different sets of inputs. The medial portion of the dorsal lateral PB, for example, receives a dense input from the general visceral parts of the NTS, whereas the lateral part of the dorsal lateral YB is innervated by all of the regions contributing to the respiratory pattern. Yet both parts of the dorsal lateral nucleus project lo hypothalamic sites concerned with the regulation of blood volume and blood pressure. The complex

H. HERBERT E T AL. relationships of PB afferents and efferents may serve as an anatomical substrate for the integration of different visceral stimuli with one another and with ongoing behavioral activity.

ACKNOWLEDGMENTS The authors thank Quan Hue Ha, Steven Price, and Yabina Herbert for excellent technical assistance. This work was supported by USPHS grant NS22835; American Heart Association Grant-in-aid 850894 and AHA-Wyeth-Ayerst Grant-in-aid 881120; and a grant from the Brain Research Foundation. H.H. was a DAAD/NATO Research Training Fellow (300/402/530/6).

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Subdivisions and neuron types of the nucleus of the solitary tract that project to the parabrachial nucleus in the hamster.

Electrophysiological characterization of reciprocal connections between the parabrachial nucleus and the area postrema in the rat.

Connections of the thalamic reticular nucleus with the contralateral thalamus in the rat.

Cholecystokinin-, galanin-, and corticotropin-releasing factor-like immunoreactive projections from the nucleus of the solitary tract to the parabrachial nucleus in the rat.

Intramedullary connections of the rostral nucleus of the solitary tract in the hamster.

Caudal ventrolateral medullary projections to the nucleus of the solitary tract in the cat.

Taste coding in the parabrachial nucleus of the pons in awake, freely licking rats and comparison with the nucleus of the solitary tract.

Thyrotropin-releasing hormone-immunoreactive projections to the dorsal motor nucleus and the nucleus of the solitary tract of the rat.

Parabrachial nucleus projection towards the hypothalamic supraoptic nucleus: electrophysiological and anatomical observations in the rat.

Projections of the parvocellular reticular formation to the contralateral mesencephalic trigeminal nucleus in the rat.

Odor-taste convergence in the nucleus of the solitary tract of the awake freely licking rat.

Lack of Intrinsic GABAergic Connections in the Thalamic Reticular Nucleus of the Mouse.

6J mouse: Subnuclear parcellation, chorda tympani nerve projections, and brainstem connections.

Connections of the nucleus accumbens.

Projections of the parabrachial nucleus in the pigeon (Columba livia).

The lateral reticular nucleus of the opossum (Didelphis virginiana). II. Connections.

The nucleus of the solitary tract and the coordination of respiratory and sympathetic activities.

Enkephalin immunoreactivity and messenger RNA in a discrete projection from the nucleus of the solitary tract to the nucleus ambiguous in the rat.

Neuropeptide organization of the hypothalamic projection to the parabrachial nucleus in the rat.

Dynorphin A-containing neural elements in the nucleus of the solitary tract of the rat. Light and electron microscopic immunohistochemistry.

Connections of the subthalamic nucleus with ventral striatopallidal parts of the basal ganglia in the rat.

Postsynaptic potentials recorded from nucleus of the solitary tract and its subjacent reticular formation elicited by stimulation of the carotid sinus nerve.

Organization of cortical, basal forebrain, and hypothalamic afferents to the parabrachial nucleus in the rat.

Non-dopaminergic neurons in the substantia nigra project to the reticular formation around the trigeminal motor nucleus in the rat.