THE JOURNAL OF COMPARATIVE NEUROLOGY 293399424 (1990)

Neuropeptides and Catecholamines in Efferent Projections of the Nuclei of the Solitary Tract in the Rat D. RICHE, J. DE POMMERY, AND D. MENETREY CNRS, Laboratoire de Physiologie Nerveuse, kquipe de Neuroanatomie Fonctionnelle, 91198 Gif-sur-Yvette (D.R.) and INSERM. Unit6 161 de Physiopharmacologie du SystPme Nerveux, 75014 Paris (J.d.P., D.M.), France

ABSTRACT This study focuses on the involvement of catecholamines and nine different peptides in efferents of the nucleus of t,he solitary tract, to the central nucleus of the amygdala, the bed nucleus of the stria terminalis, and different parabrachial and hypothalamic nuclei in the rat. A double-labeling technique was used that combines a protein-gold complex as the retrograde tracer with immunohistochemistry. Catecholaminergic projection neurons were the most numerous type observed and projected mainly ipsilaterally t o all targets studied. Most projections arose from areas overlying the dorsal motor nucleus, mainly the medial nucleus. Neurons synthesizing somatostatin, met-enkephalin-Arg-Gly-Leu, dynorphin B, neuropeptide Y, and neurotensin projected to all structures examined. Somatostatin and enkephalin immunoreactive projection cells were the most numerous. They were located in close proximity t c i each other, including all suhnuclei immediately surrounding the solitary tract, bilaterally. Most dynorphin and neuropeptide Y immunoreactive projection cells were found rostra1 to that of enkephalinergic and somatostatinergic projections, and mainly in the ipsilateral medial nucleus. Neurotensinergic projections were sparse and from dorsal and dorsolateral nuclei. Substance P and cholecystokinin contribute to parabrachial afferents. The location of substance P immunoreactive projection cells closely resembled that of enkephalinergic and somatostatinergic projections. Projecting cholecyst,okinin immunoreactive cells were observed in dorsolateral nucleus. Bombesin immunoreactive cells in dorsal nucleus projected to either the parabrachid or hypothalamic nuclei. No vasoactive intestinal polypeptide-containing cells were detected. Thus, most catecholaminergic and neuropeptidergic efferents originated from different populations of cells. I t is proposed that catecholaminergic neurons constitute the bulk of solitary efferents and that they may contribute to autonomic neurotransmission. Peptidergic neurons mainly form other subgroups of projections and may play a role in modulating the physiological state of the target nuclei. Key words: solitary tract complex, amygdala, hypothalamus, parabrachial nucleus, bed nucleus of stria terminalis, retrograde tracing, immunocytochemistry

Accepted August 28,1989. Address reprint requests to D. Menetrey, INSERM, IJnitQ161 de Physiopharmacologie du Systeme Nerveux, 2 rue d'AIQia 75014 Paris, France.

0 1990 WILEY-LISS, INC.

D. RICHE ET AL.

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The nucleus of the solitary tract (nTS) comprises a variety of neurons containing distinct neuroactive substances, including biogenic amines and neuropeptides (see references in Leslie, '85; Nakamura, '88; Palkovits, '85). Most neurons in riTS stain for catecholaminergic enzymes (Dahlstrom and Fuxe, '64; Palkovits and Jacobowitz, '74). Mixed or well-defined populations of dopaminergic, noradrenergic, and adrenergic cells have been recognized in relation to specific cytoarchitectonic boundaries (Armstrong et al., '82; Dahlstrom and Fuxe, '64; Hijkfelt et al., '74; Kalia et al., '85a,b; Ritchie et al., '82; Ruggiero et al., '85). Neuropeptides-including neurotensin, cholecystokinin, enkephalin, dynorphin, somatostatin, melanocyte-stimulating hormone, neuropeptide Y, substance P, P-inhibin, and galanin-are found in various regions (Harfstrand et al., '87c; Higgins et al., '84; Jennes et al., '82; Kubota et al., '83; Lee and Basbaum, '84; Sawchenko et al., '88a,b; Skofitsch and Jacobowitz, '85; Vincent et al., '85; Yamazoe et al., '84). Colocalizations between cells containing catecholamines and peptides often occur (Ceccatelli et al., '89; Everitt et al., '84; Guthrie and Basbaum, '84; Harfstrand et al., '87b; HGkfelt et al., '84, '85; Kawai et al., '88a; Kubota et al., '86; Melander et al., '86; Milhorn et al., '87; Murakami et al., '89),and observations of serotonin-positive (Calza et al., '85), gabaergic (Meeley et al., '85), and cholinergic (Armstrong et al., '88) cells have also been reported. Whereas catecholaminergic neurons are known to contribute to nTS projections (Day et al., '80; Kawai et al., '8%; Milner et al., '86; Moore and Guyenet, '83; Sakumoto et al., '78; Saper et al., '83; Sawchenko and Swanson, '82; Swanson and Hartman, '75), iess is known about the neuropeptidergic component. To date, direct evidence based on double-labeling studies has demonstrated somatostatin and neuropeptide Y-projections to the hypothalamus, with the latter being in part colocalized with catecholamines (Sawchenko et al., '85. '88a) and enkephalin, neurotensin, and substance P-IR projections to the parabrachial nuclei (Maley and Panneton, '88; Mantyh and Hunt, '84; Milner and Pickel, '86a,b). The possibility that other neuropeptides may contribute to solitary tract nucleus projections is suggested by the partial overlap of neuropeptidergic and efferent projection neurons in several areas of the nTS; and the fact that nuclei receiving solitary efferents are innervated by fibers containing a variety of these peptides (see refs. in Palkovits '84). In the present study, we have used a double-labeling technique combining the retrograde transport of a protein-gold complex (Rasbaum and MenBtrey, '87; Menktrey, '85) to label the cells of origin of solitary tract nucleus efferents, to with avidin-biotin immunocytochemistry (Hsu et al., %l), map the distribution of neurotransmitter-containing neurons in the nTS. Antisera against tyrosine-hydroxylase and nine different neuropeptides (somatostatin, met-enkephalin-Arg-gly-Leu, dynorphin B, neuropeptide Y, substance P, cholecystokinin, neurotensin, bombesin, vasoactive intestinal polypeptide) have been used to demonstrate their individual contributions to nTS efferents to the amygdala, hypothalamus, and parabrachial areas. The results have been presented in abstract form (Riche et al., '88).

MATERIALS AND METHODS Experiments were performed on male Sprague-Dawley rats (275-320 gj anesthetized with pentobarbital sodium (55 mg/kg/i.p.). Stereotaxic pressure injections of the retro-

grade tracer (0.25-0.5 pl) were made unilaterally through 20-40 pm diameter glass micropipettes. The retrograde tracer, a protein-gold complex. was made of wheat germ agglutinin-apohorseradish peroxidase conjugate (WGAapo-HRY, Sigma, L0390) coupled to 10 nm diameter colloidal gold (Rasbaum and Menetrey, '87). The tracer diffuses only minimally from the injection site; thus, injections could be precisely localized to the central nucleus of the amygdala, different nuclei of the hypothalamus, bed nucleus of the stria terminalis and medial parabrachial area. As shown by previous studies (Mac Kellar and Loewy, '81; Norgren, '78; Ricardo and Koh, '78; Sawchenko and Swanson, '82; Van der Kooy et al., '84), each of these sites is a major terminal field for efferents of the nucleus of the solitary tract. These targets were approached stereotaxically using coordinates from the atlas of Paxinos and Watson ('86).

Abbreviations ac

AIIy AP '49

nL

nsri, dln'l'S

drnnX dnTS

r

IIPO KF LV mnTS n corn nI nTS ox PV SCP

TS

vlnTS vnTS VPB IV

XI1 3V

anterior commissure anterior hypothalamic area area postrema cerebral aqueduct basolateral amygdaloid nucleus bcd nucleus of the stria terminalis dorsolateral nucleus dorsal motor nucleus dorsal nucleus fornix medial preoptic area Kolliker-Fuse nucleus lateral ventricle medial nucleus nucleus commissuralis intermediate nucleus nucleus of the solitary tract optic chiasma periventricular area superior cerebellar peduncle solitary tract ventrolateral nucleus ventral nucleus ventral parabrachial nucleus fourth ventricule nucleiis of the hypoglossal nerve third ventricule.

~

Figs. 1-4. Location of retrogradely labeled neurons in the nucleus of the solitary tract after injection (black areas) in various nuclei. T h e levels are from caudal (A) to rostra1 (F). Ipsilateral projection cells are shown on the right. Each dot represents one labeled cell, one section per level.

Fig. 1. Injection in the nucleus of the bed nucleus of the stria terminalis (BSTL). This injection site was 0.8 mm long and 0.5 mm wide. It was centered on the anteroventral area and confined mainly to the dorsomedial nucleus (Ju and Swanson, '89). Part of t h e tracer, however, spread to the anterior commissure (ac).

Fig. 2. Injection in the central nucleus of the amygdala ( C e ) .This injection site was 0.8 mm long and 0.5 mm wide. I t was centered mainly on the anterior portion of the nucleus, but it also involved the lateral capsular subdivision (Mac Donald, '82).

AP 7.2

BSTL

AP 8.5 A

B

C

D nTS

drnnX dmnX

vnTS

PV

' v nTS

E

F nTS

d

nTS ;

nX

Figures 1 arid 2

I

AP 7.7

J

AP 0.2

A

A

B

B

XI1

C

C

D

D

dlnTS

vlnTS

TS

'

dmnX

vnTS

dmnX

\

vnTS D\/

E

E

7

F

4--

F dmnX

A

dmnX

Figures 3 and 4

NEUROTRANSMITTERS IN SOLITARY EFFERENT'S Two days postinjection, the animals were reanesthetize6 and 160 r g of colchicine in 8 pl saline were injected into the cisterns magna. Twenty-four hours later, the animals were reanesthetized and perfused intracardially with 0.1 M phosphate-buffered saline (PBS, pH 7.4) followed by 400 ml of a fixative solution containing 0.1 M phosphate-buffered (PB) 4 SX periodate-lysine-paraformaldehyde(Mac Lean and Nakane, '74). The brains were removed and immersed in the same fixative for 4 hours at 4 O C before cryoprotection in a phosphate-buffered 30% sucrose solution, overnight. Frozen serial coronal sections (40 Gm a t the nTS level and 100 pm at the level of the injection site) were collected in PB to be silver intensified as described previously (Menetrey, '85). Sections t,hrough the injection site were mounted and lightly counterstained with neutral red, whereas those from t,he nTS were processed immunocytochemically as freefloating sections, incubated in normal serum (NS, 3 % in PBS, 0.1 M, 0.35 Triton X-100) for 30 minutes, and in one of the primary antisera a t 4°C for 16 to 48 hours. The antisera to substance P (SP),bombesin (BR), vasoactive intestinal polypeptide (VIP), neurotensin (NT), and neuropeptide Y (NPY) were purchased from Cambridge Research Biochemicals. The antiserum to cholecystokinin (CCK) was a gift from Dr. G. Tramu (TNSERM U156, Lille, France; Studler et al., '84), and those to dynorphin B (DYN) and met-enkephalin-Arg-Gly-Leu (ENK) were obtained from Dr. E. Weber (University of Oregon; Weber and Barchas, '83). All primary polyclonal antisera were preabsorbed against rat, liver acetone powder (50 mg powder in 500 ~1 of 1/200 dilution of antisera, 2 hours at 37"C, followed by centrifugation) and then brought to a final dilution (1/2,0001/3,000 in PBS containing 1 % NS and 0.3% Triton X-100). The antiserum to somatostatin (SS) was monoclonal (a gift from Dr. .J.C. Brown, University of British Columbia; Vincent et al., '85) and was used at a concentration of 5 rglml. A11 positive staining was abolished after omitting immunoreagent in the staining sequence or after preabsorption of the antisera with their respective pure homologous peptides (10 nmol per ml diluted antiserum), except for enkephalin (ENK) staining, which was reduced. The antiserum to tyrosine-hydroxylase was purchased from Institut Jacques Boy S.A. (Reims, France), and used at l/S,OOO. After incubabion the sections were washed in three changes of N S containing 0.3% Triton X-100, incubated for 1 hour a t room temperature in biotinylated secondary antiserum, washed twice, and then incubated for 2 hours in avidin-biotin-peroxidase complex (Vectastain, Vector Laboratories). Following a final wash, the sections were developed in 15 mg/ml diaminobenzidine, 0.01 % hydrogen peroxide in PBS. Staining was observed under the microscope. The sections were then air-dried, mounted, and the retrogradely silver-labeled cells examined for neuropeptide-like immunoreactivity.

Fig. 3 . Injection in the medial preoptic area (HPO). This injection site was 1 mrn long and 0.7 mm wide.

Fig. 4. Injection in the parabracial area. This injection site was 1.3 mrn lung and 0.6 mm wide. It was centered on the ventral parabrachial nucleus (VPB) but spread to the superior cerebellar peduncule (scp).

403

Results were obtained from 37 rats; 21 were used for double-labelling studies. In all experiments, immunoreactive cells were seen clearly throughout the rostrocaudal extent of the medulla, indicating effective diffusion of the colchicine. Data are from experiments in which retrograde labeling was proved to be substantial. The pattern of labeling was examined at six levels, covering the entire rostrocaudal extent of the nTS from the spinomedullary junction to approximately 2 5 mm rostral to the obex. The location of cells is based on the cytoarchitectonics of Kalia and Sullivan ('82) and Shapiro and Miselis ('85).

RESULTS Distribution of ascending solitary tract cells Because the protein-gold tracer diffuses only minimally, all injections were located unilaterally and restricted to defined nuclei. Figures 1 to 4 show the location of retrogradely labeled cells in typical cases resulting from injections i n the bed nucleus of the stria terminalis (Fig. I), central nucleus of the amygdala (Fig. 2), medial preoptic area (Fig. 3), and parabrachial nucleus (Fig. 4). In agreement with previous studies (Milner et al., '86; Milner and Pickel, '86a,b; Ricardo and Koh, '78; Van der Kooy et al., '84), retrogradely labeled cells were always found bilaterally, with ipsilateral projections outweighing the contralateral ones. Whatever the injection site, several characteristics of the labeling were observed. Labeling was seen throughout the entire rostrocaudal extent of the nTS, although most labeled cells were confined to caudal levels (A to E). At rostral levels where the dorsal motor nucleus (dmnX) shifts laterally, fewer or no retrogradely labeled cells were seen (level F). Most labeled cells were dorsal to the dmnX (levels I3 to D) in the medial nucleus (mnTS). Fewer projection cells were seen in nuclei situated close to the solitary tract (7%). Retrogradely labeled cells were particularly numerous at levels close to or just posterior to the area postrema (AP). A t the level of A P (levels C and D), retrogradely labeled cells were still observed in the mnTS, with some in subnuclei adjacent to the TS, and thus including the intermediate (nI), dorsolateral (dlnTS), dorsal (dnTS), and ventral (vnTS) nuclei. More rostrally (level E), retrograde labeling involved the periventricular region that dorsomedially forms the wall of the fourth ventricle. At this level, labeling was still observed in mnTS, nT, and dlnTS. A t rostral levels, retrogradely labeled cells (level F) were sparse and confined to the medial border of the dmnX. Additional retrogradely laheled cells were inserted ventrally along the border between the dmnX and nucleus of the hypoglossal nerve (XII, levels R and C). Other labeled cells were seen caudally in the nucleus commissuralis (n com.) at levels A and B. Projections to certain targets displayed special features, such as those to the central nucleus of the amygdala, which were almost totally ipsilateral (Fig. 2). The parabrachial area (Fig. 4) received abundant projections that were clearly bilateral at caudal levels (A to C). At levels just rostral to the area postrema, they entered the dlnTS (level E) contralatera1 t o the injected area and were still abundant even more rostrally (level F), where projections to other targets were only sparse and restricted to the medial edge of dmnX.

Distribution of solitary immunoreactive cells Figures 5 to 9 illustrate the locations of T H - (Fig. 5 ) , SSand ENK- (Fig. 6), DYN- and NPY- (Fig. 7), SP- and CCK-

D. RICHE ET AL.

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(Fig. 81, and NT- and BE-immunoreactive (IR) neurons (Fig. 9) throughout the rostrncaudal extent of the nTS. No VIP-IR cells were observed. Levels caudal to area postrema ( A to C). Perikarya were imrnunoreactive to all antisera, except NPY a t these levels. The majority of cells in the rnnTS dorsal to the dmnX contained TH-IR (Figs. 5 , 11, and 12). Except for cells in ventromedial loci (black arrow in Figs. 5C, 11A, and 12A), TH-TR cells overlapped most retrogradely labeled cells. Only a few immunoreactive cells were seen laterally in the intermediate and dorsolateral nuclei; some other cells were scattered among the dmnX. Peptidergic cells at these levels reacted for SS, ENK, DYN, CCK, SP, NT, or BB antisera, with an increase in number rostrally (level C). SS-, ENK-, and SP-IR cells were numerous and more widely distributed in the n T S than were TH-TR cells. A high proportion was seen in the most. lateral subnuclei surrounding the TS, that is, including the nI? dlnTS, vnTS, and vlnTS. Some cells inserted along the border of drnnX with the nucleus of the hypoglossal nerve. Caudal to the area postrema (level C), CCK-IR cells appear, and NT-IR neurons are seen clustered close to t.he ventral border of the area postrema. Levels including the area postrema (D). Cells immunoreactive to all antisera are seen throughout this level, including NPY-IR cells. It is a t this level that TH-IR cells begin to shift dorsolaterally to areas surrounding the TS (Fig. 12A and white arrow in Fig. 5D), thus resulting in a partial segregation with the retrogradely labeled population. TH-IR cells are seen in the mnTS, nI, dnTS, and dmnX. SS-, ENK-, DYN-, SP-, or BB-IR cells are still observed in the same areas. EN#-, SS- (open arrows in Fig. 6), and SP-IH.cells are localed in all subnuclei immediately surrounding the TS, with a particularly dense concentration of somatostatinergic cells in the dorsolateral position. In contrast to caudal levels, there is a clear increase in the number of CCK- and NT-IR cells present. Both populations gather dorsomedially to the TS, between TS and AP; they include both the dnTS and the dorsal strip (black arrows in Figs. 8D, 9D). Most cells from the latter groups lay dorsal to the retrogradely labeled neurons. Levels just anterior to area postrema (E). TH- and all peptide-immunoreact.ive cells are seen a t this level, although in different locations and numbers. TH-IR cells were still abundant in the periventricular region, mnTS, nI, and dnTS and showed a characteristic ventrolateral extension inserted between T S and dmnX (arrowhead, Fig. 5E). Most peptide-IR cells were distributed differently. SS- and SP-IR cells were widely distributed and occurred in both the periventricular region arid the subnuclei surrounding the solitary tract. SS-IR cells were predominant and could be observed far laterally. ENK-, NPY-, and CCK-IR cells were located medially. NPY-IH. cells were inserted ventrolaterally betureen TS and dmnX (arrow, Fig. 7E). DYN-IR cells were concentrated laterally mainly in vlnTS (open

Fig. 5. Location of tyrosine-hydroxylase immunoreactive (TH-IR) cells in the nucleus of the solitary tract. Only one side is represented. Each dot represents one labeled cell. The arrow in C points to a group of ventromedial immunoreactive cells that does not contribute to nTS efferents. The white arrow in D points to immunoreactive cells that border the solitary tract. Arrowheads in E and F point to imrnunoreactive cells that insert ventrolaterally between the TS and the dmnX or wrap around dmnX and nTS, respectively.

TH-IR C e l l s

A

C

nl.

dmnX

vnTS

E 4 '

drnnX

405

NEIJROTRANSMITTERS IN SOLITARY EFFERENTS arrow, Fig. 7E). NT-and BB-IR cells were gathered dorsally, and NT-IR cells were situated within or near the dnTS and dlnTS, whereas BB-IR cells were placed more medially in dnTS and dorsal strip (arrows, Fig. 9E). Levels just anterior to the NTS (F). Immunoreactive but little retrogradely labeled cells were observed at these levels, where the nTS shifts ventrolaterally from the floor of the fourth ventricle. Immunoreactive cells occurred in several different locations. SS-, ENK-, and CCK-IR cells were gathered in dmnX and laterally in nTS; TH-, DYN-, NPY-, and BB-IR cells were scattered along the medial edge of dmnX (arrowheads in Figs. 5F; 7F, and 9F). No SP- or NTIR cells were observed in the nTS a t this level. Neuroactive substances in solitary ascending tract cells. TH- and all neuropeptide immunoreactivities were colocalized with the retrograde transport. Double-labeled cells displayed black granules of silver (retrograde staining) superimposed on diffuse brown cytoplasmic immunoreactive staining (Figs. 11,12, 14, and 16). TH-immunoreactiue projections (Fig. 101. TH-IR cells constituted the bulk of the nTS projections and were organised in a similar fashion whatever the pathway studied. TH-IR projections arose from all rostrocaudal levels of the nTS, and most projected ipsilaterally. The majority of cells were observed a t levels caudal to the area postrema, where they formed dense clusters dorsal to the dmnX, including nucleus commissuralis and mnTS. Examples of TH-immunoreactive projection cells a t these levels are shown in Figures 11 and 12. More laterally, they were also seen in the intermediate nucleus (nI). At the level of the area postrema, cells were confined mainly to the region immediately surrounding the TS, including mnTS, nI, and dnTS. TH-IR projection cells were less frequent a t levels anterior to the area postrema. A projection arose from cells that inserted ventrolaterally between the T S and the dmnX (level E) or bordering the medial region of dmnx when it shifts laterally (level F). Other TH-IR projection cells were scattered in the periventricular wall. Neuropeptide immunoreactive projections. Peptidergic projections were bilateral. They were less numerous than TH-IR projection cells. Common peptidergic projections to all targets (Figs. 13-16). ENK-, SS-, DYN-, NPY-, and NT-IR cells projected to all structures studied. The peptidergic vs. catecholaminergic content varied according the projection studied. Afferents to the parabrachial area had the highest amount of peptidergic content; they were rich in ENK and, to a lesser extent, SS,DYN, and NPY. Amounts of peptidergic afferents to the hypothalamus and the bed nucleus of stria terminalis were about half that seen for parabrachial afferents; afferents to the bed nucleus of stria terminalis contained ENK, SS, DYN, and NPY in almost similar amounts, and those to the hypothalamus contained mainly ENK, SS, and NPY and, to a lesser extent, DYN. Afferents to nucleus centralis of amygdala had the lowest amount of peptidergic content, being about one third of that obtained for parabrachial afferents; they contained mainly NPY and DYN and in a lesser extent ENK and SS. NT-IR projections were always sparse. SS- and ENK-IR projection cells were numerous and located in similar regions bilaterally (Fig. 13). Most of these cells were observed in Ihe caudal aspects of the nTS extending from the medullary spinal junction to the anterior pole of the area postrema. Except for a distinct SS-IR projection to the parabrachial area arising from the ipsilateral nuclei

immediately surrounding the 'l'S (n1, vlnTS, and dlnTS a t level E), SS-IR projection neurons did not extent as far rostral as ENK-IR cells. Most SS- and ENK-IR projection cells were located in the lateral half of the complex, including mainly mnTS and nI; fewer were seen in vlnTS and dlnTS. ENK-IH. projection cells had the widest mediolateral extension. Other immunoreactive projection cells were observed ventral to the dmnX along its border with the hypoglossal nucleus (XTT). Projections were clearly bilateral. More cells projected contralaterally a t caudal levels. The location of either contralaterally projecting or laterally located immunoreactive peptidergic cells was clearly distinguished from that of TH-IR projection neurons. A partial spatial overlap for both populations was, however, evident in areas forming a dorsolateral cap over the dmnX or bordering the solitary tract. Examples of ENK-immunoreactive projection cells are shown in Figure 14. DYN- and NPY-IR projection cells were observed in similar regions (Fig. 15), mostly in the rostral aspect of the nTS, where they extended from the caudal pole of the area postrema to the more anterior pole of the nTS. Most were therefore rostral to the majority of projection cells staining for SS or ENK. DYN-IR projection cells had the most caudal extension, and NPY-IR projection neurons had the most rostral one. At all levels they included an area dorsal to the dmnx (mostly mnTS). It was a t the most rostral levels that the NPY-IR projection neurons were inserted ventrolaterally between TS and dmnX (Fig. 16) or bordered the medial edge of the dmnX (levels E and F, respectively). These locations are close to those already reported for TH-IR projection cells in these areas. Although bilateral, most DYNand NPY-IH projection cells were ipsilateral to the injection site, especially at more rostral levels. NT-IR projection cells were located dorsally and just lateral to the area postrema in dnTS, and the majority projected to the parabrachial area. Other peptidergic projections. CCK- and SP-IR cells projected only to parabrachial areas (Fig. 17). SP-IR projection cells were numerous and were located close to SSand ENK-IR cells. Most were observed mainly in the caudal aspects of the nTS. Their distribution extended from the medullary spinal junction to the anterior pole of the area postrema mainly in the lateral half of the complex, including mostly nucleus comrnissuralis, mnTS, and some subnuclei surrounding the TS (nI, dlnTS). Other SP-IR cells were seen ventral to the dmnX along its border with the hypoglossal nucleus (XII). Projections were clearly bilateral; the caudally located cells projected contralaterally, and the rostrally located cells ipsilaterally. More contralaterally projecting cells were seen for SP-than for ENK- and SS-IN cells. The location of most of SP-IR projection cells was distinguished from that of TH-IR projection cells; however, a partial spatial overlap existed in areas that surrounded the solitary tract. CCK-IH projection cells were found a t levels close to the area postrema. This projection was ipsilateral. Cells were concentrated dorsally between AP and TS, including the dorsolateral nucleus. BB-IR projection cells were retrogradely labeled from the hypothalamus or parabrachial area and were observed in the dorsal nucleus at levels close the area postrema.

DISCUSSION The present study provides data on the contribution of catecholaniines and nine different neuropeptides in solitary efferents to Ihe parabrachial area, amygdala, hypothalamus,

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D. RICHE ET AL.

SS-IR Cells

ENK-IR Cells

A n . corn

dlnTS

1

0

vnTS

E

-T

F 7

&

nTS

Figs. 6-9. Location of peptide immunoreactive cells in the nucleus of the solitarv tract. Only one side is shown. Each dot represents one labeled celi.

Fig. 6. Somatostatin (SS-IR)and enkephalin (ENK-IR) immunore active cells. The open arrows in D point to cells that surround the TS.

NEUROTRANSMITTERS IN SOLITARY EFFERENTS

407

D Y N B-IR C e l l s

NPY-IR Cells

A n.com

B

C

D

E

F

Fig. 7. Dynorphin (DYN-IR) and neuropeptide Y (NPY-IR) immunoreactive cells. The open arrow in E points to DYN-IR cells that concentrate in ventrolateral areas, and the arrow in E points to NPY-IR

cells that insert ventrolaterally between TS and dmnX. Arrowheads in F point to cells that border the dmnX and nTS.

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SP-IR C e l l s

* k-q,

CCK-IR C e l l s

D

E

PV

0

Fig. 8. Substance P (SP-IR) and cholecystokinin (CCK-IR) immunoreactive cells. The arrow in D points to dorsally located cells that gather ventrally to area postrema.

409

NEUROTRANSMITTERS IN SOLITARY EFFERENTS

BB-IR C e l l s

NT-IR C e l l s

B

C

D

&

TS

f

vnTS

dmnX

E

F

dnTS

-7

n TS

Fig. 9. Neurotensin (NT-IR) and bombesin (BB-IR) immunoreactive cells. Arrows in D and E point to cells that gather ventralaterally to

the area postrema and in dorsal nuclei, respectively. Arrowhead in F points to BB-IR cells that border the dmnX and nTS.

D. RICHE ET AL.

41 0

TH-

(0)

IR PROJECTION CELLS TO

PB

HYP.

A

C

D dmnX

E

F

Figure 10 A-F

411

NEUROTRANSMITTERS IN SOLITARY EFFERENTS

TH- (*) IR PROJECTION CELLS TO

BST

Ce

4 J U\ \ p--

drnnX

Fig. 10. Location of tyrosine-hydroxylase immunoreactive (TH-IR) projection cells following an injection in parabrachial area (PB), hypothalamus (Hyp), bed nucleus of the stria terminalis (BST),and nucleus

centralis (Ce). Ipsilateral projection cells are on the right. Each dot represents one immunoreactive projection cell, one section per level.

41 2

D. RICHE ET AL.

Figs. 11, 12. Brightfield photomicrographs illustrating tyrosine hydroxylase immunoreactivity in the nucleus of the solitary tract (frontal section, 40-pm-thick sections) a t levels posterior to t h e area postrema (levels B and C, respectively). A: General view. T h e arrows point tcl a ventromedial group of immunoreactive cells t h a t do not contribute to

nTS projections. B: Higher magnification of the boxed areas of the medial nucleus in A. Arrows point to double-labeled cells, i.e. immunoreactive cells (diffuse cell staining) containing granular silver precipitate t h a t prove retrograde transport from the injection site. Arrowheads point to immrinoreactive nonprojection cells.

and bed nucleus of stria terminalis. Catecholamines and a variety of peptides were observed in projections to all these targets. Catecholaminergic projection cells were the most numeroub and projected primarily ipsilaterally, whereas neuropeptidergic projection cells contributed to bilateral solitary efferents in different ways. In concordance with previous investigations, this study emphasizes the high density of catecholamines and the large variety of peptides that contribute to solitary efferents. Catecholaminergic projections arose mainly from mnTS and. to a lesser extent, from some subnuclei immediately surrounding the solitary tract. They densely innervated all targets. l h e density and widespread distribution of these projections plead for a major role of these projections in the well-known effect of catecholamines in cardiovascular regu-

lation (Korner et al., '87) and feeding processes (Bhakthavatsalam and Leibowitz, '86; Jiang and Oomura, '88; Lenard and Hahn, '82). The location of these catecholaminergic projection cells closely resembles those described in previous reports (Day et al., '80; Kawai et al., '88b; Milner et al., '86; Saper et al., '83; Sawchenko and Swanson, '82; Sawrhenko et al., '85). Neuropeptidergic projections arose from neurons located in lateral suhnuclei immediately surrounding the solitary tract. They reached all targets studied. Projections of caudal origin reacted mainly for SS. ENK, or SP. Their locations are similar to those reported for SS-1H projections to the hypothalamus (Sawchenko e t al., '88a) or for ENK- and SPIR projections to parahrachial area (Maley and Panneton, '88; Milner and Pickel, '86a). Projections originating in ros-

413

NEUROTRANSMITTERS IN SOLITARY EFFERENTS

Figure 12

tral regions reacted mainly for DYN or NPY. The location of the NPY -INprojection cells was close to that previously demonstrated for hypothalamus (Sawchenko et al., '85); DYN-IR projections have not been studied previously. The NT-IR projections arose from dorsal subnuclei, but they were not as dense or extending as far caudally as those to the parabrachial area reported by Milner and Pickel ('86b). This report is the first to demonstrate CCK- and BB-IR projections. Catecholaminergic and neuropeptidergic projections originated from distinct subnuclei, resulting in a partial spatial segregation of both populations. Clear spatial overlap, however, exists at rostra1 levels, between catecholaminergir and NPY immunoreactive cells. Such an area could correspond to that in which Sawchenko e t al. ('85) have demonstrated

the coexistence of catecholamine and NPY in projection cells to the hypothalamus. Multiple neuroactive substances may coexist in nTS neurons (Everitt et al., '84; Harfstrand et al., '87b; Hokfelt et al., '84; Kawai et al., '88a; Kubota et al., '86; Milhorn e t al., '87), and i t is thus very likely that some neuropeptides and/or neurotransmitters may coexist in the same projection cell. This is probable in subnuclei immediately surrounding the solitary tract, for SS-, ENK-, and SP-IR projection cells caudally, for SS-,ENK-, and catwholamine-IR projection cells at levels close the area postrema, and for T H - and CCK- or TH- and NT-IR projection cells i n dorsal subnucleus. The nTS is a major visceral afferent nucleus that is concerned with cardiovascular, respiratory, and gustatory control (Kalia et al., '84; Leslie, '85; Onai et al., '87). Different

D. RICHE ET AL.

414

SS-

(0)

& ENK- (*) IR PROJECTION CELLS TO

PB

HYP-

A

B

drnnx

Figure 13 A-F

NEUROTRANSMITTERS IN SOLITARY EFFERENTS

SS-

( 0 )

415

& ENK- (*> IR PROJECTION CELLS TO

BST

Ce

*&

nl

)) drnn X

Fig. 13. Location of somatostatin (0)and enkephalin (*) immunoreactive projection cells. Details as in Figure 10. Two sections per level.

416

D. RICHE ET AL.

Fig. 14. Brightfield photomicrographs illustrating enkephalin immunoreactivity in a n area ventromedial to the solitary tract (TS) a t a level just posterior to the area postrema (level C, frontal section, 40 p m thick section). A General view. B: Higher magnification of the boxed

area in A. Arrows point to double-labeled cells, the arrowhead to an enkephalinergic cell, and the white arrow to a projection cell, respectively.

srihnuclei have been associated with different functions. The medial nucleus has dense afferents from cardiovascular and haroreceptor origins; gastrointestinal afferent inputs project predominantly to its medial pole. The subnuclei immediately surrounding the solitary tract are mostly responsible for integrating respiratory functions, with the vlnTS and vnTS associated with pulmonary afferents and the nI with tracheal afferents. The dlnTS and dnTS nuclei, which receive baroreceptor and chemoreceptor afferents as well as the commissural nucleus, which receives dense

cardiac afferents, are more concerned with cardiovascular functions. The nTS has reciprocal projections with the hypothalamus, bed nucleus of stria terminalis, amygdala, and parabrachial nuclei that process similar information (see refs. in Smith and De Vito, '84). The neuroactive suhstances contained in the nTS efferents consequently could be important in coordinating several autonomic functions (see references in Gardiner and Bennett, '89; Korner et al., '87). The present study combines the tracing method with imniunocytochemistry of a wide range of neuroactive

NEUROTRANSMITTERS IN SOLITARY EFFERENTS substances and provides information relevant to the neurochemical organization of these neuronal networks. Considering the wide distribution of the origins of these projections, it appears that catecholaminergic and neuropeptidergic solitary efferents may have functional roles in modulating-simultaneously-respiratory, cardiovascular, and feeding processes a t the level of their respective targets. Endogeneous opioid projections consisted of enkephalinand dynorphin-IR cells and reached all targets. Opioids act as feeding stimulators. The neural substrates of opioidinduced feeding include the hypothalamus and amygdala (Gosnell, '88; Gosnell eta]., '86; Grandison and Guidotti, '77; Leihowitz and Hor, '82; Mac Lean and Hoebel, '83; Stanley et al., 1989; Tepperman and Hirst, '83; Woods and Leibowitz, '85). This effect is coincident with an increase in blood pressure and heart rate (Saunders and Thornhill, '87). Opioids microinjected i n the anterior hypothalamus also induce hyperthermia (Martin and Morrison, '78). A role for the solitary tract nucleus projections demonstrated in this study in the processes modulated by opioids, a t either hypothalamic or amygdalar levels, is thus highly probable. Somatostatin-IR cells projected to all targets. Somatostatin is a potent regulator of feeding acting to reduce food intake. A central site of action is the dorsal hypothalamic area (Brown, '83; Ho et al., '89; Iguchi et al., '84), and its action is through the regulation of adrenal epinephrine secretion (Fischer and Brown, '80) and via interaction with bombesin (Brown, '83). A role in the regulation of oxytocin release at hypothalamic levels has also been envisaged (Sawchenko et al., '88a). It is probable that the solitary hypothalamic projections we have shown are part of the somatostatinergic neuronal network involved in such effects. Somatostatin also acts in central cardiovascular control mechanisms (Gardiner and Bennett, '89). Microinjection of somatostatin in the central nucleus of the amygdala produce increases of mean arterial pressure and decreases of heart rate without affecting plasma catecholamine concentrations (Brown and Gray, '88), an effect that could be partly dependent on the somatostatinergic solitary tract nucleus projections. Bombesin-IK projections reach the parabrachial area and the hypothalamus. Bombesin acts as a feeding inhibitor a t both hypothalamic and amygdalar levels (Bellinger and Bernardis, '84; Kyrkouli et al., '87; Stuckey and Gibhs, '82). Hypothalamic injection of bombesin increases blood glucose, free fatty acids, corticosterone (Brown, '83; Gunion et al., '89; Iguchi e t al., '84), and plasma concentration of epinephrine (Brown, '83) and inhibits vagus-stimulated gastric acid secretion (Okuma et al., '87). Its action on plasma concentration of glucose and epinephrine at hypothalamic levels is prevented by somatostatin (Brown, '83). The bombesin-IR projections to hypothalamus we have observed could have a role in the regulation of those effects. Bombesin also acts in the cardiovascular sphere (Gardiner and Bennett, '89). If microinjected into the central nucleus of the arnygdala, it induces an increase of arterial pressure, but it does not alter heart rate (Brown and Gray, '88). The paucity of hombesin-IR fibers in nTS projections to amygdala does not plead fnr a role of nTS efferents in such an effect. Cholecystokinin-IR cells projected to the parabrachial area. Cholecystokinin acts in attenuating feeding response, and this effect is vagus dependent (Smith et al., '81) and probably exerted by altering the animals reactivity to food taste (Hsiao and Deupree, '83; Waldbillig and O'Callaghan,

417 '80). The cholecystokinin-IR projections demonstrated here could be involved in this effect, as they originated from a solitary area that is densely innervated from the upper alimentary tract (Altschuller et al., '89) and that is connected to a parabrachial area that includes a taste area (Norgren and Leonard, '73). The paucity of cholecystokinin-1R fibers in nTS efferents to hypothalamus indicates that the solitary-hypothalamic connections are probably not involved in the regulation of food intake that cholecystokinin may exert at the hypothalamic level (Myers et al., '86). Neuropeptide Y-IR projections reach all targets. Harfstrand e l al. ('87b) have shown that NPY-IR cells in the nTS lay in the site of termination of aortic and carotid sinus nerve afferent fibers. NPY-IR cells in this location are ideally situated for receiving monosynaptic input from baroreceptor aff'erents and for playing a key role in the central integration of cardiovascular reflexes. The NPY-like projection from nTS we have demonstrated to hypothalamic, limbic, and parabrachial areas could be part of that network. They could be involved in the blood pressure regulation exerted by the neuropeptide Y at hypothamic levels (Harland et al., '88; Martin et al., '88). A role in food intake a t hypothalamic levels may also be envisaged (Morley et al., '85; Stanley and Leibowitz, '85). Neurotensin-IR projections reach all targets but are sparse. The central administration of neurotensin produces a variety of behavioral alterations. Neurotensin is a feeding inhibitor that acts a t hypothalamic levels to induce satiety (lguchi et al., '84); it also acts as an hypotensor when injected intracerebroventricularly although its central level of action is still unknown (Gardiner and Bennett, '89). As neurotensin-IR cells in solitary complex are located in areas that receive afferents from the alimentary tract (Altschuler et al., '89) or aortic afferents (Higgins et al., '84), the neurotensin-IR solitary efferents we have demonstrated are ideally situated to act in the control of those feeding and cardiovascular behaviours. Neurotensin also acts as a modulator of body temperature or nociception at hypothalamic levels (Kalivas et al., '82b; Martin et al., '80), another effect that could be dependent on nTS efferents. Neurotensin may also produce antinociception at amygdalar levels (Kalivas et al., '82a,b), but this effect seems more relevant to the connections of the amygdala with stria terminalis. Substance P-IR cells project to parabrachial area. The pressor effect this substance has following intraventricular injection (Gardiner and Bennett, '89) could depend partly on such neuroanatomical connections. The action of suhstance P on glucoregulation a t hypothalamic levels does not seem to depend on a solitary tract nucleus projection (Iguchi et al., '84). That catecholaminergic and most neuropeptidergic neurons seem to be distinct subgroups in nTS efferents also applies to spinal ascending tracts (Leah et al., '88). I n both cases, peptidergic efferents are minor in number and restricted mostly to specific areas. In the spinal cord, there is a large contingent of peptidergic neurons that are interneurons or short projection neurons. One hypothesis is that spinal and nTS projection systems are organized in a similar manner. Both are divisible into neurotransmitter and peptide-containing groups of cells that convey information in a parallel fashion. Indeed, it is becoming increasingly evident that many neuropeptides are involved in modulatory functions (Harfstrand et al., '87a; Leah et al., '88). Theoretically peptidergic projections could modulate the physiological state of the target nuclei by regulating blood flow and

D. RICHE ET AL.

41 8

DYN-

(0)

& NPY- (*) IR PROJECTION CELLS TO

PI3

HYP.

A

= aXI1

A

C

D

E

F Figure 15 A-F

419

NEUROTRANSMITTERS IN SOLITARY EFFERENTS

DYN-

( 0 )

& NPY- (*) IR PROJECTION CELLS TO

Ce

BST

dmnX

-7

7

j G r n n x

nTS

Fig. 15. Location of dynorphin (0)and neuropeptide Y 1). imrnunoreactive projection cells. Details as in Figure 10.Two sections per level.

420

D. RICHE ET AL.

421

NEUROTRANSMITTERS IN SOLITARY EFFERENTS

SP-& CCK-IR PROJECTION CELLS TO PARABRACHIAL AREA

A

B

C

dlnTS

vlnTS

vnTS

Fig. 17. Location of substance P (left) and cholecystokinin (right) immunoreactive projection cells to parabrachial area. Details as in Figure 10. Two sections per level.

D. KICHE ET AL.

422 energy metabolism. Through such supporting functions, they might perhaps modulate convergent nonpeptidergic afferent neurotransmission.

ACKNOWLEDGMENTS The authors are indebted to P. Sanderson for her kindness in preparing the English text and are also grateful to A. Menktrey for making illustrations and M. Cayla for typing the k x t . This study was supported by INSERM funds. D. MenCtrey and D. Riche are supported by “Centre National de la Recherche Scientifique.”

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