Calcitonin gene-related peptide-like immunoreactivity and its relation with neurotensin- and corticotropin-releasing hormone-like immunoreactive neurons in the bed nuclei of the stria

Brain Research Bulletin, Vol. 27, pp. 617624. 0 Pergamon Press plc, 1991. Printed in the U.S.A. 0361s9230/91 $3.00 + .oO

Calcitonin Gene-Related Peptide-Like Immunoreactivity and Its Relation With

Neurotensin- and Corticotropin-Releasing

Hormone-Like Immunoreactive Neurons in the

Bed Nuclei of the Stria Terminalis in the Rat

GONG JU

Department of Neurobiology, Institute of Neurosciences, The Fourth Military Medical University Xian, The People’s Republic of China 710032

Received 2 January 1991

JU, G. Calcitonin gene-related peptide-like immunoreactivity and its relation with neurotensin- and corticotropin-releasing hormone-like immunoreactive neurons in the bed nuclei of the snia terminalis in the rat. BRAIN RES BULL 27(5) 617-624, 1991. -The distribution of calcitonin gene-related peptide (CGRP)-like immunoreactive (LI) nerve cells and terminals (BST) was studied in the rat. Only the fusiform nucleus was found to consistently contain CGRP-LI neurons. The CRGP-LI terminals had a wide distribution in the BST, being most numerous in the oval and the fusiform nuclei. In the oval nucleus the CGRP-LI terminals formed characteristic “baskets.” Since the oval nucleus was known to be studded with neurotensin (NT)- and corticotropin- releasing hormone (CRH)-LI neurons, the relationship of the CGRP-LI nerves to NT- and CRH-LI neurons was investigated by means of a double immunostaining technique. A part of the CGRP-LI baskets were found to contain NT- or CGRP-LI neurons. The possible involvement of the CGRP/NT or CRH relationship in reciprocal connection between the BST and the parabrachial nucleus and in cardiovascular regulation is discussed.

Bed nucleus of the stria terminals Immunohistochemistry Rat

Calcitonin gene-related peptide Neurotensin Corticotropin-releasing hormone

THE bed nuclei of the stria terminals (BST) is an important component of the limbic system. It has been differently parcel- lated by different authors [for review see (12, 18, 32)]. The present article follows the nomenclature of a recent study by Ju and Swanson (12). The BST contains a great variety of neuropeptides. The distribution of some of them in the BST has been better studied, including neurotensin, corticotropin-releasing hormone, gala&, substance P, cholecystokinin, somatostatin, Met-enkephalin and vasoactive intestinal polypeptide, and each has a characteristic distribution (13, 17, 18, 32). Others have not yet been fully investigated, calcitonin gene-related peptide (CGRP) being one of them, though its presence in the BST has been demonstrated earlier (14,22). The present study, therefore, investigated the distribution of CGRP-like imrnunoreactiv- ity in the BST. It was found that in the oval nucleus of the BST, the CGRP-LI axons formed many basket-like terminals suggesting a close relationship with neurons in this nucleus. Since the oval nucleus is known to be densely packed with neurotensin (NT)- and corticotropin-releasing hormone (CRH)-LI neurons (13,18), the relationship of CGRP-LI terminals to NT- and CGRP-LI neurons was also studied.

METHOD

Tissue Preparation

Twelve adult male Sprague-Dawley rats were used, weighing 200-220 grams. Gperations and perfusion-fixation were per- formed under pentobarbital anesthesia (35 mg/kg IP). In some of the animals, 120 pg of colchicine dissolved in 20 pl of saline was injected intracerebroventricularly, 24-28 hours prior to sacrifice of the animals to enhance immunostaining of the cells. The rats were perfused via the ascending aorta with 150 ml of warm physiological saline followed by 800 ml of ice-cold 4% paraformaldehyde in phosphate buffer (pH 7.4) for 1.5 h. The brains were sectioned either on a freezing sliding microtome (30-50 pm) or in a cryostat (12-15 pm), mostly in coronal place. Two of the animals were cut horizontally.

Immunohistochemistty

Antisera. Anti-CGRP and anti-CRH were kindly donated by Dr. Vale (The Salk Institute, California). Anti-NT was kindly donated by Dr. Brown (The Salk Institute). All these antibodies

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ABOVE AND FACING PAGE

FIG. 1. Distribution of CGRP-LI terminals in the BST in rat 89LHL12. (A-H) Coronal sections arranged rostro- caudallv. For details of the brain sections see (12). ac, anterior comrnissure; AD, anterior dorsaf area; AL, anterior lateral area; AV, anterior ventral area; CP, caudate putamen; Fu, fusiform nucleus; fx, fomix; GP, globus pallidus: ic. internal capsme; If, interfascicutar nucleus; Ju, juxtacapsular nucleus; Mg, magnocellular nucleus; MP, medial preoptic nucleus; Ov, oval nucleus; PD. posterior dorsal nucleus; Pr, principal nucleus; Ps, parastrial nucleus, PVN. paraventricular nucleus; Rh, rhomboid nucleus; sm, stria medullaris; Tr, transverse nucleus; V. ventral nucleus; *. cerebral ventricle.

were raised in the rabbit. For distribution study the sections were stained with antise-

rum against CGRP. To study the relationship between CGRP-LI terminals and NT- and CRH-LI cells, consecutive sections were alternatively stained with antisera against CGRP, NT, and CRH in one animal, and double immunostaining was conducted in 6 cases with anti-CGRP combined with either anti-NT or anti- CRH.

Single imrnu~s~a~~i~g. The sections were incubated in anti- CGRP (1:3,000), anti-NT (1:3,000), or anti-CRH (1:2,000) for 3 days at 4’C, then stained with ABC technique (9) and visual- ized by glucose oxidase-DAB-nickel method (27). The final re-

action product was black in color. Doubie immu~osfainjng. The sections were first immuno-

stained with anti-CGRP serum as described in the previous para- graph. They were then incubated with antiserum against either NT or CRH for 2-3 days, at 4”C, and processed with ABC technique using DAB as chromogen so that the stained elements appeared brown, contrasting fairly sharply with the first immunostaining.

Controls for antisera specificity. The antisera were preincu- bated overnight at 4°C with the homologous synthetic peptides (1 mg.ml -’ of NT or CRH; lop7 M of CGRP). The immuno- stainings were completely blocked.

CGRP-LIKE IMMUNOREACTIVITY IN BST 619

To facilitate identification of the subdivisions of the BST, sections in some cases were counterstained with cresyl violet or neutral red.

The methods of sectioning and staining for each animal are summarized in Table 1.

RESULTS

According to Ju and Swanson (12), the BST is composed of a posterior and an anterior division. The anterior division can be further divided into anterior lateral, anterior dorsal, and anterior ventral areas. Within each of them several nuclei can be identified. The nuclei which appear in this article are: In the anterior lateral area, the oval nucleus (Ov) which occupies the dorsolat- eral part of this area (Fig. lB, C); the juxtacapsular nucleus (Ju), located ventrolateral to the oval nucleus next to the internal capsule (Fig. lB, C); the rhomboid nucleus (Rh), caudal to the oval nucleus lying between the internal capsule and the ventromedi- ally coursing stria terminalis (Fig. lE), and the transverse nucleus (Tr) in the caudal part of the anterior lateral area, in which the cells are transversely arranged. In the anterior ventral area,

the fusiform nucleus (Fu), which appears in coronal sections as a spindle-shaped cells mass at the ventral lateral border of the anterior half of this area (Fig. lB, C); magnocellular nucleus (Mg), adjacent to the posterodorsal nucleus (PD), rather ill defined on coronal sections but more clearly identifiable on hori- zontal sections (Fig. ID-F); and the ventral nucleus (V), the ventral most part of the caudal half of the anterior ventral area (Fig. lD-H). In the posterior division, the principal nucleus (Pr) (Fig. lE-H), an elongated nucleus densely packed with small neurons, and the interfascicular (If) nucleus lateral to it.

CGRP-Like Immunoreactivities

CGRP-like immunoreactivity in the BST occurred mainly in tine varicose nerve fibers and terminals (for brevity they will be referred together as terminals). Only relatively few CGRP-LI cells were identified.

CGRP-LI Nerve Cells

In the BST CGRP-LI cells were consistently seen only in the fusiform nucleus, where a moderate number could be identified in colchicine-treated cases (Fig. 2). Individual cells also ap-

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TABLE I

METHODS OF SECTIONING AND STAINING FOR EACH ANIMAL

Animals Sectioning Colchicine Immunostaining Counterstaining

89Al Fro C 40 p,m + NT,CGRP,CRH(consec) 89A2 Fro C 30 p,m t CGRP/NT CGRPXRH 89A3 Fro C 50 pm + CGRP/NT CGRPKRH 89A4 CryC 12&m t CGRP/NT CGRPKRH 89LHL3 Fro C 40 pm CGRP C’V 89LHL4 Fro H 40 km CGRP (‘V 89LHL9 Fro C 40 km + CGRP (‘V 89LHLlO Fro H 40 km _ CGRP NR 89LHL12 Fro C 40 km _ CGRP CV 89LHL13 Fro C 40 pm + CGRPiNT CGRPKRH 89C1 CryC15pm + CGRP/NT CGRPKRH 89C2 Fro C 40 km t CGRPiNT CGRPKRH

CGM’/, double immunostaining with; consec. consecutive; Cry, cryostat; CV, cresyl violet; Fro, frozen; NR, neutral red,

peared occasionally in the oval nucleus.

CGRP-W Nerve Fibers

When traced rostro-caudally in coronal sections, the CGRP-Ll terminal area in the nucleus accumbens was found to continue into the anterior end of the BST as a dense vertical band curv- ing around the medial border of anterior commissure (Fig. 4). At the anterior part of the BST this band bordered the medial and ventral sides of the anterior commissure.

The CGRP-LI terminals were found mainly in the anterior division of the BST, most numerous in the oval nucleus and the fusiform nucleus (Fig. 1).

In the anterior dorsal area only a small amount of CGRP-LI terminals were found. At its anterior part, more were seen in the central region (Fig. 1A).

In the anterior lateral area there was a wealth of CGRP-LI terminals. The oval nucleus was the area where the densest accumulation of CGRP-LI terminals were found in the BST (Figs. lB, C; 8B). In certain places this densely stained area extended medially into the anterior dorsal area (Fig. 1B). Also strands of CGRP-LI fibers were seen to continue from the oval nucleus laterally into the medial part of the caudate putamen (Figs. 1 A, B). It was characteristic of the oval nucleus that the CGRP-LI terminals formed many baskets (Fig. 3). Occasionally, the baskets showed short processes. The juxtacapsular nucleus situated ventrolateral to the oval nucleus contained only scattered terminals, contrasting sharply with the oval nucleus (Figs. lB, C; 8B). A small to moderate number of CGRP-LI terminals were located in the rhomboid nucleus (Figs. lD, 5). A small number of terminals could be identified in the transverse nucleus (Figs. IG, H). Into the caudal ventral part of the anterior lateral area extended the dense immunostaining of the fusiform nucleus of the anterior ventral area (Figs. lD-G), which could be. further traced ventrolaterally along the ventral border of the globus pallidus to the claustrum and amygdala (Figs. ID-H).

CGRP-like immunoreactivity in the anterior ventral area was, in general, weak to moderate, except in the fusiform nucleus, where CGRP-LI terminals were fairly prominent (Fig. lB, C; 6, 7). Unlike the oval nucleus the CGRP-LI terminals in the fusiform nucleus did not show basket formations. The immunoreactive terminal area of the fusiform nucleus extended caudally into the anterior lateral area (Figs. 1D-G). A small to moderate amount of CGRP-LI terminals were unevenly distributed in the magnocellular and ventral nuclei.

The posterior division of the BST was weakly immunoreactive (Figs. lE-H). Only scattered nerve fibers could be identified in the caudal part of the principal nucleus. Somewhat more fibers were found in its rostra1 half. The interfascicular nucleus was more intensely labeled (Figs. 1 F-H).

Relation Between CGRP-LI Nerve Terminals and NT- and CRH- LI Nerve Cells

Consecutive sections. In rat 89A1, consecutive sections were stained with antisera against CGRP, NT, and CRH. The oval nucleus was densely innervated with CGRP-LI terminals (Fig. 8B). It also contained a wealth of NT- and CRH-LI nerves cells and terminals (Figs. 8A, C). By comparison of consecutive sections it was evident that the CGRP-LI terminal area partially overlapped the NT- and CRG-LI cell areas.

Double immunostaining. At the center of CGRP-like immunoreactivity in the oval nucleus the immunostaining was so dense that the relationship of CGRP-LI terminals with NT- and CRG-LI cells was often masked. At less densely stained areas the CGRP-LI terminal baskets could be found to surround NT-L1 (Fig. 9) or CRH-LI cells (Fig. lo), contacting their somata. Oc- casionally, CGRP-LI terminals were seen to line along their proximal dendrites. Often NT- or CRH-LI cells were seen naked close to dense CGRP-LI terminals (Fig. 10).

In the fusiform nucleus the CRH-LI cells were located among dense CGRP-LI terminals, but they did not show the kind of close relationship with the CGRP-LI terminals they had in the oval nucleus.

DISCUSSION

It has repeatedly been proved that double immunoprecipitate staining with antisera raised in the same animal species is possible (1, 11, 20). Provided the first peroxidase reaction is well done so that enough precipitate gathers around HRP-IgG com- plexes, there will be little cross-immunostaining. The possibility of cross-staining was even less a problem in the present study. Because in the oval nucleus there were virtually no CGRP-LI cells, rendering the identification of the brown immunostained NT- and CRH-LI cells an easy practice.

CGRP is a 37 amino acid polypeptide, widely distributed in the central nervous system, including the BST (14,22). How- ever, no detailed account of its distribution pattern in the BST is available. The present study shows that the BST has a fairly rich

CGRP-LIKE IMMUNO~ACTIVITY IN BST 621

PIGS. 2-5.

PlG. 2. CGRP-LI nerve cells and terminals in the fusiform nucleus. Bar= 50 urn. PIG. 3. CGRP-LI terminals in the oval nucleus. Note the formation of terminal baskets (arrows). Bar=20 p,rn. FIG. 4. CGRP-LI fibers in the transitional zone between the nucleus accumbens and the BST. ac, anterior commissure. Bar= 50 p,m, FIG. 5. CGRP-LI terminals in the rhomboid nucleus (Rh). ic, internal capsule; st, stria texminals. Bar= 100 pm.

innervation of CGRP-LI terminals, more in its anterior division, particularly the oval nucleus and the fusiform nucleus. As is often the case (13), the distribution of the immunoreactive terminals does not always strictly observe the cytoarchitectural confines of the nuclei. The dense accumulation of CGRP-LI terminals in the oval nucleus contrasts sharply with virtually negative stain-

ing in the nearby juxtacapsular nucleus, which further jur itified their identification by McDonald (16) and Ju and Swanson (12). The CGRP-LI terminals in the oval nucleus of the BST form characteristic baskets. This situation bears some resemblar me to the CGRP-LI terminals in the lateral septal nucleus. In the latter many CGRP-LI terminals were found to gather around cell bod-

PlGS. 6-8.

FIG. 6. CGRP-LI terminals in the anterior part of the fusiform nucleus (Fu), corresponding to the level in Fig. 1B. Bar= 100 pm. FIG. 7. CGRP-LI terminals in the posterior part of the fusifonn nucleus (Fu), corresponding to the level in Fig. 1C. Bar= 100 pm. FIG. 8. Consecutive sections cutting through the oval nucleus and the juxtacapsular nucleus (Ju) and stained with antisera against NT (A), CGRP (B), and CRH (C). Bar= 100 pm.

ies and the proximal dendrites, so much so that they even dis- played silhouettes of neurons. Kawai et al. (14) have made the same observation in the lateral septal nucleus. But basket formations of CGRP-LI terminals in the BST apparently escaped their notice. Recently, Gustafson and Greengard (5) have also re- ported a dense accumulation of immunoreactive axons with peri- cellular arrays in the lateral dorsal BST, an area corresponding to our oval nucleus. They, however, have found only scattered axons in other parts of the BST. Whereas fairly dense terminals

have been identified in the fusiform nucleus in the present study. There are many other peptidergic terminals in the BST as well, containing CRH, NT, substance P, cholecystokinin, vasoactive intestinal popypeptide, or Met-enkephalin (13,32). The CGRP-LI terminals are unique for their basket formation. The formation of terminal baskets would suggest close synaptic contacts between CGRP-LI terminals and cells in the oval nucleus. The oval nucleus is densely packed with medium sized neurons (12), in which a large amount of NT-L1 and CRH-LI cells have been

CGRP-LIKE IMMUNOREACTIVITY IN BST 623

FIGS. 9-10.

FIG. 9. CGRP/NT double immunostaining. Note an NT-L1 cell em- braced by CGRP-LI terminals (arrow). Bar= 10 pm. FIG. 10. CGRPKRH double i~unos~n~. Note a CRH-LI cell in contact with CGRP-LI terminals (arrow) and a naked CRH-LI cell (open arrow). Bar= 10 pm.

identified (13, 18, 25, 32), many co-containing these two peptides (10, 25). Double i~unos~ning in this study proved that some NT-L1 and CRG-LI neurons are surrounded by CGRP-LI terminal baskets.

The fusiform nucleus is a well-identified nucleus both cytoar- chitecturally and chemo~chit~t~ally (12,13). An area corresponding to Ju and Swanson’s (12) fusiform nucleus has also been indicated in Paxinos and Watson’s atlas (21) without being nominated. It can also be easily recognized in Schober et al.‘s

(24) illustrations. It has been defined as ventral lateral subnu- cleus by Moga et al. (18), but at its rostral part they seemed to have included a larger area (their Fig. 9A) than JU and Swanson (12). This does not seem to be supported by the distribution of CGRP-like immunoreactivity shown in the present study. The fusiform nucleus is the only nucleus in the BST that has been found in the present study to consistently contain CGRP-LI neurons in any quantity. Occasionally scattered CGRP-LI cells oc- cur in the oval nucleus. In Kawai et al’s (14) mapping study the hippocampal formation and the amygdaloid complex are the only telencephalic structures that contain CGRP-LI cells. The fusiform nucleus is also rich in CGRP-LI terminals. The presence in it of CRH-LI neurons confirms Ju and Swanson’s find- ing (13). The cells lie in the mass of CGRP-LI terminals, suggestive of inputs from the latter, though terminal baskets have not been found.

The BST has wide connections with the telencephalon and the brainstem (4, 8, 18, 23, 26, 28-31). The only telencephalic structure that contains a substantial amount of CGRP-LI neurons, as identified by Kawai et al. (14), is the gyms dentatus of the ventral hippocampal formation. However, the major hippocampal input of the septal region is not to the BST (30), and it is the subiculum that has been found to project to the BST (29). It is, therefore, likely that the CGRP-LI terminals in the BST mainly originate from brainstem structures. Among the candidates the parabrachial nucleus has been demonstrated to have a wealth of CGRP-LI neurons in the rat (14, 22, 26) and project to the BST (23.26). Using combined retrograde tracing and immunohistochemical technique, it has been demonstrated by Gu and Ju (unpublished), that a large number of CGRP-LI neurons in the lateral parabrachial nucleus do project to the oval and fusiform nuclei of the BST. It is interesting to note that the NT- and CRH-LI neurons in the area corresponding to the oval nucleus have recently been shown to project to the parabrachial nucleus (18). It is, therefore, likely that the connections between CGRP-LI terminals and NT-, CRH-LI neurons in the oval nucleus may close the circuit of the reciprocal connections between the parabrachial nucleus and the BST. Similarly, there are a few CGRP-LI cells in the nucleus of the solitary tract (14), which is reciprocally connected with the BST (28), and Gray et al. (5) have found that some of the NT- and CRH-LI neons in the dorsal part of the lateral division of the BST, corresponding in position to the oval nucleus, project to the nucleus of the solitary tract and the dorsal motor nucleus of the vagus nerve. Both the parabrachial nucleus and the dorsal vagal complex are known to be related to cardiovascular functions (2, 6, 7, 17, 19). There is evidence suggesting that the BST may also be involved (15). In this connection, it is interesting to note that intracerebroven- tricular injection of CGRP has been found to increase arterial blood pressure and heart rate (3). This, of course, can be the result of the involvement of a variety of structures. However, the oval nucleus may well be one of the candidates.

This study was supported by the National Natural Sciences Founda- tion of China. The skillful technical assistance of Miss H. L. Liu and Ms. F. M. Li is gratefully acknowledged.

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Calcitonin gene-related peptide-like immunoreactivity and its relation with neurotensin- and corticotropin-releasing hormone-like immunoreactive neurons in the bed nuclei of the stria