Visualization of an efferent projection route of the hypothalamic rat arcuate nucleus through the stria terminalis after labeling with carbocyanine dye (DiI) or proopiomelanocortin-immunohistochemistry

ELS EVIER

Neuroscience Letters 172 (1994) 134-138

,, ,

Visualization of an efferent projection route of the hypothalamic rat arcuate nucleus through the stria terminalis after labeling with carbocyanine dye (DiI) or proopiomelanocortin-immunohistochemistry R a b i a M a g o u l a'*, P h i l i p p e C i o f i b, G 6 r a r d T r a m u a aLaboratoire de Neurocytochimie Fonctionnelle, URA CNRS 339, UniversitO de Bordeaux L Avenue des facuttOs, 33405 Talence cedex, France bU.156 INSERM, Lille, France Received 5 January 1994; Revised version received 21 March 1994; Accepted 21 March 1994

Abstract The hypothalamic arcuate nucleus is known to be reciprocally connected to various limbic regions, such as the bed nucleus of the stria terminalis (BST). The route of this reciprocal connection, in particular with the BST, remains unknown. In order to visualize this pathway, we used the fluorescent tracer carbocyanine dye (DID, that was inserted in the arcuate nucleus in fixed and dissected brains. This allowed us to label an arcuate-BST pathway DiI-labeled coursing through the stria terminalis. Immunohistochemistry for the arcuate-derived peptide adrenocortico-tr0pin (ACTH) revealed the presence of ACTH-immunoreactive axons in the stria terminalis. Together, these results provide arguments in favour of the existence of an arcuatofugal projection to the BST via the stria terminalis. Key words: ACTH; Amygdala; Arcuate nucleus; Bed nucleus; DiI; Preoptic area; Proopiomelanocortin; Stria terminalis

The arcuate nucleus (ARC) is located in the mediobasal hypothalamus immediately dorsal to the median eminence with which it shares a common arterial supply [1]. The ARC is considered to lie outside the blood-brain barrier, with an extracellular continuum separated from that of the rest of the hypothalamic nervous tissue by a palisade of tanycytes restricting diffusion of portal blood-borne substances within its limits [11]. In addition to receiving potential humoral inputs, the ARC is also connected to a variety of limbic nervous system regions [2]. The ARC therefore appears to be exquisitely situated to play a pivotal role in integrating and redistributing both humoral and nervous information. If a sum of data is available concerning the afferent connections of the ARC, knowledge of its efferent connections is comparatively poor and essentially derived from immunohistochemical mappings of the projections of the mediobasal hypothalamus proopiomelanocortin (POMC)-containing neurons [7], with no comprehensive

* Corresponding author. 0304-3940/94/$7.00 © 1994 Elsevier Science Ireland Ltd. All rights reserved SSDI 0304-3940(94)00244-5

neuroanatomical tract-tracing studies, This may be explained by the difficulty in reliably approaching the ARC from dorsal conventional routes using stereotaxy in such a small animal as the laboratory rat, given the narrow mediolateral and dorsoventral extent of the nucleus and its close vicinity to the third ventricle. To circumvent this problem, we have attempted to label these efferent connections by implanting within the ARC under direct visual control in dissected fixed brains, crystals of the fluorescent liposoluble tracer DiI (1,1'-dioctadecyltetramethylindo-carbocyanine perchlorate). We describe here the distribution of labeled structures observed in some limbic regions with particular attention to the visualization of a projection route through the stria terminalis. Six young (150 g) male Wistar rats (Iffa-Credo, Lyon, France) were anesthesized and perfused with 500 ml of 0.1M phosphate buffer (PB, pH7.4) containing 4% paraformaldehyde. Brains dissected out in toto were then immersed for 2 h in the same fixative at room temperature. In three brains, the ARC was exposed at its mid-rostrocaudal level by a single transversal cut. Small

R. Magoul et al./Neuroscience Letters 172 (1994) 134 138

135

Fig. 1. a: photomicrograph showing the carbocyanine dye (Dil) implantation site in the arcuate (ARC); DiI-labeled nerve fibers seem to exit the ARC ventrolaterally. Retrogradely labeled cell bodies are seen in the ARC itself and in the adjacent hypothalamic ventromedial nucleus, b,c: fluorescent nerve fibers from the ARC travel in the dorsal supraoptic commissure (CSD) to either enter the amygdala (Am, arrow) or to run dorsally (arrowheads). d: similarly, and after ACTH-immunohistochemistry, positive fibers in the CSD either enter the amygdaloid nuclei (Am) or course dorsally, e: several nerve fibers are DiI labeled in the ipsilateral stria terminalis (st) and the ventral margin of the fimbria (fi). f: after ACTH immunohistochemistry, the stria terminalis (st) shows oblically sectioned immunoreactive fibers (arrowheads). 3 V, third ventricle; LV, lateral ventricle; OT, optic tract. Bars = 200/lm.

136

R. Magoul et al./Neuroscience Letter.s" 172 (1994) 134 138

Fig. 2. a,c: a considerable fiber contingent which is earbocyanine dye (DiI)-labeled appears within the bed nucleus of the stria terminalis (BST) at anterior (a) and posterior (c) level, surrounding and delineating the anterior commissure (ac) and the fornix (f); few retrogradely labeled cell bodies are observed. 3V, third ventricle; LV, lateral ventricle, b,d: ACTH irnmunohistochemistry visualizes a dense network of fibers in the same regions (b, anterior and d, posterior BST) Bars = 200/am.

crystals of DiI (Molecular ~obes, Eugene, Oregon) were implanted into the fight ARC (in its ventromedial region) in the three anterior halves, and also in the ARC mid-rostrocaudal portion by a ventral approach (at the level of the infund~alar sulcus) in the three other unsliced brains, after which tissues were immersed in fixative and transport was allowed to proceed for 5 months in the dark at 37°C. Brains were then cut coronally into thick slabs (2 mm) that were recut coronally at 50-80/~m in PB using a Vibratome (Lancer). The Vibratome sections were collected in PB, mounted on slides, coverslipped under a PB/glycerol (1/9 v/v) mixture and observed under an epifluorescence photomicroscope (Leitz) with the ill-

ter cube for rhodamine. In addition to DiI-labeled brains, three other brains from adult male rats were fixed as above, coronally cut on a freezing microtome (25/Lm) and processed for immunoperoxidase-diaminobenzidine visualization of the POMC fragment ACTH~ 24 using a previously described antiserum [14] and protocol including silver intensification [3]. Analysis of the implantation sites showed that after 5 months of transport DiI had diffused throughout the rostrocaudal extent of the ARC without spreading into adjacent areas (Fig. la). Similar results were obtained in whole or sliced brains. DiI is known to be transported in both anterograde and retrograde directions [5,10], and

R. Magoul et al. / ~v\,uroscience Letters 172 (1994) 134 138

labeling was accordingly found both in numerous emerging fibers from the ARC as well as in some cell bodies located at a distance from it in regions with established projections to the ARC such as the bed nucleus of the stria terminalis, the preoptic-anterior hypothalamus continuum and the medio-cortical amygdala [2,4,8,9,13,17]. The relative paucity of these DiI-labeled cell-bodies in comparison to the flamboyant labeling of nerve fiber tracts throughout the studied forebrain regions, however, indicates that Dil had preferentially been transported in the anterograde direction over the 5 month period adopted. This is also suggested by the fact that perikarya retrogradely labeled with Dil in this study were in the above-mentioned areas of much lower numbers than after retrograde tracing from the ARC of HRP or WGAapoHRP-gold complex, as reported [2,4,9,17] and observed by us [8]. A minor contingent of labeled structures was commonly observed in areas contralateral to the implantation site. Our micrographs and following descriptions are focused on labeled pathways between the ARC and the bed nucleus of the stria terminalis (BST). Numerous DiIlabeled nerve fibers were seen to leave the ARC by a ventrolateral route (Fig. la), to course along the base of the hypothalamus up to the dorsal supraoptic commissure (Fig. lb,c) before splitting into two streams oriented, the frst one to the medial amygdala (an apparent terminal field) (Fig. lb,c) and the second one to the stria terminalis (Fig.lc) in which numerous labeled axons were observed at every rostrocaudal levels (Fig. le). Labeled axons were also conspicuous in the ventral margin of the fimbria. The BST was observed to contain very dense networks of fluorescent fibers throughout its rostrocaudal extent with streams arching medially around the anterior limb of the anterior commissure (Fig. 2a). Abundant fluorescent fibers were also present in the preoptic area. The subiculo-hippocampal formation was devoid of Dil-labeled nerve cell bodies and fibers. The distribution and apparent orientation of ACTHimmunoreactive (ir) axons in the forebrain areas investigated were in perfect agreement with previous descriptions [7] and in addition closely matched those of DiI-labeled fibers in respective regions (compare Figs. lc with ld, 2a with 2b, 2c with 2d). Especially, obliquely sectioned ACTH-ir axons were constantly observed within the stria terminalis (Fig. le,f). This reinforces the view that the majority of DiI-labeled fibers in the amygdala, stria terminalis and BST originate from the ARC. Our observations suggest that these arcuate efferent projections use the following route: they exit the nucleus ventrolaterally, pass below the ventromedial nucleus, course towards and enter the ventral amygdalofugal pathway where they give off a branch terminating within the medial amygdala while the remaining axons enter the stria terminalis to finally arborize in the BST and preot>

137

tic regions. The DiI-labeled fibers observed contralateral to the implantation site may arise from intra-arcuate crossed projections made through the subependymal zone of the median eminence where running fluorescent fibers were commonly observed. The existence of other efferent routes from the ARC to the anterior hypothalamus using the periventricular continuum or strial pathways in opposite direction (i.e., first to preoptic regions through the medial forebrain bundle, then to BST, stria terminalis and finally amygdala) cannot be neglected although our images suggest they would rather represent minor components. It is at present premature to ascribe a precise role to the projections evidenced in this study, although they appear parallel to diencephalic pathways of the vomeronasal system, an integrated complex sexually dimorphic in its organization and controlling reproductive endocrinology and behavior [12]. The fact that neuromodulators, such as POMC-derived peptides, neuropeptide Y, substance P and enkephalins, produced by projectionneurons of the ARC [2,7,15,16], are known to be deeply involved in regulating reproductive processes [6], suggests that the ARC projections visualized here constitute a multi-signaling pathway and reflects well the complexity of the organization and of the integrational potentialities of the medial basal hypothalamus. [l] Ambach, G. and Palkovits, M., The blood supply of the hypothalamus. In R Morgan and J. Panskeep {Eds), Handbook of the Hypothalamus: Anatomy of the Hypothalamus, Vol. 1, Marcel Dekker, New York, 1979, pp. 267 377. [2] Chronwall, B., Anatomy and physiology of the neuroendocrine arcuate nucleus, Peptides 6, Suppl. 2 (1985) 1 II. [3] Ciofi, P., Fallon, J.H., Croix, D., Polak, J.M. and Tramu, G., Expression of neuropeptide Y precursor-immunoreactivity in the hypothalamic dopaminergic tubero-infundibular system during lactation in rodents, Endocrinology, 128(1991 ) 823 834. [4] Gruber, K., McRae-Deguerence, A., Wilkin, L.D., Mitchell, L.D. and Johnson, A.K., Forebrain and brainstem afferents to the arcuate nucleus in the rat: potential pathways for the modulation of hypophyseal secretions, Neurosci. Lett., 75 (1987) 1 5. [5] H6nig, M.J. and Hume, R.I., Dil and DiO: versatile fluorescent dyes for neuronal labelling and pathway tracing, Trends Neurosci., 12(1989) 333 341. [6] Kalra, S.R and Kalra, RS., Steroid-peptide interactions in the endocrine brain: reproduction. In M. Motta led.), Brain Endocrinology, 2nd edn., Raven, New York, 1991, pp. 177 216. [7] Khachaturian, H., Lewis, M.E., Tsou, K. and Watson S.J., /~endorphin, c~-MSH, ACTH, and related peptides. In Bj6rklund, A. and H6kfelt, T. (Eds), Handbook of Chemical Neuroanatomy: GABA and neuropeptides in the CNS, Vol. 4, Part 1, Elsevier, Amsterdam, 1985, pp. 216 272.. [8] Magoul, R., Onteniente, B., Benjellouu. W. and Tramu, G., Tachykinergic afferents to the rat arcuate nucleus. A combined immunohistochemical and retrograde tracing study, Peptides, 14 (1993) 275 286. [9] Palkovits, M. and Zaborszky, L., Neural connections of the hypothlamus. In RJ. Morgane and J. Panksepp (Eds.), Handbook of the Hypothalamus, Marcel Dekker, New York, 1979, pp. 379 510. [10] Papadopoulos, G.C. and Dori, 1., Dil labeling combined with

138

[11] [12]

[I 3]

[14]

R. Magoul et al./~Y~uroscience Letters 172 11994,, 134 13,~

conventional immunocytochemical techniques for correlated light and electron microscopic studies, J. Neurosci. Methods, 46 0993) 251-258. R6thelyi, M., Diffusional barrier around the hypothalamic arcuate nucleus in the rat, Brain Res., 307 (1984) 355-3358. Segovia, S. and Guillamon, A., Sexual dimorphism in the vomeronasal pathway and sex differences in reproductive behaviors, Brain Res. Rev.,18 (1993) 51-74. Swanson, L,W., An autoradiographic study of the efferent connections of the preoptic region in the rat, J. Comp. Neurol., 167 (1976) 227-256. Tramu, G. and Dubois, M.P., Comparative cellular localization of corticotropin and melanotropin in Lerot adenohypophysis. An

immunohistochemical study, Cell. Tissue Res., 183 (1977) 557 569. [15] Tsuruo, Y., Kawano, H., Hisano, S., Kagotani, Y.. Daikoku, S.. Zhang, T. and Yanaihara, N., Substance P-containing neurons innervating LHRH-containing neurons in the septo-preoptic area of rats, Neuroendocrinology, 53 (1991) 236-245. [16] Yamano, M., Tohyama, M., Shiosaka, S., Shiotani, Y., lnagaki, S. and Kito, S., Leucine-enkephalin-like immunoreactive fibers in the medial preoptic area of the rat: their distribution and origin, Exp. Brain Res., 60 (1985) 535- 541. [17] Zaborszky, L., Afferent Connections ot" the Medial Basal Hypothalamus, Springer, Berlin, 1982, 103 pp.