Cell Tissue Res (1992) 269:21-27

Cell&Tissue Research 9 Springer-Verlag 1992

The origin of the luteinizing hormone-releasing hormone (LHRH) neurons in newts (Cynops pyrrhogaster): the effect of olfactory placode ablation Shizuko Murakami 1, Saka~ Kikuyama 2, and Yasumasa Arai 1 i Department of Anatomy,Juntendo UniversitySchoolof Medicine,Hongo, Tokyo, 113 Japan z Department of Biology,School of Education,WasedaUniversity, Shinjuku,Tokyo, 169 Japan Received December 30, 1991 / AcceptedMarch 6, 1992

Summary. Neurons containing luteinizing hormone-releasing hormone (LHRH) are first detected in newt embryos (Cynops pyrrhogaster) in the olfactory epithelium and ventromedial portion of the olfactory nerve, after which they sequentially appear in the intracerebral course of the terminal nerve at prometamorphosis, and in the septo-preoptic area at postmetamorphosis. In adults, however, LHRH-immunoreactive cells are rarely seen in the nasal region, and their distribution shifts into the brain, suggesting their migration. In order to ascertain the origin and possible migration route of these neurons in newt larvae, the effect of unilateral or bilateral olfactory placodectomy on the L H R H neuronal system has been studied. Removal of the olfactory placode results in the absence of LHRH-immunoreactive cells in the nasal and brain regions of the operated side, whereas the subsequent growth and the LHRH-immunoreactive cellular distribution in the contralateral side are identical to those of normal larvae. Following bilateral placodectomy, no L H R H immunoreactivity is detected on either side of the olfactory-brain axis. These results suggest that LHRH neurons of the newt, Cynops pyrrhogaster, originate in the olfactory placode and then migrate into the brain during embryonic development. Key words: LHRH-containing neurons - Olfactory placode, origin - Terminal nerve - Development - Cynops pyrrhogaster (Urodela)

In various vertebrates, neurons containing the luteinizing hormone-releasing hormone (LHRH) are primarily found in the septo-preoptic area and hypothalamus; they have also been detected in the olfactory system including the terminal nerve (TN) (Demski 1984; Demski and Schwanzel-Fukuda 1987; Silverman 1988). Recent studies on the development of the L H R H neuronal system in mammalian and avian species have indicated that

Correspondenceto: Y. Arai

LHRH-positive cells are detectable in the nasal region prior to their appearance in the brain, and that the distribution of the main cell population containing LHRH shifts from the olfactory region to the brain along the TN or the olfactory nerve (ON) as development progresses (Schwanzel-Fukuda and Pfaff 1989; Wray et al. 1989; Ronnenkleiv and Resko 1990; Daikoku etal. 1991; Murakami etal. 1991; Norgren and Lehman 1991). Based on these findings, it has been suggested that the L H R H neurons originate in the olfactory placode and migrate into the brain along the TN and/or the ON during development. In adult amphibians, many LHRH neurons are distributed along the course of the TN (Wirsig and Getchell 1986; Muske and Moore 1988); those in the TN and septo-preoptic are a form a distinct anatomical continuum (Muske and Moore 1988). This finding has lead to the speculation that the septo-preoptic L H R H neurons are derived embryologically from the TN and/or the olfactory placode (Muske and Moore 1988). In this regard, in Rana catesbeiana, LHRH-immunoreactive (-ir) fibers can be detected in the TN around the onset of metamorphosis (Muske and Moore 1990). However, no detailed information is yet available on the L H R H neurons in the developing amphibian olfactory system. The purpose of this study has been to investigate the development of the LHRH neuronal system in the olfactory and brain regions of the newt. Furthermore, the olfactory placode in the newt embryo was surgically removed to ascertain the embryonic origin of the L H R H neurons and their possible migration route from the nose to the brain.

Materials and methods Fertilized eggs were obtained by injectingadult femalenewts (Cynops pyrrhogaster) containing stored sperm in their spermathecae with 25 IU human chorionic gonadotropin (Gonatropin, Teikoku Hormone Mfg, Tokyo)and I IU prolactin(Prolactin, TeikokuHormone Mfg) every day for 5 days. The developing embryos were kept at 23~ in tap water. After hatching, they were fed with

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Fig. 1. Scanning electron micrograph of a unilaterally placodectomized newt larva sacrificed 1 month postoperatively. Note the lack of the left nostril

Tubifex. To study the development of the LHRH neuronal system in this species in detail, 29 larvae at stages 26-29, 51, 55, or 58-59 (Okada and Ichikawa 1947), 12 newly metamorphosed juvenile newts, and 4 adult newts were sacrificed for LHRH immunohistochemical staining. Twenty-eight embryos at stages 26 or 29 underwent olfactory placode surgery, since the olfactory placode begins to invaginate to form the early olfactory pit during these stages. The operations were carried out in a gum-coated Petri dish filled with Holtfreter's solution that contained MS222 diluted 1:5000. The left olfactory placode or both olfactory placodes were excised using finely sharpened steel needles. The larvae were subsequently kept in Holtfreter's solution for 3 days and then transferred into tap water in separate glass containers. Placodectomy resulted in the absence of the nostril on the operated side (Fig. 1). The larvae were sacrificed for LHRH immunostaining at prometamorphosis (stages 58-59) or postmetamorphosis. For immunohistochemistry, animals were decapitated and fixed in Bouin's solution without acetic acid overnight at 4~ C, after which they were immersed for a few days or several weeks in 0.1 M phosphate buffer (pH 7.4) containing 20% sucrose. Serial sections of each entire head were cut at thickness of 16 pm on a cryostat in transverse, sagittal, or horizontal planes, and mounted on glass slides that had been coated with egg white. The LHRH-producing neurons were immunohistochemically stained by the avidin-biotin peroxidase complex (ABC) method using a commercial kit (Vector Laboratories, Burlingame, Calif., USA). Sections were pretreated for the suppression of endogeneous peroxidase with 1% H202 in methanol, rinsed in phosphate-buffered saline (PBS), and incubated with a monoclonal antibody LRHt3 (HAC-MM02-MSM84) diluted 1:2000 in PBS containing 1% bovine serum albumin and 2% normal horse serum for 48 h at 4 ~ C. LRH13, which was kindly provided by Dr. Wakabayashi, has been found to demonstrate a high LHRH specificity and a wide cross-reactivity (Park and Wakabayashi 1986). The peroxidase complex was visualized by exposure to 3,3'-diaminobenzidine tetrahydrochloride and H202, after which the sections were counterstained with methyl-green, dehydrated, and mounted in Canada balsam. Specificity of the immunoreactions was determined by omitting the first antibody LRH13, and by pre-incubation of LRH13 with synthetic LHRH (1 pg/ml; Peninsula Laboratories, Calif., USA), In these cases, no immunoreaction was found in the nasal and brain regions. Results

Normal development of L H R H neuronal system N o L H R H i m m u n o r e a c t i v i t y in the e p i t h e l i u m o f the o l f a c t o r y p i t o r in a n y o t h e r a r e a s was d e t e c t e d in emb r y o s at stages 2 6 - 2 9 ( t a i l - b u d stage) or at stage 51.

Fig. 2A-C. LHRH-ir cells in the nasal region at premetamorphosis (stage 55). A A cluster of LHRH-ir cells in the ventromedial portion of the olfactory nerve (ON); cross section. B Sagittal section. C A lightly stained LHRH-ir cell in the basal portion of the olfactory epithelium; sagittal section. OE Olfactory epithelium; ON olfactory nerve; OB olfactory bulb. A Interference contrast optics. x 2 5 0 ; B , C x400 A t stage 55 ( p r e m e t a m o r p h o s i s stage), h o w e v e r , a cluster o f L H R H - i r cells was f o u n d in all i n s p e c t e d l a r v a e in the v e n t r o m e d i a l p o r t i o n o f the O N , a d j a c e n t to the o l f a c t o r y b u l b (Fig. 2A, B). These L H R H - i r cells were r o u n d o r f u s i f o r m ; their processes were either u n i p o l a r o r b i p o l a r , a n d were o r i e n t e d p a r a l l e l to the l o n g axis o f the O N . These p o s i t i v e n e u r o n s a p p e a r e d to be associated w i t h the p e r i p h e r a l c o u r s e o f the T N , the o r i e n t a tion o f these p o s i t i v e n e u r o n s a s s u m i n g a s i m i l a r p o s i tion in the O N in o t h e r species ( H e r r i c k 1906; M c k i b b e n 1911). I n the o l f a c t o r y e p i t h e l i u m , a small n u m b e r o f

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Fig. 3. A LHRH-ir cells in the ventromedial portion of the O N at prometamorphosis (stage 58, sagittal section). OE Olfactory epithelium; OB olfactory bulb. Interference contrast optics, x200. B LHRH-ir cells in the ventromedial portion of the O N in a postmetamorphic animal (sagittal section). LHRH-ir and LHRH-negative ceils are seen. The processes of the LHRH-ir cells extend from the O N into the OB. O N Olfactory nerve, x 400. C LHRH-ir cell (arrow) and fibers (arrowhead) in the intracerebral course of the TN in a prometamorphic larva at stage 58 (sagittal section). An LHRH-ir cell at the ventral surface of the forebrain can be seen

just caudal to the OB. Interference contrast optics, x 200. D Two LHRH-ir cells located more caudally on the ventral surface of the forebrain in this sagittal section at stage 58. D' High magnification of the area indicated by the arrow in D. A C Anterior commissure; L T lamina terminalis. D • 30; D' x 400. E An LHRH-ir cell body in the basal portion of the O E in a prometamorphic larva at stage 59 (cross section), x 400. F Two LHRH-ir cells in the lamina propria in a postmetamorphic newt, just beneath the basal portion of the OE. Interference contrast optics. • 500

lightly stained L H R H - i r cells were seen in the basal portion o f the epithelium, or j u s t b e n e a t h the e p i t h e l i u m (Fig. 2 C). T h e m o r p h o l o g y of these n e u r o n s was similar to those seen in the O N . F u r t h e r m o r e , in one o u t o f 5 larvae, a few L H R H - i r cells were f o u n d in the v e n t r a l

surface o f the r o s t r a l - m o s t p o r t i o n of the olfactory b u l b ; n o L H R H - i r cells were observed within the b r a i n s o f the other larvae. In p r o m e t a m o r p h i c larvae (stages 58-59), a small n u m b e r of L H R H - i r cells were f o u n d in the basal por-

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tion of the olfactory epithelium and, occasionally, just beneath the olfactory epithelium (Figs. 3 E, 6A). The majority of the LHRH-ir cells, which appeared to be associated with the TN, were found in the ventromedial portion of the ON adjacent to the olfactory bulb, where they were observed to intermingle with nonreactive cells in the ON (Fig. 3A). In the brain, a small number of LHRH-ir cells and fibers were found in the ventral portion of the olfactory bulb and also more caudally, on the basal surface of the forebrain (Fig. 3 C, D). LHRH-ir cells along the intracerebral course of the TN were detected in half of the larvae examined. However, no LHRH-ir cells were detected in the septo-preoptic area at these stages, although LHRH-ir fibers were distributed in the septo-preoptic region, in several instances. At postmetamorphosis, one or two LHRH-ir cells were still found in the nasal mucosa (Fig. 3F), and a considerable number of LHRH-ir cells were detected in the ventromedial portion of the ON (Figs. 3 B, 6 C). In all animals, LHRH-ir cells were found on the ventral surface of the forebrain throughout the course of the TN, and in the septo-preoptic area. Some LHRH-ir cells were present in the ventral forebrain just caudal to the lamina terminalis (Fig. 6 E). In adult newts, LHRH-ir cells were rarely seen in the nasal region, and LHRH-ir fibers were confined to the ventromedial portion of the ON (through which TN fibers are thought to pass). The LHRH-ir cells seen in the TN were concentrated in the proximal ON near the junction with the olfactory bulb. Furthermore, a number of LHRH-ir cells were located on the rostral forebrain surface along the course of the TN (Fig. 4); they were continuous with the major population of the septo-preoptic LHRH-ir cells lying close to the ventromedial surface of the brain (Fig. 5). This LHRH-ir cellular distribution pattern in adult newts was consistent with previous reports concerning LHRH-ir cellular distribution in adult urodeles (Nozaki et al. 1984; Wirsig and Getchell 1986; Muske and Moore 1988). Changes in the

Stage5

~

Fig. 5. Topographic distribution of LHRH-ir cells in the olfactory and forebrain regions in the newt. The solid dots represent L H R H ir neurons. AC Anterior commissure; G glomerular layer; OB olfactory bulb ; OE olfactory epithelium; ON olfactory nerve; POR preoptic recess; V third ventricle

topographic distribution of the LHRH-ir cells during the course of development are schematically summarized in Fig. 5.

Histological investigation following olfactory placode surgery Unilateral or bilateral placodectomy at stages 26 or 29 was successfully accomplished in 20 out of 28 larvae; compete ablation of the olfactory placode resulted in a permanent loss of the olfactory organ (Figs. 1, 6B, D, F). In the nasal region of the operated side, the nasal sac, the olfactory epithelium, the ON, and the TN bundles were not seen. Moreover, the glomerular structures in the olfactory bulb did not develop, and a reduction in the size of the olfactory bulb was noted. However, the other brain regions did not seem to be affected by the operation, and the development of the diencephalon in the placodectomized newts appeared to be normal. The results in the animals in which placode ablation was incomplete and olfactory structures developed were excluded from this paper.

Effects of olfactory placode ablation on the L H R H neuronal system Fig. 4. LHRH-ir cells (arrows) in a sagittal section through the brain of an adult newt located in the ventromedial olfactory nerve (ON) near the junction with the olfactory bulb (OB) and at the ventral surface of the rostral olfactory bulb. Arrowheads Stained fibers. Interference contrast optics, x 75

Observations in the animals that underwent complete removal of the left olfactory placode are summarized in Fig. 6. In all 3 unilaterally operated larvae at stage 26, reared until stages 58-59 (prometamorphosis), no

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Fig. 6A-F. Comparison of L H R H immunoreactivity between controls (A, C, E) and unilaterally placodectomized animals (B, D, F). A Horizontal section through the nasal region and the forebrain (FB) in a prometamorphic control. OE Olfactory epithelium; arrows LHRH-ir cells, x ]20. B Horizontal section through the same level in a unilaterally placodectomized animal. LHRH-ir cells only occur on the intact side (left, arrows). No LHRH-ir cells are seen on the operated side (right). x 120. C Cross-section through the nasal region and the forebrain in a postmetamorphic control animal. LHRH-ir cells are seen in the O N adjacent to the olfactory bulb on both sides. • 80. D In this newt subjected to a unilateral

operation, the distribution of the LHRH-ir cells on the unoperated side (left) is similar to that of the control, whereas no LHRH-ir cells are visible on the operated side (right). Black structures in the upper corners are the pigment layer of eye balls whose structural organization was artificially deteriorated in the process of sectioning; * sectioning artifact, x 60. E Cross-section through the septo-preoptic level of the brain in a postmetamorphic control. Note the bilateral appearance of the LHRH-ir cells in the septopreoptic area. x 320. F Cross section through the septo-preoptic area. Note the absence of LHRH-ir cells on the operated side (right). x 320

26 LHRH-ir cells and/or fibers were detected in either the nasal region or in the brain on the operated side (Fig. 6 B). In the undisturbed contralateral side of unilaterally operated larvae, however, LHRH-ir cells were observed in the nasal region and at the ventral surface of the rostral forebrain, i.e., along the intracerebral course of the TN, as in unoperated normal animals (Figs. 3 C-D, 6A-B). In all 4 larvae that were subjected to a unilateral operation at stage 26 and subsequently reared until postmetamorphosis, no LHRH-ir cells were detected in the nasal and brain regions of the operated side, whereas they were clearly seen along the course of the TN and in the septo-preoptic area of the unoperated side, as found during the course of normal development (Fig. 6 C-F). In all 13 larvae subjected to bilateral ablation of the olfactory placode at stages 26 or 29, no LHRH-expressing neurons were found in the olfactory-forebrain axis on either side, at either prometamorphic (10 larvae) or postmetamorphic (3 newts) stages.

Discussion

This study in the newt, Cynops pyrrhogaster, has shown that LHRH-ir neurons first appear in the olfactory epithelium and in the ventromedial portion of the ON prior to their appearance in the telencephalic and diencephalic portions of the brain, and that the location of the LHRH-ir cells changes gradually and progressively from the olfactory epithelium to the ventral forebrain area during the course of development. As far as is known, this is the first description of the ontogeny of the L H R H neuronal system in the developing Qlfactory-forberain axis in urodeles and the first experimental attempt to analyze this process. LHRH-ir cells are detectable in the placode of embryonic mouse and chick (SchwanzelFukuda and Pfaff 1989; Wray et al. 1989; Murakami et al. 1991; Norgren and Lehman 1991); however, the initial appearance of LHRH-ir cells in the newt is delayed until the time of differentiation of the olfactory epithelium. Nevertheless, the spatial-temporal appearance of the LHRH-ir cells during embryonic development in the newt closely resembles that observed in mammals and chicks, in that the L H R H neurons first appear in the medial olfactory placode and then migrate across the nasal septum to enter the forebrain with the TN and/or the ON (Schwanzel-Fukuda and Pfaff 1989; Wray et al. 1989; Murakami et al. 1991 ; Norgren and Lehman 1991). As has been shown in this study, the distribution pattern of LHRH-ir cells within the developing forebrain is similar to that of the LHRH-ir ganglion cells in the TN of an adult newt, forming a single, anatomically continuous distribution along the course of the TN (Muske and Moore 1988). Since the peripheral course of the TN fibers in amphibian larvae has been found to intermingle with the ventromedial fibers of the ON (Herrick 1906; Mckibben 1911), the LHRH-ir cells observed in the ventromedial portion of the ON of our

animals may correspond to these TN components in the ON. It is therefore suggested that the LHRH-ir neurons in the developing newt originate within the nasal structures and migrate into the brain together with the TN. This hypothesis is strongly supported by the results of experiments conducted in which olfactory placodes were surgically removed. Removal of one olfactory placode at stages 26 or 29 results in the complete absence of LHRH-positive cells in the olfactory-brain axis on the operated side at the pro- and the postmetamorphic stage, although the progressive development of the L H R H neuronal system is not disturbed on the unoperated side. Furthermore, the LHRH-ir ganglion cells of the TN and the septo-preoptic LHRH-ir neurons disappear following placodectomy, again suggesting that these LHRH-positive neurons originate in the olfactory placode of the newt. It has been reported that the surgical removal of the olfactory placode results in a reduction in the size of the telencephalon and/or in the absence of the olfactory bulb on the operated side (Burr 1916; Clairambault 1976). In the present study, we have also observed a reduction in the size of the anterior telencephalon, presumably because of a reduction in the size of the olfactory bulb. However, other more posterior brain regions were not affected. Therefore, based on the above considerations, the lack of LHRH-ir cells in our placodectomized animals is probably not the result of some nonspecific effect of the surgery, but is directly caused by the removal of the progenitor cells of the L H R H neurons in the placode and/or the absence of the ON and TN, which may provide these neurons with a migration route. In amphibians, the distribution pattern of L H R H neurons in the preoptic area and hypothalamus is said to be correlated with the maturation of the brain-pituitary-gonadal axis (Crim 1985; D'Aniello etal. 1991). Furthermore, in a study of the postmetamorphic developmental changes in the L H R H neurons in Rana esculenta, the full distribution pattern of the LHRH neurons has been found to be attained at the completion of spermatogenesis (D'Aniello et al. 1991). Although we have not performed cell counts, more LHRH-positive cells seem to be found in the septo-preoptic area of adult newts than in newly metamorphosed juvenile newts. It is possible that neurons that originate from other sources may begin to synthesize LHRH during postmetamorphic development. Actual migration of LHRH-producing neurons from the olfactory to the diencephalic site is not definitively proven by our experiments. However, it is at least shown that attainment of a normal adult pattern of distribution of such neurons depends on an intact pre-existing relationship to the olfactory system. There is a clear need for further study of this interesting phenomenon.

Acknowledgements. We would like to thank Dr. K. Wakabayashi, Gunma University, for his generous supply of LRH13. We are grateful to Dr. A. Gorbrnan, University of Washington, for his critical reading of the manuscript and to Dr. K. Kawamura,Waseda University, for his help in taking the scanning electron micrograph. This research was supported by Grants-in-Aidfor Scientific

27 Research from the Japanese Ministry of Education, Science and Culture.

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