Monoamine fluorescence in the median eminence of the Japanese quail

Cell Tiss. Res. 164, 425-434 (1975) - 9 by Springer-Verlag 1975

Monoamine Fluorescence in the Median Eminence of the Japanese Quail, Coturnix coturnix japonica, following Medial Basal Hypothalamic Deafferentation* M. Nozaki and H. Kobayashi Misaki Marine Biological Station, University of Tokyo, Misaki, Miura-shi, Kanagawa-ken, Japan

M. Yanagisawa and T. Bando Department of Pharmacology, School of Medicine, Juntendo University, Hongo, Bunkyo-ku, Tokyo, Japan

Summary. Monoamine fluorescence was examined in the ventral hypothalamus of the Japanese quail, C o t u r n i x c o t u r n i x j a p o n i c a after medial basal hypothalamic deafferentation. In sham-operated control birds, numerous yellowgreen fluorescent fibers were observed in the median eminence and the nucleus tuberis. In the area of the paraventricular organ, a number of fluorescent fibers and cell bodies were observed. In birds with deafferented hypothalami, fluorescence disappeared both in the median eminence and the nucleus tuberis. In the area of the paraventricular organ, which was within the area of deafferentation, fluorescence of neuronal perikarya did not change, but fluorescent fibers decreased markedly in number. Disappearance of monoamine fluorescence in the median eminence and the nucleus tuberis is discussed in relation to the tanycyte absorptive function and gonadal development. Key words: Median eminence - Japanese quail, C o t u r n i x c o t u r n i x j a p o n i c a Monoamine fluorescence - Hypothalamic deafferentation - Tanycyte absorption.

Introduction The tanycytes of the median eminence of mammals and birds absorb peroxidase injected into the third ventricle (Kobayashi et al., 1972; L6ranth and Schiebler, 1974). In the Japanese quail, this absorption of peroxidase by the tanycytes is markedly augmented following medial basal hypothalamic deafferentation (Nozaki, 1975). Since it has been suggested that monoaminergic fibers form synaptoid contacts with the tanycytes of the median eminence (Matsui, 1966 a, b; Kobayashi et al., 1970), it is assumed that the augmentation of the tanycyte Send offprint requests to." Prof. Hideshi Kobayashi, Misaki Marine Biological Station, University

of Tokyo, Misaki Miura-shi, Japan 238-02. * Supported by Grants from the Ministry of Education to Professors T. Bando and H. Kobayashi, and a Grant from the Ford Foundation to Prof. H. Kobayashi.

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a b s o r p t i o n following d e a f f e r e n t a t i o n m a y be a t t r i b u t e d to the t r a n s e c t i o n o f the m o n o a m i n e r g i c fibers r u n n i n g to the m e d i a n eminence. In an a t t e m p t to support this hypothesis, the authors have looked for monoamine fluorescence in the m e d i a n e m i n e n c e after m e d i a l basal h y p o t h a l a m i c d e a f f e r e n t a t i o n . In addit i o n , c h a n g e s in f l u o r e s c e n c e in t h e n u c l e u s t u b e r i s a n d t h e p a r a v e n t r i c u l a r o r g a n were also noted.

Material and Methods Female Japanese quail (Coturnix coturnix.japonica) at the age of three weeks were obtained from a commercial source. They were reared under a short-day photoperiod of 8L 16D (light from 0800 to 1600). Three weeks thereafter, the birds were divided into two groups: one group was subjected to the medial basal hypothalamic deafferentation and another was subjected to sham-operation as control birds. The details of the operation have been described in a previous paper (Nozaki, 1975). The deafferented area and the nuclei located within this area are shown in Fig. 1. On the day of the operation, birds were transferred to a long-day photoperiod of 16L 8D (light from 0800 to 2400). Two weeks after the operation, the birds were killed and monanaine fluorescence was examined in the hypothalamus. Fluorescent histochernistry. Three birds with complete hypothalamic deafferentation and five with sham-operation were sacrificed by decapitation two weeks after exposure to a long-day photoperiod. Immediately after removing the hypothalamus, together with the pituitary gland, the tissue was placed on a small metal plate and immersed in isopentane-cooled liquid nitrogen. All tissues were dehydrated in vacuo at - 3 7 ~ C for 6 days. According to the technique of Falck and Hillarp (1962), the freeze-dried tissues were exposed to formaldehyde gas which had been kept at a relative humidity of 70% at 80~ C for two hours. Tissues were then infiltrated with paraffin at 60~ C for 45 minutes in vacuo. As control preparations, the hypothalami of three sham-operated birds were subjected to the procedures mentioned above and those of the remaining two sham-operated birds

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Fig. 1. Schematic drawing of the area isolated by the transection (thick line). A H adenohypophysis, A M E anterior median eminence, H A M nucleus hypothalamicus anterior medialis, H M P nucleus hypothalamicus posterior medialis, H I nucleus hypothalamicus inferior, M M nucleus mamillaris medialis, N T nucleus tuberis, OC optic chiasma, P M R posterior median eminence, P N pars nervosa, P V nucleus paraventricularis, PVO paraventricular organ

M o n o a m i n e Fluorescence in the Quail Median Eminence (Deafferentation)

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were treated with the same procedures without exposure to paraformaldehyde gas. Transverse sections were cut at 10pro and m o u n t e d on fluorescent-free slides with a mixture of Entellan and xylol. The preparations were observed under a Zeiss fluorescence microscope and photographed on Fuji X-ray film. The exciting light was delivered from an O s r a m H B O 200 high-pressure mercury lamp and was filtered through Schott BG 12 and Zeiss 47 plus 50 as the primary and secondary filters.

Fig. 2. Transverse section through the ventral hypothalamus at the level of the anterior median eminence of a control bird. Intense fluorescence is seen in the median eminence (ME), nucleus tuberis (NT) and paraventricular organ (PVO). III, third ventricle. • 100

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Results and Discussion

Yellow-green fluorescence was very strong in the median eminence, nucleus tuberis and paraventricular organ of control birds (Fig. 2). In paraformaldehydeuntreated preparations of control birds, there was no fluorescence in these regions. Therefore, the fluorescence observed in this study is considered to be induced by monoamine(s).

Median Eminence o f Sham-Operated Birds 1. Anterior Median Eminence. In the subependymal, fiber and reticular layers of the anterior median eminence, there were numerous thick fibers with "beaded" fluorescent varicosities. These fluorescent fibers were mostly concentrated in the lateral portions of the median eminence (Figs. 3, 4). Some thick fluorescent fibers appeared to continue to a fluorescent area of the nucleus tuberis (Figs. 2, 3 and 4). Some thick fluorescent fibers in the fiber and reticular layers of the anterior median eminence ran toward the palisade layer (Fig. 3). In the palisade layer, numerous fine fluorescent fibers were observed. They ran perpendicularly toward the ventral surface of the median eminence (Figs. 2, 3). 2. Posterior Median Eminence. Thick fluorescent varicose fibers were observed in the subependymal layer of the posterior median eminence (Fig. 5). Their number was not as great as in the anterior median eminence. In the fiber and reticular layers of the posterior median eminence, thick fluorescent varicose fibers were not numerous (Fig. 5) compared with the anterior median eminence (Figs. 3, 4). In the palisade layer, the number of fine fluorescent fibers was much smaller in the posterior median eminence than in the anterior median eminence. Thus, the distribution of the thick and fine fluorescent varicose fibers varied in the anterior and the posterior median eminence, and also among the layers of the median eminence. These findings coincide generally with the observations of Sharp and Follett (1968, 1970) in Japanese quail, of Soest et al. (1973) and of Calas et al. (1974b) in other avian species. Calas et al. (1974b) reported that fluorescence in the subependymal layer of the duck is due to noradrenaline, and Calas et al. (1974a) suggested that fluorescent fibers in the palisade layer contain serotonin. In the present study, a differentiation between fluorescence due to noradrenaline or serotonin has not been made.

Fig. 3. Transverse section through the anterior median eminence of a control bird. Two types of varicose fibers can easily be distinguished in the median eminence: 1) thick, " b e a d e d " varicose fibers distributed in the subependymal (S), fiber (F) and reticular (R) layers; 2) fine fluorescent fibers distributed in the palisade layer (P). x 200 Fig. 4. Transverse section through the anterior median eminence of a control bird. Thick fluorescent fibers continue to the fluorescent area of the nucleus tuberis (NT). Ill, third ventricle, x 200

Fig.

5. Transverse section through the posterior median eminence of a control bird. Thick varicose fibers are not numerous. Fluorescent terminals (arrows) are in close contact with non-fluorescent cell bodies of the nucleus tuberis (NT). III third ventricle, x 200

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Median Eminence o f Birds with Deafferented Hypothalami

In birds with deafferented hypothalami, only very few fluorescent terminals were observed in the subependymal, fiber and reticular layers both of the anterior (Fig. 7) and posterior median eminence (Fig. 6). Fine fluorescent fibers disappeared completely in the palisade layer (Fig. 7). These findings indicate that the perikarya of monoaminergic neurons, the axons of which penetrate into the median eminence are mostly located outside the medial basal hypothalamic area. In a previous paper Nozaki (1975) reported that, in the Japanese quail, the tanycyte absorption of intraventricularly administered peroxidase is augmented following medial basal hypothalamic deafferentation. It is possible that the augmentation of absorption is due to transection of the monoaminergic fibers originating from the neuronal perikarya located outside the medial basal hypothalamus. The present fluorescent study shows that monoaminergic fibers running toward the median eminence were transected by hypothalamic deafferentation. In the rat and pigeon median eminence it has been demonstrated that there are synaptoid contacts between monoaminergic fibers and tanycytes (Matsui, 1966a, b; Kobayashi et al., 1970; Gfildner, 1973). Soest et al. (1973) have also suggested that catecholamine-containing fluorescent terminals in the subependymal layer have a synaptoid relation to glial elements. Considering all the observations mentioned above, it is likely that monoaminergic fibers have an inhibitory action on the tanycyte absorptive function. Thus, when these fibers are transected, the tanycyte function is released from this inhibition.

Regions Outside the Median Eminence 1. Nucleus tuberis and Ovarian Growth. Numerous "beaded" varicose fibers were observed in the area of the nucleus tuberis. They were in close contact with non-fluorescent cell bodies (Figs. 2, 4 and 5). No fluorescent cell bodies were found in the tuberal nuclei. Sharp and Follett (1970) and Soest et al. (1973) were also unable to observe fluorescent perikarya in the basal tuberal nuclei of birds (cf. Oksche and Farner, 1974). After medial basal hypothalamic deafferentation, only a few fluorescent varicose fibers originating from the paraventricular organ were observed in the nucleus tuberis (Fig. 6). These findings suggest that most of the neuronal perikarya with monoamine-containing afferents to the nucleus tuberis are located outside the hypothalamic deafferented area. In birds with deafferented hypothalami, the gonodal development was completely inhibited even under a long-day photoperiod. Ovarian weight was less than 50 mg in experimental birds and about 400 mg in control birds. These results coincide with those obtained in the Japanese quail subjected to hypothalamic deafferentation (Wada, 1974; Davies and Follett, 1974; Nozaki, 1975). It has been suggested that failure of gonadal development under a long-day photoperiod in experimental birds is due to the transection of fibers transferring photoperiodic information from photoreceptor(s) or the transection of the fibers con-


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Fig. 6. Transverse section through the ventral hypothalamus at the level of the posterior median eminence of a bird with deafferented hypothalamus. White broken line indicates the knife cut. Note small amount of fluorescent material in the median eminence (ME) and the nucleus tuberis (NT). At the level of this section, the paraventricular organ (PVO) was within the isolated area (see Fig. 1). x tO0

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Fig. 7. Transverse section through the anterior median eminence of a bird with deafferented hypothalamus. Fluorescence not present in the median eminence. III, third ventricle, x 200 Fig. 8. Transverse section of the paraventricular organ. Fluorescent cells (F) extend processes into the third ventricle (IIl). x 320


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trolling production of gonadotrophin-releasing hormone in the nucleus tuberis (Wada, 1974). Monoaminergic as well as cholinergic fibers are suggested to be involved in controlling the function of the nucleus tuberis (see Wada, 1974). The present study indicates that the inhibition of gonadal development is apparently correlated to a marked decrease in fibers showing monoamine fluorescence in the area of the nucleus tuberis. The possibility exists that failure of gonadal development is due to the transection of monoaminergic fibers controlling the function of the nucleus tuberis. 2. Paraventricular Organ. The area of the paraventricular organ revealed the most intense fluorescence, and the cell bodies containing monoamines were seen only in this region of the medial basal hypothalamus (Figs. 2 and 8). Fluorescent cell bodies in contact with the ependymal cells of the paraventricular organ extend processes into the third ventricle (Fig. 8). After medial basal hypothalamic deafferentation, there was a considerable decrease in the number of fluorescent fibers in the area of the paraventricular organ, which was included in the deafferented area. This can be clearly seen by comparing Fig. 6 with Fig. 2. There was no remarkable change in the fluorescence of cell bodies after deafferentation (Fig. 6). The functional relationship between afferent monoaminergic fibers and fluorescent neurons of this organ is not known at the present time.

References Calas, A., Alonso, G., Arnauld, E., Vincent, J.D. : Demonstration of indolaminergic fibers in the median eminence of the duck, rat and monkey. Nature (Lond.) 250, 241-243 (1974a) Calas, A., Hartwig, H.G., Collin, J.P.: Noradrenergic innervation of the median eminence. Microspectrofluorimetric and pharmacologicalstudy in the duck, Anas platyrhynchos. Z. Zellforsch. 147, 491-504 (1974b) Dadies, D.T., Follett, B.K.: Hypothalamic deafferentation in Coturnix quail and its blockade of photoperiodically induced testicular development. Gen. comp. Endocr. 22, 359 (1974) Falck, B., Hillarp, N.A., Thieme, G., Torp, A. : Fluorescence of catecholamines and related compounds condensed with formaldehyde. J. Histochem. Cytochem. 10, 348-354 (1962) Giildner, F.H.: Charakteristika der neuro-gli6sen synapsen~ihnlichen Kontakte in der Eminentia mediana der Ratte. Verh. anat. Ges. (Jena) 67, 270-283 (1973) Kobayashi, H., Matsui, T., Ishii, S.: Functional electron microscopy of the hypothalamic median eminence. Int. Rev. Cytol. 29, 281 381 (1970) Kobayashi, H., Wada, M., Uemura, H., Ueck, M. : Uptake of peroxidase from the third ventricle by ependymal cells of the median eminence. Z. Zellforsch. 127, 545 551 (1972) L6ranth, Cs., Schiebler, T.H. : lJber die Aufnahme von Peroxydase aus dem 3. Ventrikel der Ratte. Elektronenmikroskopische Untersuchungen. Brain Res. 67, 1-11 (1974) Matsui, T.: Fine structure of the median eminence of the rat. J. Fac. Sci., Univ. Tokyo, Sect. IV 11, 71-96 (1966a) Matsui, T. : Fine structure of the posterior median eminence of the pigeon, Colurnba livia domestica. J. Fac. Sci., Univ. Tokyo, Sect. IV 11, 49-70 (1966b) Nozaki, M.: Tanycyte absorption affected by the hypothalamic deafferentation in the Japanese quail, Coturnix coturnix japonica. Cell Tiss. Res. (in press) Oksche, A., Farrier, D.S. : Neurohistological studies of the hypothalamo-hypophysial system of Zonotrichia leucophrys garnbelii (Ayes, Passeriformes). With special attention to its role in the control of reproduction. Adv. Anat. Embryol. Cell Biol. 48(4), 1-136 (1974) Sharp, P.J., Follett, B.K.: The distribution of monoamines in the hypothalamus of the Japanese quail, Coturnix coturnix japonica. Z. Zellforsch. 90, 245-262 (1968)

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Sharp, P.J., Follett, B.K.: The adrenergic supply within the avian hypothalamus. In: Aspect of neuroendocrinology (W. Bargmann and B. Scharrer, eds.), p. 95-103. Berlin-Heidelberg-New York: Springer 1970 Soest, S.W., Farner, D.S., Oksche, A. : Fluorescence microscopy of neurons containing primary catecholamines in the ventral hypothalamus of the White-crowned Sparrow, Zonotrichia leucophrys gambelii. Z. Zellforsch. 141, 1 17 (1973) Wada, M. : Blockade of photoperiodically induced testicular growth by hypothalamic deafferentation in Japanese Quail, Coturnix coturnixjaponica. Gen. comp. Endocrin. 24, 113-120 (1974)

Received August 10, 1975