Cell and Tissue Research

Cell Tiss. Res. 190, 417-434 (1978)

9 by Springer-Verlag 1978

Localization of Monoamines in the Forebrain of Two Salmonid Species, with Special Reference to the Hypothalamo-Hypophysial System M. Terlou*, B. Ekengren** and K. Hiemstra* *

Zoological Laboratory, State University of Utrecht, The Netherlands

** Department of Zoology, University of Stockholm, Sweden

Summary. In the salmon and trout aminergic cell bodies were found in the nucleus recessus lateralis (NRL) and the nucleus recessus posterioris (NRP), both of which are situated near the third ventricle. Three cell types could be distinguished. Type 1 produces a green and type 2 a yellow fluorescence. The former type probably contains dopamine and the latter 5-hydroxytryptamine. Both types possess intraventricular protrusions in contact with the cerebrospinal fluid. The third cell type produces a less intense blue-green fluorescence; relatively few cells of this type have thick processes in contact with the ventricle. In addition, large fluorescent cells were found in the salmon, dorsal from the caudal part of the NRL. The various parts of the N R L and NRP are interconnected by thick bundles of nerve fibers; tracts leaving the nuclei could be traced for short distances only. The cells of the nucleus praeopticus (NPO), those o f the medial part and to a much lesser extent also of the lateral part of the nucleus lateralis tuberis (NLT) have an aminergic innervation which probably originates from the N R L and/or NRP. All parts of the neurohypophysis contain many monoaminergic fibers, with aminergic material concentrated at the neuro-adenohypophysial interface. Fibers were not observed to penetrate the basal lamina. In the salmon and trout the fibers have a similar distribution, but differ in the intensity of fluorescence, being high in the salmon and low in the Sendoffprint requeststo: Dr. M. Terlou, ZoologicalLaboratory, Transitorium 3, Padualaan 8, Utrecht, The Netherlands; or B. Ekengren, Department of Zoology, University of Stockholm, Box 6801, S11386, Stockholm, Sweden Acknowledgements. Dr. M. Terlou acknowledges the travel grant received from the Netherlands Organization for theAdvancementofPure Research(Z.W.O.). Dr. B. Ekengrenwas supported by grants from the Swedish Natural Research Council (no. 2124-037). The authors thank Prof. Dr. P.G.WJ. van Oordt for his stimulating commentsand interest, Mrs. V. Lundin and Miss. Y. Lilliemarckfor technicalassistance, Miss B. Meyerhoferfor preparing the drawings (Stockholm), and Messrs. H. van Kooten, E. van der Vlist and J.J. van der Vlis for preparing photographs and diagrams (Utrecht). They also wish to thank Mrs. S.M. McNab and Dr. L. Boomgaartfor correcting the English text

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M. Terlou et al. trout. Only in the trout have fluorescent cells been found in the adenohypophysis and very occasionally in the neurohypophysis. A number of these cells are basophilic and show a PAS-positive reaction. Key words: Monoamines Formaldehyde-induced

- Hypothalamo-hypophysial system fluorescence.

-

Teleosts

-

Introduction

In the diencephalon of several vertebrate species Kappers (1920/1921) and Charlton (1928) identified a "modified ependyma", which was later found to be composed of an ependymal and a neuronal part. Since this time, numerous articles dealing with different classes of vertebrates (mammals excluded) have been published on the monoaminergic nature of the neuronal part. In amphibians, reptiles and birds this ependymal structure is known as the paraventricular organ (PVO) or organon vasculosum hypothalami (for review, see Vigh, 1971; Baumgarten, 1972; Terlou and Ploemacher, 1973). In addition to a PVO, at least some species of amphibians have a nucleus infundibularis dorsalis (NID) (Terlou and Ploemacher, 1973; Chacko et al., 1974; Prasada Rao and Hartwig, 1974; cf. Goossens, 1977). Generally, the hypothalamus of teleosts contains two paired aminergic nuclei which are comparable to the PVO: the nucleus recessus lateralis (NRL) and the nucleus recessusposterioris (NRP) (trout: Bertler et al., 1963; Weiss, 1970; goldfish: Baumgarten and Braak, 1967; Baumgarten, 1972; eel: Lefranc et al., 1969, 1970; l'Hermite and Lefranc, 1972; Fremberg et al., 1977; roach: Ekengren, 1975b; three gobiid species: Honma and Honma, 1970; Ekengren, 1973; Swanson et al., 1975; see also Vigh, 1971, Vigh and Vigh-Teichmann, 1973). In addition, Lefranc et al. (1969, 1970) and l'Hermite and Lefranc (1972) reported localizing monoaminergic perikarya in the floor of the preoptic recess in the eel, and Ekengren (1975b) reported the same for the roach (cf. Fremberg et al., 1977). Furthermore, Lefranc and coworkers have found formaldehyde-induced fluorescent cells in the caudolateral part of the telencephalon and in the geniculate nucleus. In the eel, Fremberg et al. (1977) found a group consisting of about a dozen cells located just caudal to the commissura transversa." the nucleus hypothalami anterior. In the NRL and NRP, tracts originate that can be traced in different directions, some running towards the two peptidergic nuclei (the nucleus praeopticus, NPO, and the nucleus lateralis tuberis, NLT) and others toward the hypophysis (see above-mentioned literature). In particular, the monoaminergic supply of information to the hypophysis, has received much attention. Electron microscopically, the "type B" fibers, which are thought to be at least partly monoaminergic, were demonstrated in the hypophysis of various species of teleosts (for review, Ball and Baker, 1969; Ball et al., 1972; Zambrano et al., 1972; Peter, 1973; Holmes and Ball, 1974). The presence of monoamine oxidase (MAO) in nerve fiber tracts which penetrate the pituitary in Anguilla japonica and Oryzias latipes indicates its monoaminergic innervation (Urano, 1971). This was corroborated for Gasterosteus aculeatus by Foll6nius, using 3H-noradrenaline (1968, 1970) and 6-hydroxydopamine (1972). With the Falck-Hillarp fluorescence technique, monoaminergic fibers were found in the hypophysis of a few teleost species (Iturriza, 1967; Bern et

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al., 1 9 7 1 ; Z a m b r a n o , 1 9 7 0 ; P o l e n o v e t al., 1 9 7 2 ; E k e n g r e n , 1 9 7 5 a ) . F r e m b e r g a n d Meurling (1975) were the first to demonstrate convincingly that the hypophysis of t h e eel p o s s e s s e s a m o n o a m i n e r g i c i n n e r v a t i o n . The present study deals with the distribution of monoamine-containing structures in the forebrain of the Atlantic salmon and the rainbow trout and the relation of these structures to the known peptidergic neurosecretory centers, the NPO and the NLT, and the hypophysis.

Materials and Methods

Fish. Rainbow trout (Salmo gairdneri) ranging in age from 6 months to 2 years and varying in weight from 50 g to 300 g, were obtained from a hatchery at Vaassen (Holland). The fish were kept in aquaria for several weeks under a natural light-dark regime, on a black background and in running tap water (average temperature 11 ~C), before being sacrificed. Salmon (Salmo salar) approximately 2 years old, with an average weight of 50 g and an average length of 200 ram, were kindly supplied by the Salmon Research Institute at ~dvkadeby (Sweden). For comparison, two small juvenile animals approximately 1 year old (90mm - 5 g) and three sexually mature 3 year old animals (300 mm - 500 g) were included in the experiments. The animals were kept in aquaria containing tap water for a few days before being sacrificed. Atlas. In order to pinpoint the distribution of monoamine-containing perikarya and fibers, an atlas was prepared, a portion of which is included in the present article. To that end, a selection was made from histological material of a number of transversely sectioned brains of 1-3 year old rainbow trout and 1-5 year old Atlantic salmon. Drawings were produced from one series of sections of a 3 year-old sexually mature trout by means of a camera lucida. For neuroanatomical terminology the reader is referred to Goldstein (1905), Holmgren (1920), Bergquist (1932), Nieuwenhuys (1963) and Baumgarten and Braak (1967).

Histochemical Procedure Trout. Three hours before being sacrificed 25 fishes of both sexes were injected intraperitoneally with nialamide (Sigma, 25-100mg/kg) and placed on a white background in an attempt to increase the amount of monoamines in the fibers running towards the hypophysis (see Fremberg and Meurling, 1975). Five fish served as controls for the nialamide treatment. Animals were anesthetized in MS 222 (0.035 %) and decapitated. The brains were cut out with the pituitary still attached, frozen in liquid propane cooled in liquid nitrogen, and then freeze-dried in a Pearse tissue dryer model I for 3 days. The Falck-Hillarp reaction was carried out with paraformaldehyde (relative humidity 40-60 ~ ) for 1.5 to 3 h, and the material was then embedded in paraffin in a vacuum oven (Falck and Owman, 1965; Bj6rklund et al., 1972). Sections were dry mounted, embedded in paraffin oil or entellan, and examined under a Zeiss fluorescence microscope equipped with an epi-illumination system (beam splitter 420 rim, excitation narrow band filters 405 rim, and a barrier filter LP 418). In order to identify certain fluorescent cells in the adenohypophysis, some freeze-dried sections after examination in the fluorescence microscope were stained either with Azan according to Heidenhain or with periodic acid Schiff(PAS) according to Hotchkiss/McManus. Slides were coated with a thin layer of albumin glycerin to prevent the sections from being washed off during the staining procedure. Salmon, Six of the 17 animals were injected intraperitoneally with nialamide (25-100 mg/kg) 3 h before the experiment. The subsequent procedure was as described for the trout, with the exception of the freeze-drying step; the salmon tissue was freeze-dried for 1 week according to the cold finger method. The sections were examined with a Zeiss standard RA microscope equipped with a Philips CS W-4 mercury lamp, a Zeiss 1.2/1.4 dark field condensor, excitation filters BG38 and BG12, and barrier filters 50 and 44. A few freeze-dried'brains of trout and salmon, which had not been exposed to formaldehyde vapor served as controls for the specificity of the Falck-Hillarp reaction.

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Results

O b s e r v a t i o n s a p p l y i n g to the A t l a n t i c s a l m o n as well as the r a i n b o w t r o u t will be p r e s e n t e d first (Figs. 1, 2). A s e p a r a t e section deals with differences between these t w o species o f salmonids. P r e t r e a t m e n t o f the a n i m a l s with n i a l a m i d e caused a slight to m o d e r a t e increase in fluorescence, p a r t i c u l a r l y in the fiber systems. T h e general d i s t r i b u t i o n o f m o n o a m i n e - c o n t a i n i n g structures d i d n o t change, a n d the localization o f m o n o a m i n e s in the v a r i o u s age g r o u p s r e m a i n e d the same. Telencephalon

S c a t t e r e d fluorescent fibers were o b s e r v e d t h r o u g h o u t the telencephalon. C o n t r a r y to the findings o f L e f r a n c et al. (1969, 1970) a n d l ' H e r m i t e a n d L e f r a n c (1972) for the eel, the presence o f a "'nucleus telencephali p o s t e r i o r " c o u l d n o t be e s t a b l i s h e d in the s a l m o n a n d trout. In s o m e cases, at the p o s i t i o n o f this nucleus, which is l o c a t e d d o r s o - l a t e r a l l y in the t e l e n c e p h a l o n in the a r e a o f nucleus taeniae, unspecific fluorescent cells were noted. A t the level o f the a n t e r i o r commissure, which is c o m p l e t e l y nonfluorescent, the density o f fluorescent fibers was higher in the ventral p a r t o f the brain. T h e fibers c a n be t r a c e d in c a u d a l direction u p to the area p r a e o p t i c a where they merge with diencephalic fiber systems.

Diencephalon

Area praeoptica A t the level where the optic c h i a s m a is a t t a c h e d to the brain, the m e d i a l p a r t o f the f l o o r o f the p r e o p t i c recess is thickened. This floor is d e e p l y p e n e t r a t e d by several extensions o f the recess. In this a r e a a n d laterally a r o u n d the recess, the fluorescent fibers f o r m a n e t w o r k a r o u n d the p e r i k a r y a , some o f which represent the m o s t

Figs. 1a-f and 2a-i. Distribution of formaldehyde-induced fluorescence in the forebrain of a sexually mature rainbow trout, shown in a series of schematic drawings of transverse sections. Perikarya are indicated in solid black and nerve fibers as dots. Except for the fluorescent cells in the hypophysis, the drawings apply to the Atlantic salmon also. Fig. 1a at the level o f the praeoptic area. Distance between the sections: Fig. la-f. a,b: 400gin; b,c: 800pro; c,d: 100gm; d,e: 300gm; e,f: 200jim. Fig.2a-i. Fig. 1 f-Fig. 2a: 200 ~tm; b,c: 100 ~m; c,d: 100 gm; d,e: 300 pro; e,f: 100 gm; f,g: 200 gm; g,h: 100 gin; h, i: 100~m. Abbreviations: AS aqueduct of Silvius; B thick bundles of fluorescent fibers; BV blood vessel; CHcommissura horizontalis; Eepiphysis; FC fluorescent cells corresponding to the pars isthmi of Fremberg et al. (1977); FLL fasciculus longitudinalis lateralis; FR fasciculus retroflexus; LL lobus lateralis; L M lobus medius; LPR lateral part of the preoptic recess; ACES mesencephalon; NE nucleus entopeduncularis; N L T pars lateralis of the nucleus lateralis tuberis; NPO nucleus praeopticus; NRL nucleus recessus lateralis; NRP nucleus recessus posterioris; O C optic chiasma; OF optic fibers; P I pars intermedia; PPD proximal pars distalis; PR preoptic recess; RL recessus lateralis; RLL recessus lobi lateralis; RP recessus posterior: RPD rostral pars distalis; SCO subcommissural organ; SD saccus dorsalis; SV saccus vasculosus; TC third cell type; TEL telencephalon; TL torus lateralis; TLo torus longitudinalis; TO tectum opticum; TM tractus mesencephalo-cerebellaris anterior; TST tractus saccothalamicus; V third ventricle

Monoamines in the Forebrain of Salmonids

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Fig. 3. Transverse section o f the infundibular area of the Atlantic salmon. Fluorescent fibers are running through the nucleus lateralis tuberis (NLT). Note the varicosities associated with some cell bodies o f the medial part of the N L T (open circles). The lateral part (NLTpl) is almost devoid of fluorescent terminals. RI recessus infundibuli, x 200 Fig. 4a. Transverse section o f the anterior hypothalamus o f the rainbow trout, showing a network of fluorescent fibers around cell bodies of the nucleus praeopticus(NPO). Vthird ventricle, x 200. b Detail of cell bodies of the N P O (,), nearly enclosed by fluorescent fibers, x 485

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rostral cells of the NPO (Terlou and Ekengren, in preparation). The entire NPO, which is situated rostrally in the floor and at the extreme lateral parts of the preoptic recess and borders caudally on the dorsal and medial lining of the preoptic recess, is heavily infiltrated by varicose fluorescent fibers. These fibers are in close contact with the perikarya of the neurosecretory cells and indicate their monoaminergic innervation (Figs. 1 a, b; 4a, b). A great number of ascending fibers occupy the area lateral to the NPO and can be traced in a caudal direction up to the area of the monoamine-producing nuclei in the hypothalamus (Fig. 1 b-f). Contrary to the findings of Fremberg et al. (1977), the nucleus entopeduncularis is never found to be fluorescent. The presence of a "nucleus recessus praeoptici", as observed in the eel (Lefranc et al., 1969, 1970; l'Hermite and Lefranc, 1972; cf. Fremberg et al., 1977) and the roach (Ekengren, 1975b), could not be established. Hypothalamus Monoamine-Containing Nuclei. The hypothalamus possesses two well-developed

monoamine-containing nuclei, situated on either side of the ventricular system: the nucleus recessus lateralis (NRL) and the nucleus reeessus posterioris (NRP). The NRL begins a few hundred microns rostral to the lateral recess of the infundibular ventricle, and extends caudally, following the ventricular lining. The rostral part is equivalent to the pars dorsomedialis in the roach (Ekengren, 1975b) and the PVO pars anterior in the eel (Fremberg et al., 1977). In the rostral part the ventricular wall shows a paired furrow orientated from rostro-dorsal to caudo-ventral and connecting the medial ventricle with the lateral recess (Fig. 2 a-c). Somewhat more caudal from this connection, the ventricular wall develops a characteristic system of furrows which comprises part of the NRL. At this level the recessus laterales lose contact with the main ventricle in the mesencephalon and continue as the ventricle in the lobi laterales (Fig. 2c-e). Caudally, the furrows are situated more laterally and the ventricular wall becomes gradually flattened. The NRL follows the dorsal wall of the ventricle. Between the end of the N R L and the beginning of the NRP, an area is found essentially free of fluorescent cells (between Fig. 2e and 2f). The NRP begins medially (cf. PVO pars isthmi of Fremberg et al., 1977) and borders the entire posterior recess, which more caudally has the shape of a butterfly (Fig. 2f-i). In the saccus vasculosus, fluorescence has not been noted. Generally, the cytology of the NRL is similar to that of the NRP. The fluorescent cells are arranged in two to four layers, and have an apical process which penetrates the ependymal layer to form a knob-like ending in the ventricle. Axons originate from the basal parts. Two types of cells can be distinguished based on the color of the fluorescence. Most cells have a green to yellow-green fluorescence; a few cells are yellow-orange. This difference could be readily noticed in the intraventricular protrusions. In addition to the NRL and NRP, a group of neurons in the ventral wall of the lobus medius possesses other fluorescent microscopical characteristics. The rostral end is situated opposite the furrows in the dorsal wall of the recessus lateralis (TC in Fig. 2d). Caudally these neurons are present in the medial wall of the recessus lobi

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lateralis (Fig. 2e-f) and in the ventral wall of the caudal extensions of the lateral recess (Figs. 2e; 8). This localization coincides with that of the nucleus inferior tuberis (cf. Baumgarten and Braak, 1967), but not all cells of this nucleus show fluorescence. Caudally in the most ventral wall of the posterior recess similar cells were observed (TC in Fig. 2g; Fig. 6). These are generally grouped close together and differ from the cells of the NRL and NRP. They are larger, less fluorescent and blue-green in color. Moreover, the fluorescent content shows a thread-like appearance. Relatively few cells have a rather thick process contacting the cerebrospinal fluid (Figs. 6, 8). Based on the fluorescence microscopical characteristics of these cells and their localization in the ventratwall of the ventricular system, it seems justifiable to regard them as a separate group of monoaminergic neurons. It would be premature, however, to classify these cells as a separate nucleus; additional electron microscopical information is required to prove conclusively that these cells differ from the two cell types of NRL and NRP. Therefore, for the time being they will be considered as a third cell type of the hypothalamic monoamine-producing complex consisting of NRL and NRP.

Fiber Tracts Lateral to the dorsomedial part and dorsal to the lateral part of the NRL many fluorescent fibers traverse the nonfluorescent periventricular gray and form welldefined bundles. These bundles come into contact with each other in front of the level of the dorsal furrows in the ventricular wall (Fig. 2c, d) and extend caudally, dorsal to the NRL (Fig. 9). In the area between the NRL and NRP, these fiber bundles divide into smaller ones, which are connected with similar bundles of the NRP (Fig. 2e-h; Fig. 9). Less well-defined tracts leave the NRL and run in various directions. From the rostral part fibers were traced dorsally, especially in the subependymal fibrous layer, and rostrally towards the area praeoptica. Other fibers continue laterally to the junction of the torus lateralis and tectum opticum (Fig. 2a-i), where they form a tract orientated rostro-caudally. This tract could be traced in caudal direction into the mesencephalon, where fibers split off in the tegmentum (Fig. 2h, i). Many fibers are present throughout the lobus medius of the hypothalamus, with a higher density ventromedially from the region of the horizontal commissure. Just behind the horizontal commissure these fibers pass close to the cells of the NLTpars lateralis (Fig. 1d-f); some of them show varicosities in contact with the cell. However, in contrast to the observations reported by Weiss (1976), the main part of the pars lateralis of the NLT does not appear to receive a significant input of fluorescent fibers (Fig. 3), as was already mentioned for the roach (Ekengren, 1973). Instead, the ventro-medially located periventricular gray is penetrated by fluorescent fibers at many sites, some fibers extending as far as the cerebrospinal fluid. Fibers coming from rostral and caudal directions are abundant near the hypophysial stalk. Monoaminergic fibers were also found in the lobi laterales, around the perirecessal gray and in close contact with larger perikarya in the fibrous parts. Thick bundles of fluorescent fibers originate from the NRP; caudally they

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occupy nearly the entire area around the posterior recess (Fig. 2g-i), except for dark silhouettes representing the non-aminergic bundles of the tractus sacco-thalamicus from the saccus vasculosus. The thick bundles are running in a rostral direction, where they split into smaller bundles that come into contact with the bundles o f the NRL. Generally, fibers are distributed over the entire ventromedial hypothalamus (Fig. 2c-f). The axons of the above-mentioned third type of fluorescent cells are part of the network of fluorescent fibers, particularly in the area where these cells are localized. It should be stressed that, contrary to what has been described for the eel (Lefranc et al., 1969, 1970; l'Hermite and Lefranc, 1972) and the goldfish (Baumgarten and Braak, 1967), it has not been possible in the case o f the trout and the salmon to trace with certainty the exact origin and the terminal areas of the fibers and bundles. Innervation of the Hypophysis In the salmon, many fibers were observed to enter the hypophysial stalk and to spread out over the entire neurohypophysis. A great number of beaded terminals are present at the neuro-adenohypophysial interface (Fig. 5). The strongest fluorescence was noted in the region bordering the rostralpars distalis (RPD) and in the deep invaginations of neurohypophysial tissue in the pars intermedia (PI). The fluorescence was less intense opposite the proximal pars distalis (PPD) and the remaining parts of the PI. No fluorescent fibers were observed to penetrate between the glandular cells of the adenohypophysis. In the trout the distribution of the monoamine-containing fibers was generally the same as in the salmon, but the intensity of the fluorescence was much lower. In both species the fibers entering the hypophysis are not arranged in welldefined tracts, and their origin could not be established. Most probably they originate in the N R L and/or the NRP. Difference between the Two Salmonid Species The minor difference observed between the two species are more quantitative than qualitative. In the salmon, large green fluorescent cells are present dorsal to the Fig. 5. Transverse sectionof the hypophysisof the salmonat the levelof the proximal pars distalis. Many fluorescent fibers terminating at the neuro-adenohypophysial interface. N neurohypophysis; A adenohypophysis; RI recessus infundibuli, x 100 Fig. 6. Transverse section of part of the nucleus recessus posterioris (NRP) of the rainbow trout. Strongly fluorescent cells in the dorsal lining of the posterior recess (RP) and weakly fluorescent cells (= possible third cell type) in the ventral lining (arrows). • 200 Fig. 7. Transverse section through the caudal part of the nucleus recessus lateralis of the salmon. Large fluorescent cells dorsal to the nucleus, x 125 Fig. 8. Transversesectionof the caudal part of the nucleus recessuslateralis (NRL) of the rainbowtrout, at the levelof Figure. 2e. Fluorescentcell types 1 and 2 borderingthe dorsal wall of the extensionsof the lateral recess, the possible third cell type located in the ventral wall. Relatively few cells with thick intraventricular protrusions (arrows). RL recessus lateralis, x 150

Fig. 9. Sagittal section of the nucleus lateralis (NRL) and the nucleus recessus posterioris (N/he) of the rainbow trout, showing perikarya and thick connecting fiber tracts (B). H hypothalamus; HT hypothalamus; RL recessus lateralis; RP recessus posterior; T tegmentum of the mesencephalon, x 120 Fig. 10. Sagittal section of the hypophysis of the rainbow trout, showing single fluorescent cells, in neurohypophysial tissue (N) and in the adenohypophysis close to strands of neurohypophysial tissue. F follicles o f the rostral pars distalis. • 200

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caudal part of the NRL (Fig. 7), but these cells have not yet been recognized in the trout. Another difference is the presence of a variable number of green fluorescent cells in the hypophysis of the trout (Figs. I e-f, 2a~:l, 10). These are found mainly in the RPD and PPD, and some only occasionally in the PI. Most of these cells are situated in the endocrine parenchyma, along the neurohypophysial digitations, and many of them having a protrusion to the basal lamina. Some cells are located separately in the neurohypophysial tissue. Subsequent staining of the fluorescence microscopical preparations with Azan according to Heidenhain show the fluorescent cells to be identical with blue basophilic cells. Not all these cells, however, show fluorescence and not all fluorescent cells show basophilia. Application of the periodic acid-Schiff (PAS) reaction shows only a number of the fluorescent cells to be PAS positive. Fluorescent cells have not yet been observed in the hypophysis of the salmon.

Discussion

A. Telencephalon The presence of fluorescent nuclei in the telencephalon, as described for the eel (Lefranc et al., 1969), could not be established for the salmon or the rainbow trout. Fremberg et al. (1977), who studied the eel, were also unable to confirm the results of Lefranc.

B. Diencephalon a) Nuclei. The general structure of the monoamine-containing nuclei in the two salmonids is in agreement with the corresponding descriptions for other teleost species. In this paper the terminology NRL and NRP, as proposed by Baumgarten and Braak (1967), has been used for the nuclei, without further subdivision as made by other authors (Vigh, 1971; Swanson et al., 1975; Fremberg et al., 1977). The minor differences reported for teleost species might be attributed to differences in the development (i.e. the extensions) of the ventricular system in the hypothalamus. This might be true in the case of other vertebrate classes as well (Kappers, 1920/21; Charlton, 1928; Diepen, 1962; Vigh, 1971; Vigh and VighTeichmann, 1973). As in other teleosts, a green and a yellow fluorescent CSF-contacting cell type was found in the NRL and NRP (Vigh, 1971 ; Baumgarten, 1972; Ekengren, 1975 b; Fremberg et al., 1977). The green cell produces a catecholamine, probably dopamine, and the yellow one most probably produces 5-hydroxytryptamine (5HT) or a 5-HT-like substance (Fremberg et al., 1977). Electron microscopically, only in the goldfish were indications found for the presence of two cell types, after the fish had been treated with p-chlorophenylalanin, a drug that selectively depletes 5-HT (Baumgarten, 1972). In other species only one type has been found (Vigh and Vigh-Teichmann, 1973; Ekengren, 1975b). In other vertebrates, however, two types have been distinguished (Baumgarten et al., 1969-Lacerta; Peute, 1969Xenopus; cf. Terlou and Ploemacher, 1973).

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The third cell type in the NRL and NRP of the two salmonids is different from the other two types in size, intensity of fluorescence, and in its relation to the cerebrospinal fluid. Relatively few of these cells are in contact with the ventricle. Although smaller, this cell type has some characteristics in common with the large fluorescent cells (LFC), which were reported to contain noradrenalin (Fremberg et al., 1977). It is tempting to assume that these cells are in contact with cells of the other fluorescent types of the NRL and NRP. Spectral analysis of fluorescent terminals close to these cells indicates the presence of noradrenalin (Fremberg et al., 1977). The presence of probable monoaminergic synapses on cells of the NRL/NRP or corresponding nuclei in other vertebrates supports this possibility (Baumgarten et al., 1969; Peute, 1969; Vigh and Vigh-Teichmann, 1973). A thorough electron microscopical investigation of the two salmonids is now in progress.

b) Innervation of the NPO and NLT. The presence of many beaded fluorescent fibers in close contact with the perikarya of the entire NPO clearly points to its monoaminergic innervation. This is in accordance with the situation in Carassius auratus (Baumgarten and Braak, 1967), Salmo truttafario (Weiss, 1970), Leuciscus rutilus (Ekengren, 1973, 1975b), and Anguilla anguilla (Fremberg et al., 1977). In some species a monoaminergic innervation of the NPO has been indicated by other techniques, such as monoamine oxidase (Weiss, 1970; Urano, 1971) and electron microscopy (Mfiller et al., 1971; Ekengren, 1973; Vigh-Teichmann et al., 1976). For the two salmonids the possibility of a monoaminergic innervation of the NPO has to be confirmed by electron microscopical investigations using, for example, false transmitters such as 5-hydroxydopamine (Tranzer and Thoenen, 1967; Richards and Tranzer, 1970) or a special fixative for catecholamines (Richards and Tranzer, 1974). The present study shows that the lateral part receives less aminergic fibers than the more rostrally and medially situated cells of the nucleus. This is in agreement with electron microscopical results for the rainbow trout (Foll6nius, 1963), electron and fluorescence microscopical results for the roach (Ekengren, 1973) and fluorescence microscopical observations in a gobiid fish, Gillichthys mirabilis (Swanson et al., 1975). Weiss (1970, 1976) reported, however, a well-developed monoaminergic innervation of the NLT in the brook trout. Although the fluorescent fibers innervating the NPO and NLT may originate in the NRL and NRP, it has not been possible to trace them with certainty. Lesioning experiments are planned to establish the origin of these fibers. c) The Hypophysis. All parts of the neurohypophysis of the two species contain many monoaminergic fibers, which has also been reported for some other species (Polenov et al., 1972; Ekengren, 1975a; Fremberg and Meurling, 1975; Swanson et al., 1975). Fibers were never observed to penetrate the basal lamina; this has been confirmed by electron microscopical investigations (Foll6nius, 1965; Fridberg and Ekengren, 1977; Peute, in preparation). Instead of directly innervating the adenohypophysial cells, the nerve fibers are in contact with the extensive perivascular system (PVS). Nerve fibers endings and PVS together form a kind of median eminence (Fridberg and Ekengren, 1977). All substances released into the PVS can reach and affect the endocrine cells.

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Fluorescent parenchymal cells have been found only in the hypophysis of the rainbow trout. The negative results for the salmon m a y be explained by seasonal variations, since the animals were obtained in winter and early spring and the trouts throughout the year. Cells showing a specific fluorescence were reported for Salmo truttafario (Weiss, 1970), Percafluviatilis and Tinca tinca (Weiss and R/ihle, 1969) and for Leuciscus rutilus (B~ge et al., 1974). In contrast to our results, Weiss and R~hle (1969) did not observe any fluorescent cells in the rainbow trout. According to Foll6nius (1967) such cells m a y incorporate labeled noradrenalin. There are various opinions on the type of cell that is involved: in Gasterosteus acidophils of the proximal pars distalis (PPD) (Foll6nius, 1967), in other teleosts m a n y but not all PAS-positive cells in the PPD (Weiss and Riihle, 1969), and in the roach a specific cell type that ultrastructurally contains dense core granules o f about 85 nm (B~ige et al., 1974). In the rainbow trout it is not certain whether the fluorescence is restricted to a special cell type, but m a n y of the cells show a basophilic and PAS-positive reaction. Additional work will be concentrated on the identity of these cells. In this respect it is noteworthy that after modifications of the Falck-Hillarp technique fluorescent cells in the mammalian hypophysis were reported that appear to contain some kind of tryptamine (Bj6rklund and Falck, 1969). The presence of amines in other endocrine polypeptide hormone-producing cells is known as well: cells of the A P U D series of Pearse (1969) ( A P U D = A m i n e Precursor Uptake and Decarboxylation). So far, information concerning their function is scarce; they m a y play a role in synthesis, storage and/or release of the polypeptide hormones (Owman et al., 1973). The present study has been carried out to locate and describe the position of aminergic nuclei and fiber tracts in the diencephalon, especially in the hypothalamo-hypophysial complex of salmonids. The results should serve as a structural basis for future studies of a more functional nature on the hypothalamohypophysial complex.

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Accepted April 10, 1978