Cell Tiss. Res. 168, 343-360 (1976)
Cell and Tissue Research 9 by Springer-Verlag 1976
Retinohypothalamic Pathway in the Duck (Anas platyrhynchos) * * * N. Bons Laboratoire de Neuroendocrinologie, ERA 85 du CNRS, PhysiologieAnimale, Universit6 de Montpellier II, Montpellier, France
Summary. Retinohypothalamic connections were studied in the duck after unilateral optic nerve transection using both light and electron microscopic techniques. Degenerated endings of optic fibers were found only in a circumscribed part o f the anterior hypothalamic area, i.e. the ventral region of the contralateral suprachiasmatic nucleus. Images o f degenerating boutons were observed in frozen sections (method according to Johnstone-Bowsher), and their presence confirmed by electron microscopic examination. These degenerating boutons make synaptic contacts with dendrites or dendritic spines o f neurons o f the suprachiasmatic nucleus. In the same material, the decussation of the optic chiasma was studied with the light microscope. Uncrossed retinal fibers were found in the marginal optic tract, the basal optic root and occasionally also in the isthmo-optic tract. Key words: Retinohypothalamic connections - Suprachiasmatic nucleus Optic chiasma - Duck.
Introduction The influence of photic (or photoperiodic) stimuli on the activity o f gonads was first demonstrated by R o w a n (1925) and Bissonnette (1930) in the starling and by Benoit (1934) in the duck. Since that time, this has been confirmed in a large variety of avian species. More recent experimental evidence has demonstrated that the anterior pituitary and the hypothalamus are essential links in the photo-gonadal response Send offprint requests to: Dr. No611e Bons, Laboratoire de Physiologie Animale, Universit6 de
Montpellier II, F-34060 Montpellier, France. * Dedicated to Professor Dr. W. Bargmann on the occasion of his 70th birthday ** Supported by the DGRST and "European Training Program Brain and Behaviour Research" I wish to express my gratitude to Professor Andreas Oksche, who repeatedly offered me the scientific facilities at the Department of Anatomy of the University of Giessen, and who provided me with valuable neuroanatomical suggestions throughout the progress of these studies.
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mechanism (for literature, see Benoit and Assenmacher, 1970; Farner, 1973). Moreover, the direct influence of light on the hypothalamus of blinded ducks was shown to stimulate the gonadotropic axis (Benoit et al., 1950). These results speak in favour o f the involvement ofextraretinal receptors (ERP) in the gonadal response. Experiments with the house sparrow have demonstrated convincingly the role o f the highly sensitive ERP in a number of physiological regulations. Therefore, the question has been raised whether or not the classical retinal photoreceptors are actually involved in the non-visual biological function of light (Menaker, 1971). There exist, however, some pertinent data which favour the concept o f a dual influence of photoperiodic information via both retinal and extraretinal photoreceptors. At low light levels, intact ducks were shown to have a significantly greater testicular response than experimentally blinded animals (Benoit et al., 1953). It is interesting to note that unoperated short-day adapted quail (6L (1 lux) 18D) responded to a four week exposure to long day conditions (18L (5 lux) - 6D) by an increase in testicular weight which was twice that o f the blinded congeners. The plasma concentration of testosterone was unchanged after five weeks of long day conditions in blinded quail, while it was doubled in intact controls (Bons et al., 1975). Therefore, the participation of the retina in the neuroendocrine regulation of birds cannot be disregarded. This physiological evidence has provided renewed interest in the problem o f the anatomical pathways between the retina and the hypothalamus (for reviews, see Nauta and Haymaker, 1969; Oksche, 1970; Oksche and Farner, 1974). On the other hand, lesion experiments have shown that, in addition to the classical infundibular gonadotropic area o f the hypothalamus (Follett, 1973), a second area located in the upper anterior hypothalamus might also be involved in the photoperiodic regulation of the gonads (Assenmacher, 1958, in duck; Ralph and Fraps, 1959, in chicken; Oliver, 1972; and Davies and Follett, 1974, in quail), thus placing emphasis on a circumscribed region of the hypothalamus near the optic chiasma. Keeping in mind physiological parameters, we initiated a series of anatomical investigations concerned with the problem of a possible retinohypothalamic nervous route in the duck. Although the primary concern was not to map the pathways of the primary and accessory optic tracts, we begun by a reinvestigation o f the optic tract (posterior to the decussation). This became important as some of our preliminary results were found to disagree with the data presented in the classical literature on the topic. The main studies were then focussed on the search for retinohypothalamic connections by exploring the optic pathways and possible terminals of degenerating nerve fibers within the hypothalamus after unilateral transection of one optic nerve.
Material and Methods
Male ducks (Anas platyrhynchos) were used in the present investigation. The right optic nerve was completelycut. Animals werethen sacrificedunder sodium pentobarbital (0.4 ml/kg) anaesthesia 4.5 h to 21 days postoperativelyby an intracarotid perfusion of 9% 0 saline followed by fixative. The material used for light microscopywas fixed 5, 8, 13, 14 and 21 days after nerve transection by perfusion with 10% formol in saline and either stored for two and a half months in fixative
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and subsequently dehydrated, cleared and embedded in paraffin, or stored for one week in fixative and then sectioned on a freezing microtome. Six and 12 gm thick paraffin sections were cut in frontal, sagittal and oblique planes. In the latter, the plane of the section was always parallel to the plane of the optic nerves in front of the optic chiasma. The course of degenerating fibers was traced in serial paraffin sections by silver impregnation (modified procedure based on the methods of Nauta and Nauta-Gygax). The general neuroanatomy and nuclear pattern of the diencephalon and the mesencephalon was analyzed in paraffin sections stained with Cresylviolet (Fernstr6m, Kltiver-Barrera), Eryochrom cyanin R (Page), and with the Bodian silver method. The brains with degenerating optic nerves were sectioned on a freezing microtome at 20 or 30 lain,and serial sections were made in a frontal or sagittal plane. In order to analyze the degenerating nerve fibers, the Fink-Heimer, Hjorth-Simonsen and Johnstone-Bowsher techniques were used. However, the latter method was superior to the others in order to demonstrate the nerve endings. Therefore, it was used for routine work. Adjacent sections to that which had been treated by the silver method were stained using the Page technique. In all cases, the degenerating optic fibers were easily identified on the basis of their increased argentophilic staining; however, the finer retinal fibers were better seen in the hypothalami of animals eight days after operation. This period of survival was then respected in further experimental work. For comparative purposes serial sections of the brains of Coturnix coturnix japonica, Columba livia and Pica pica were prepared. For electron microscope studies, the ducks were perfused 4.5, 7, 18, 23, 28, 46, 70 h and 8 days after the operation with 9%o saline followed by 4% paraformaldehyde solution. Small blocks of the anterior hypothalamus, contralateral and ipsilateral to the sectioned optic nerve, were post-fixed without washing in a 2% osmic acid solution (Westrum and Lurid, 1966) and stained with uranyl acetate during dehydratation in ethanol (Kokko and Rechardt, 1968: procedure VIII), and embedded in Epon or Araldite (Luft, 1961). The brains of one sham-operated animal (sacrificed 45.5 h after injury) and those of unoperated animals were examined using the same techniques.
Results A. Optic chiasma (cf. Bons, 1969) The optic nerves o f the d u c k are c o m p o s e d o f fascicular a r r a n g e m e n t s o f myelinated a n d u n m y e l i n a t e d fibers. E a c h optic nerve enters the chiasmatic region a n d divides into five o r six large b u n d l e s which cross the m i d l i n e a l t e r n a t i n g with the b u n d l e s o f the c o n t r a l a t e r a l nerve. The m a j o r i t y o f the optic fibers decussate in the optic chiasma. However, a small g r o u p o f d e g e n e r a t i n g fibers does n o t intersect a n d forms two postchiasmatic fascicles which project to the ipsilateral side. This is clearly seen in the obliquely sectioned b r a i n (Fig. 1). Both u n c r o s s e d optic fascicles represent 1.2% o f the d i a m e t e r o f the optic tract. O n e o f the lateral fascicles (showing a n o v a l - s h a p e d profile o f 140 p,m/40 lam) leaves the c e n t r o - d o r s a l part o f the c h i a s m a and, instead o f crossing it, unites with the dorsal division o f the c o n t r a l a t e r a l medial optic tract. Initially, the fibers o n the surface o f these ipsilateral fascicles r u n a m o n g the c o n t r a l a t e r a l fibers. Some o f t h e m reach the t e r m i n a l p o r t i o n o f the tract a n d m a k e c o n t a c t with the a n t e r i o r v e n t r a l part o f the lateral geniculate nucleus. A n o t h e r part of this ipsilateral fascicle rejoins the lateral a n d v e n t r o l a t e r a l part of the m a r g i n a l optic tract a n d projects into the tectum. T h e m o s t dorsal fibers of these lateral fascicles s u r r o u n d the external three f o u r t h s o f the lateral a n t e r i o r n u c l e u s a n d form, together with a large c o n t r a l a -
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Fig. 1. Optic system of the duck. Schematic representation of uncrossed optic fibers in the optic chiasma and the optic projections. Hatched fascicles belong to the right optic nerve, white fascicles to the left optic nerve. ROB, basal optic root; ECN, ectomammillarynucleus; EN, external nucleus; ION, isthmo-opticnucleus; lOT, isthmo-optictract (black, in order to emphasize its mainly retinopetal character); LAN, lateral anterior nucleus; LG, lateral geniculate nucleus; MOT, marginal optic tract; OC, optic chiasma; ON, optic nerve; SSN, superficial synencephalic nucleus; T, tectum; TG, tectal grey
teral fascicle, the isthmo-optic tract. The uncrossed (degenerated) fibers of this isthmo-optic tract are present only in the lateral part of the tract, because the medial part, which runs directly dorsal to the lateral geniculate nucleus, has no degenerated fibers. Both the lateral and medial parts come from the isthmo-optic nucleus which is dorsal and posterior in relation to the optic chiasma. In this context it should not be overlooked that the isthmo-optic tract o f birds is mainly formed by efferent (retinopetal) fibers. The second ipsilateral fascicle, more posterior than the first, breaks off laterally from the postero-ventral part of the chiasma, about 1 mm behind the first fascicle. This second ipsilateral fascicle splits up into the contralateral basal optic root (200 pm in diameter). This is clearly seen when the basal optic root is completely separated from the marginal optic tract (Fig. 6). All ipsi- and contralateral fibers end in the ectomammillary nucleus (Figs. 4, 5). However, most o f the ipsilateral fibers end in the ventral internal ridge of the ectomammillary nucleus. In fact, each of the three parts of the optic tract: the marginal optic tract (i.e., lateral optic tract), the isthmo-optic tract (i.e., medial optic tract) and the basal optic root are, in the duck, made up not only of contralateral optic fibers but they contain also some ipsilateral optic elements. B. Retinohypothalamic Connections (cf. Bons and Assenmacher, 1969, 1973;
Bons, 1974) I. Operated Animals 1. Nauta-stained Paraffin Sections. The great majority of the optic fibers which are posterior to the optic chiasma project into the thalamus and the mesencepha-
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Fig. 2. Series of frontal sections through the duck brain at different levels of the anterior division of the suprachiasmatic nucleus (A D). LAN, lateral anterior nucleus; LFB, lateral forebrain bundle; LGN, lateral geniculate nucleus; LOT, left optic tract (with degenerated fibers); PR, preoptic recess; P VN, paraventricular nucleus; SCN, suprachiasmatic nucleus; SO C, supraoptic commissure; SONm, medial part of the supraoptic nucleus; SONI, lateral part of the supraoptic nucleus. The highly stippled part of the SCN represents the region where degenerated endings of retinal fibers were found
lon. However, another group o f degenerated crossed fibers are directed towards the anterior part of the brain. These fibers remain lateral to the optic tract just behind the chiasma and form a short tract with an elliptic profile (600 p,m/ 200 tam in diameter). The fibers of the short tract are scattered throughout its cross-sectioned area; in a transversal plane more than 60 degenerated fibers were counted. This tract projects completely into the suprachiasmatic nucleus (Figs. 2, 3). The suprachiasmatic nucleus is limited laterally by the lateral part of the supraoptic nucleus and medially by the preoptic recess and, in its rostral region, by the medial part o f the supraoptic nucleus. After unilateral surgical interruption of the optic nerve, these degenerating retinal fibers appear to be thinner than classical degenerating optic fibers showing a caudal route (Figs. 7, 8). Black-colored, fragmented and swollen structures show a scattered pattern. N o r m a l non-degenerated fibers are light brown and regular.
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Fig. 3. Anterior hypothalamusof the duck. Diagram showing different nuclei. Arrow, projection area of retinohypothalamicfibers. LFB, lateral forebrain bundle; LO T, left optic tract; PR, preoptic recess; PVN, paraventricular nucleus; ROT, right optic nerve; SCN, suprachiasmatic nucleus; SMT, septomesencephalictract; SOC, supraopticcommissure;SONm, medialpart of the supraoptic nucleus; SONI, lateral part of the supraoptic nucleus
2. Impregnations According to Johnstone-Bowsher. This material of the duck
brain not only showed numerous degenerating fibers (Figs. 9, 10) but also degenerating endings in the left suprachiasmatic nucleus. Generally these changes were observed eight days after transection of the right optic nerve. These degenerated endings represent three different types: a) Irregularly-shaped, bulb-like boutons which are heavily silver impregnated and also show a short preterminal segment of the degenerated axon. They contact neuronal perikarya approximately 30 ~tm in diameter (Fig. 14). b) Neurofibril-containing argyrophilic ring-like boutons (not larger than 2.5 lam in diameter) which contact thin, poorly stained dendrites (Fig. 11). c) Degenerated endings forming a fine basket around the perikarya of a neuron (Fig. 12). 3. Preparations Treated According to Bodian. The results of the preparations
treated by the Bodian method are similar to the previously described results considering the site and the terminals of the retinohypothalamic fibers as well as their contact with dendrites and cell bodies. The fiber images obtained by using this method are finer than those found in preparations of the JohnstoneBowsher type (compare Fig. 11 and Fig. 13). 4. Electron Microscopic Study. The area of the anterior hypothalamus shown
by light microscopy to be the suprachiasmatic nucleus (see Figs. 2, 3) as well as the area where the degenerating boutons and the degenerated fibers were seen were observed with the electron microscope. In the suprachiasmatic nucleus, contralateral to the lesioned axons, different signs of degeneration were observed 4.5, 7, 18, 23, 28, 46, 70 h and 8 days after the operation. The ventral part of the nucleus contains numerous myelinated fibers. Eighteen hours after the interruption of the optic nerve, the axoplasm of some fibers shows shrinkage and is electron dense (Figs. 27, 29), however, the myelin
Figs. 4-8. Nauta impregnations of paraffin sections 8 days after unilateral transection of the optic nerve in the duck Fig. 4. Ipsilateral ectomammillary nucleus with several degenerated fibers (arrows) Fig. 5. Contralateral ectomammillary nucleus with numerous degenerated fibers Fig. 6. Ipsilateral basal optic root with two degenerated fibers (arrows) Figs. 7 and 8. Degenerated fibers (short arrows) leaving the contralateral optic tract (or) and running to the suprachiasmatic nucleus; n, neurons of the suprachiasmatic nucleus. Scale markers: 10 ~m
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Figs. %14. Suprachiasmatic nucleus of the duck 8 days after contralateral optic nerve transection. Johnstone-Bowsher impregnation, frozen sections Figs. 9 and 10. Degenerated fibers (arrows') Figs. 11, 12 and 14. Degenerated endings (arrows) in contact with a dendrite (d) (Fig. 11) and with neuronal perikarya (n) (Figs. 12, 14) Fig. 13. Degenerated ending (arrow) between neuronal perikarya, Bodian impregnation. Scale markers: 5 lam
Figs. 15-19. Electron micrographs o f the contralateral suprachiasmatic nucleus of the duck after unilateral optic nerve resection Figs. 16, 18 and 19. Animal sacrificed 4.5 h after the operation Figs. 15 and 17. Control animal: a, axonal arborization; b degenerated bouton; a r r o w s , synaptic contacts. Scale markers : 330 n m
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lamellae are usually still intact. Whereas in the degenerating optic nerve and optic tract (Fig. 31) numerous mitochondria are devoid of cristae and show a dark matrix, which is considered to be the first obvious sign of axonal degeneration, no mitochondria are recognized in sections of degenerating retinohypothalamic fibers. Some of the degenerating preterminal and synaptic elements of the retinal axons are observed in the ventral portion of the suprachiasmatic nucleus somewhat earlier, i.e. 4.5 h after the operation. The degeneration is characterized in the axonal arborization (i.e., preterminal portion) by a very electron dense axoplasmic matrix masking the mitochondria (Fig. 18). This opacity results from the marked increase of neurofilaments which run mainly parallel to the long axis of the cytoplasm. Degeneration in terminals is characterized by progressive darkening of the presynaptic cytoplasm and of the mitochondria, as well as by the presence of a small number of spheroidal synaptic vesicles and by numerous flattened vesicles forming clumplike arrangements (Figs. 16, 19-26). Even 4.5 h after transection of the optic nerve, a distinct increased electron density of the postsynaptic membrane thickening can be clearly seen. Seventy hours after the contralateral operation all signs of degeneration described are present, as well as considerable increase in axoplasmic density of the synaptic endings (Fig. 28). One degenerating bouton displayed a strange appearance (Figs. 22 24). It contained a concentric lamellar formation which resembles a myelin sheath in the process of degeneration. This lamellar body may indicate the decomposition of membranes rich in phospholipids. All the degenerating terminals were found to be in contact with dendritic spines and dendrites. Within the suprachiasmatic nucleus, the astrocytes show a reactive transformation during the course of degeneration of the optic fibers. Their perikarya become greatly enlarged, their processes longer and more numerous. The latter extend toward the basal lamina of the capillaries. In the distal portion, near the capillaries, the processes contain more glycogen granules than in the proximal portion which contains bundles of glial filaments. The proximal divisions of the astrocytic processes, situated close to the perikaryon, subsequently engulf the degenerating optic terminals. The phagocytic glial cells also undergo changes after optic nerve transection. Their hypertrophied somata contain numerous long cisternae of granular endoplasmic reticulum. II. Sham-operated and Control Animals No characteristic spontaneous degeneration of retinohypothalamic fibers was observed in the corresponding parts of the ipsilateral suprachiasmatic nucleus. Degenerating (or degenerated) fibers were also lacking in the suprachiasmatic nuclei of the control and the sham-operated animals sacrificed 45.5 h after the operation. This confirms the results obtained at the light microscopic level where only one spontaneous degenerated nerve ending was found in three sections of the suprachiasmatic nucleus. Furthermore, it was impossible to determine if it is belonged to a retinal axon. Glial cells of both the control and sham-operated animals showed no sign of phagocytic activity. The cell bodies of the astrocytes are inconspicuous,
Figs. 20-25. Degenerated endings in the suprachiasmatic nucleus of the duck 4.5 h (Figs. 20, 21, 25) and 70 h (Figs. 22 24) after contralateral optic nerve transection, b, Degenerating bouton; d, dendrite; thick arrows, synaptic contacts; thin arrows, concentric array of membranes. Scale markers: 330 nm
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and possess fine cell processes. Phagocytic glial cells are only occasionally seen. Their cytoplasm forms a thin rim around an irregularly-shaped nucleus.
Discussion
A. Evidence of Uncrossed Nerve Fibers in the Avian Optic Chiasma Most investigators of the avian visual system have concluded that all optic fibers decussate in the optic chiasma (Perlia, 1889; Edinger and Wallenberg, 1889; Harris, 1904; Huber and Crosby, 1929; Bltimcke, 1961; Cowan etal., 1961 ; Karten and Nauta, 1968; Hartwig, 1970). Only Cowan and his colleagues did not exclude the possibility that a few fibers may continue into the optic tract on the ipsilateral side. These authors observed a single degenerating bouton in the lateral geniculate nucleus of the pigeon, ipsilateral to the enucleated eye. However, they felt that there were no real ipsilateral fascicles. Further, Ferreira-Berrutti (1951) and Knowlton (1954) have clearly demonstrated the existence of a partial decussation of the optic fibers in the chick embryo. Our results in the adult duck are in agreement with these two earlier reports on the chick embryo. The optic fibers which remain ipsilateral terminate in the lateral geniculate nucleus, the lateral anterior thalamus, the external nucleus, the superficial and synencephalic nucleus and deep into the tectal gray matter. Another group of ipsilateral retinal fibers run into the deeper part of the marginal optic tract and end within the mesencephalon. Uncrossed optic fibers of the basal optic roots which terminate in the ipsilateral ectomammillary nucleus seem to be characteristic of the duck optic system. Nauta and Van Straaten (1947) suggested that ipsilateral optic fibers are also a component of the basal optic roots of mammals. In fact, uncrossed optic fibers are found in all groups of Tetrapods. In several mammalian species there is evidence for uncrossed retinal fibers. In the three other classes of tetrapods (amphibians, reptiles and birds) there is a lack of uncrossed fibers in certain species; however, in every class an incomplete decussation in the optic chiasma was observed. The urodele, Cryptobranchus allegeheniensis (Riss et al., 1963), possesses uncrossed optic fibers which project into the preoptic area. This finding was confirmed in Ambystoma tigrinum by Jackway and Riss (1972). These results are in contradiction to the observation of Gruberg (1969) who was not able to find ipsilateral degenerating optic fibers in the same urodele species. These negative results are probably due to the lower temperature at which the operated animals were kept and to a different staining procedure employed. Earlier results of Herrick in Ambystoma (1925) and Necturus (1949) may be interpreted as demonstrating the presence of an uncrossed retinohypothalamic and pretectal tract. In the frog, Rana pipiens, two ipsilateral optic fascicles exist, one running to the tectum, the other terminating in the pretectal area (Knapp et al., 1965). Armstrong (1950, 1951), and more recently Burns and Goodman (1967), Ebbesson (1970), Reperant (1972, 1973), Halpern and Frumin (1973), Northcutt and Butler (1973) have observed retinal fibers which fail to cross in the chiasma
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Figs. 26-31. Contralateral suprachiasmatic nucleus of the duck after unilateral transection of the optic nerve. Degenerating boutons 70h (Figs. 26, 28) and 8 days (Fig. 30) after the operation. Degenerating myelinated fibers in the same region 4.5 h (Fig. 27) and 18 h (Fig. 29) after the operation. Degenerated optic fibers in the contralateral optic tract, 18 h after the operation (Fig. 31). a, Axon; b, degenerating or degenerated bouton; d, dendrite; g, glial process; arrows, synaptic contacts. Scale markers: 330 nm
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and project to the ipsilateral diencephalon and pretectum in different species of lizards and snakes. To date, in only one species of turtles (Emys orbicularis) have uncrossed optic fibers been found (Kosareva, 1967). It appears, therefore, that uncrossed optic fibers are not only characteristic of the mammalian optic system and related to the stereoscopic vision, since they can be found in some amphibians, reptiles and birds. However, there is no evidence that uncrossed retinal fibers occur in the teleost or the elasmobranch.
B. Retinohypothalamic Pathway In our first description of retinohypothalamic connections in ducks with unilaterally transected optic nerves (Bons and Assenmacher, 1969), the Nauta-stained paraffin sections showed some very fine degenerating fibers in the anterior hypothalamus. This area which was called the "supraoptic region" was by no means restricted to the supraoptic nucleus but included a large anterior hypothalamic area with numerous scattered extensions of the supraoptic nucleus (Assenmacher, 1958). Later, Crosby and Showers (1969) and Oksche (1970) identified this circumscribed region of the anterior hypothalamus as the suprachiasmatic nucleus of birds. Using the subtle Johnstone-Bowsher impregnation method, we were able to outline the precise projection of retinal fibers and to trace their terminals within the suprachiasmatic nucleus. This nucleus is bordered on its lateral side by the lateral part of the supraoptic nucleus, and, on its medial side a) rostrally by the medial part of the supraoptic nucleus, and b) caudally by the preoptic recess and the paraventricular nucleus (Bons and Assenmacher, 1973). Finally, an electron microscopic study on degenerating fibers (Bons, 1974) within the anterior hypothalamus displayed in the contralateral suprachiasmatic nucleus not only degenerating fibers and axonal arborizations but also degenerating presynaptic profiles and postsynaptic membranes. These electron microscopic results prove that a circumscribed region of the anterior hypothalamus of the duck (suprachiasmatic nucleus) receives direct retinal input. The results obtained in the duck with silver impregnation methods (Bons and Assenmacher, 1969, 1973) are in agreement with the reports of Bliimcke (1961) and Brugi (1937) in the domestic fowl. However, in the chicken retinohypothalamic fibers seem to be more numerous than in duck. Meier (1973) employed autoradiography in a study concerned with the terminals of the retinohypothalamic fibers in birds. Labelled amino acids were incorporated into retinal neurons and subsequently transported along their axons to the terminals. Meier described a retinal projection to the anterior hypothalamic region contralateral to the labelled retina in Columba livia and Coleus monedula. On the basis of electron microscopic observations Hartwig (1974) reported the existence of degenerated optic terminals in the suprachiasmatic nucleus of unilaterally retinectomized Passer domesticus. However, in the house sparrow degenerated myelinated axons did not occur in this region. On the other hand, in recent studies on retinohypothalamic endings in several
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mammals and anurans, electron microscopic techniques and autoradiographic tracing have been applied (see Conrad and Stumpf, 1974). In the mammals studied, i.e. rat, hamster, guinea pig, rabbit, cat and monkey (O'Steen and Vaughn, 1968; Moore et al., 1971 ; Moore and Lenn, 1972; Hendrickson et al., 1972), these techniques revealed clearly that the retinohypothalamic tracts terminate on dendritic branches of neurons in the ventral portion of the suprachiasmatic nuclei. These results show that the only difference existing between birds and mammals is that in birds the retinal axons project only into the contralateral suprachiasmatic nucleus, whereas, in mammals the retinal axons project to both the ipsi- and contralateral suprachiasmatic nuclei. Vullings and Kers (1973) applied similar techniques in Rana temporaria. They found ipsilateral connections between the retina and the preoptic nuclei. Several recent investigations have demonstrated retinal projections to the hypothalamus of amphibians (Jackway and Riss, 1972), reptiles (Butler and Northcutt, 1971; Halpern and Frumin, 1973; Northcutt and Butler, 1974), and mammals (Karamanlidis and Magras, 1972, 1974). However, these observations were made with the light microscope using specimens stained with different silver methods. These results were partly contradictory. The body of evidence presented in recent investigations on the optic system leads us to the conclusion that retinal projections to the anterior hypothalamic region exist in all systematic groups of vertebrates. We suggest that the divergent results obtained in previous studies are due to the different silver stains applied and to the variety of surgical techniques (unilateral bulbectomy, unilateral retinectomy, uni- and/or bilateral transection of the optic nerve; cf., Nauta and Haymaker, 1969). We conclude that a direct retinohypothalamic pathway exists in birds, mammals, amphibians and presumably also in reptiles. This pathway projects to the area of the suprachiasmatic nucleus or its homologues. This area of the anterior hypothalamus plays an essential role in the photoperiodic control of the gonadotropic mechanism of birds. In mammals, the suprachiasmatic nuclei are involved in cyclic regulations leading to ovulation (Clattenburg, et al., 1972), in the control of the diurnal rhythm of the adrenal cortex (Moore and Eichler, 1972) and of several rhythmic behavioural patterns (Stephan and Zucker, 1972). Whether or not this parvocellular region of the anterior hypothalamus contains secretory neurons producing the hypothalamic adenohypophysiotropic hormones (see Barry et al., 1973) will require further investigation. In the house sparrow, Passer domesticus, it is rich in secretory perikarya of different types (Oksche and Farner, 1974; Oksche and Hartwig, 1975). In the rostral hypothalamus retinal inputs to neuroendocrine and/or other hypothalamic systems might be integrated and modulated. However, the exact physiological role of the retinohypothalamic connection is still open to discussion.
References
Armstrong, J.A.: An experimental study of the visual pathways in a reptile (Lacerta vivipara). J. Anat. (Lond.) 84, 146-167(1950) Armstrong, J.A.: An experimental study of the visual pathways in a snake (Natrix natrix). J. Anat. (Lond.) 85, 275-288 (1951)
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Assenmacher, I. : Recherches sur le contr61e hypothalamique de la fonction gonadotrope pr6hypophysaire chez le Canard. Arch. Anat. micr. Morph. exp. (Paris) 47, 447-572 (1958) Barry, J., Dubois, M.P., Poulain, P. : LRF producing cells of the mammalian hypothalamus. A fluorescent antibody study. Z. Zellforsch. 146, 351-366 (1973) Benoit, J. : Activation sexuelle obtenue chez le Canard par l'6clairement artificiel pendant la p6riode de repos g6nital. C.R. Acad. Sci. (Paris) 199, 1671-1672 (1934) Benoit, J. : Actions de divers 6clairements localis6s dans la r6gion orbitaire sur la gonado-stimulation chez le Canard mille impub6re. Croissance testiculaire provoqu6e par l'6clairement direct de la r6gion hypophysaire. C.R. Soc. Biol. (Paris) 127, 909-914 (1938) Benoit, J., Assenmacher, I. (eds.): La photor6gulation de la reproduction chez les Oiseaux et les Mammif6res. C.N.R.S., Paris, p. 588 (1970) Benoit, J., Assenmacher, I., Walter, F.X. : Dissociation exp6rimentale du r61e des r6cepteurs superficiel et profond dans la gonadostimulation hypophysaire par la lumi6re chez le Canard. C.R. Soc. Biol. (Paris) 147, 186-191 (1953) Benoit, J., Walter, F.X., Assenmacher, I.: Contribution il l'6tude du r6fiexe opto-hypophysaire gonadostimulant chez le Canard soumis il des radiations lumineuses de diverses longueurs d'onde. J. Physiol. (Paris) 42, 537-541 (1950) Bissonnette, T.H. : Modification of mammalian sexual cycles: Reactions of ferrets (Putorius vulgaris) of both sexes to electric light added after dark in November and December. Proc. roy. Soc. B 110, 322-336 (1932) Blfimcke, S.: Vergleichend experimentell-morphologische Untersuchungen zur Frage einer retinohypothalamischen Bahn bei Huhn, Meerschweinchen und Katze. Z. mikr.-anat. Forsch. 67, 469-513 (1961) Bons, N. : Mise en 6vidence du croisement incomplet des nerfs optiques au niveau du chiasma chez le Canard. C.R. Acad. Sci. (Paris), 268, 2186-2188 (1969) Bons, N.: Mise en 6vidence au microscope 61ectronique, de terminaisons nerveuses d'origine r&inienne dans l'hypothalamus ant6rieur du Canard. C.R. Acad. Sci. (Paris) 278, 319 321 (1974) Bons, N., Assenmacher, I. : Pr6sence de fibres r6tiniennes d6g6n6r6es dans la r6gion hypothalamique supra-optique du Canard apr6s section d'un nerf optique. C.R. Acad. Sci. (Paris) 269, 1535 1538 (1969) Bons, N., Assenmacher, I. : Nouvelles recherches sur la voie nerveuse r&inohypothalamique chez les Oiseaux. C.R. Acad. Sci. (Paris) 277, 2529-2532 (1974) Bons, N., Jallageas, M., Assenmacher, I. : Influence des r6cepteurs r6tiniens et extra-r6tiniens dans la stimulation testiculaire de la Caille par les "jours longs". J. Physiol. (Paris) 71, No. 2, 265266 A (1975) Brugi, G. : Reperti istologici sperimentali nel pollo a conferma dell esistenza di diretti connessioni del tratto ottico con la zona anteriore dell'ipotalamo. Monit. zool. ital. 48, 264~268 (1937) Burns, A.H., Goodman, D.C. : Retinofugal projections of Caiman sclerops. Exp. Neurol. 18, 105 115 (1967) Butler, A.B., Northcutt, R.G. : Retinal projections in Iguana iguana and Anolis earolinensis. Brain Res. 26, 1-13 (1971) Clattenburg, R.E., Singh, R.P., Montemurro, D.G. : Post coital ultrastructural changes in neurons of the suprachiasmatic nucleus in the rabbit. Z. Zellforsch. 125, 448-459 (1972) Conrad, C.D., Stumpf, W.E.: Retinal projections traced with thaw-mount autoradiography and multiple injections of tritiated precursor or precursor cocktail. Cell Tiss. Res. 155, 283-290 (1974) Cowan, W.M., Adamson, L., Powell, T.P.S.: An experimental study of the avian visual system. J. Anat. (Lond.) 95, 545-553 (1961) Crosby, E.L., Showers, M.J.L.: Comparative anatomy of the preoptic and hypothalamic areas. In: The hypothalamus (W. Haymaker, E. Anderson and W.J.H. Nauta, eds.), pp. 61-135. Springfield, Ill.: Ch.C. Thomas 1969 Davies, D.T., Follett, B.K.: Hypothalamic deafferentation in Coturnix quail and its blockade of photoperiodically induced testicular development Gen. comp. Endocr. 22, 359 (1974) Ebbesson, S.O.E.: On the organization of central visual pathways in vertebrates. Brain Behav. Evol. 3, 178-194 (1970) Edinger, L., Wallenberg, A. : Untersuchungen fiber das Gehirn der Tauben. Anat. Anz. 15, 245-271 (1898)
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Farner, D.S. (ed.): Breeding biology of birds. Nat. Acad. Sci. Washington D.C., p. 513 (1973) Ferreira-Berrutti, P. : Experimental deflection of the course of the optic nerve in the chick embryo. Proc. Soc. exp. Biol. (N.Y.) 76, 302-303 (1951) Fink, R.P., Heimer, L. : Two methods for selective silver impregnation of degenerating axons and their synaptic endings in the central nervous system. Brain Res. 4, 369 374 (1967) Follett, B.K.: The neuroendocrine regulation of gonadotropin secretions in avian reproduction. In: Breeding biology of birds (D.S. Farner, ed.), pp. 209-243. Nat. Acad. Sci. Washington, D.C., 1973 Gruberg, E.R. : Functional organization of the tectum of the tiger salamander Ambystoma tigrinum. Technical report 17, Biological Computor Laboratory. Engineering Experiment Station, University of Illinois, Urbana, Ill. 1969 Halpern, M., Frumin, N. : Retinal projections in a snake, Thamnophis sirtalis. J. Morph. 141, 359-381 (1973) Harris, W. : Binocular stereoscopic vision in man and other vertebrates, with its relation to the decussation of the optic nerves, the ocular movements and the pupil light reflex. Brain 27, 107-147 (1904) Hartwig, H.G. : Das visuelle System yon Zonotrichia leucophrys gambelii. Neurohistologische Studien auf experimenteller Grundlage. Z. Zellforsch. 106, 556-583 (1970) Hartwig, H.G. : Electron microscopic evidence for a retinohypothalamic projection to the suprachiasmatic nucleus of Passer domesticus. Cell Tiss. Res. 153, 89-99 (1974) Hendrickson, A.E., Wagoner, N., Cowan, W.M.: An autoradiographic and electron microscopic study of retino-hypothalamic connections. Z. Zellforsch. 135, 1-26 (1972) Herrick, C.J. : The amphibian forebrain. III. The optic tracts and centers of Ambystoma and the frog. J. comp. Neurol. 39, 433489 (1925) Herrick, C.J.: Optic and postoptic systems of fibers in the brain of Necturus. J. comp. Neurol. 75, 487 544 (1941) Hjorth-Simonsen, A. : Fink-Heimer silver impregnation of degenerating axons and terminals in mounted cryostat sections of fresh and fixed brains. Stain Technol. 45, 199-204 (1970) Huber, G.C., Crosby, E.C. : The nuclei and fibre paths of the avian diencephalon, with consideration of telencephalic and ertain mesencephalic centers and connexions. J. comp. Neurol. 48, 1-223 (1929) Jackway, J.S., Riss, W. : Retinal projections in the tiger salamander Ambystoma tigrinum. Brain Behav. Evol. 5, 401-442 (1972) Johnstone, G., Bowsher, D.: A new method for the selective impregnation of degenerating axon terminals. Brain Res. 12, 47-53 (1969) Karamandilis, A.N., Magras, J. : Retinal projections in the domestic ungulates. I. the retinal projections in the sheep and the pig. Brain Res. 44, 127 145 (1972) Karamandilis, A.N., Magras, J. : Retinal projections in the domestic ungulates. II. the retinal projections in the horse and the ox. Brain Res. 66, 209-225 (1974) Karten, H., Nauta, W.J.H.: Organization of retinothalamic projections in the pigeon and owl. Anat. Rec. 160, 373 (1968) Knapp, H., Scalia, F., Riss, W. : The optic tracts of Rana pipiens. Acta neurol, scand. 41, 325-355 (1965) Knowlton, V.Y. : Abnormal differentiation of embryonic avian brain centers associated with unilateral anophthalmia. Acta anat. (Basel) 58, 222-251 (1964) Kokko, A., Rechardt, L.: Block-staining for electron microscopy of nervous tissue. Ann. Med. exp. Fenn. 46, 123-131 (1968) Kosareva, A.A.: Projection of optic fibers to visual centers in a turtle (Emys orbicularis). J. comp. Neurol. 130, 263-276 (1967) Luft, J.H. : Improvments in epoxy resin embedding methods. J. biophys, biochem. Cytol. 9, 409~,14 (1961) Meier, R.E. : Autoradiographic evidence for a direct retino-hypothalamic projection in the avian brain. Brain Res. 53, 417 421 (1973) Menaker, M.: Synchronization with the photic environment via extraretinal receptors in the avian brain. In: Biochemistry (M. Menaker, ed.). Nat. Acad. Sci., Washington D.C., pp. 315-332 (1971) Moore, R.Y.: Retinohypothalamic projection in mammals: a comparative study. Brain Res. 49, 40 3~,09 (1973)
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Moore, R.Y., Eichler, V.B. : Loss of circadian adrenal corticosterone rhythm following suprachiasmatic lesions in the rat. Brain Res. 42, 201-206 (1972) Nauta, W.J.H.: Uber die sogenannte terminale Degeneration im Zentralnervensystem und ihre Darstellung durch Silberimpr/ignation. Schweiz. Arch. Neurol. Psychiat. 66, 353-376 (1950) Nauta, W.J.H., Gygax, P.A.: Silver impregnation of degenerating axon terminals in the central nervous system: 1 technique, 2 chemical notes. Stain Technol. 26, 5 11 (1951) Nauta, W.J.H., Haymaker, W. : Retino-hypothalamic connections. In: The hypothalamus (W. Haymaker, E. Anderson and W.J.H. Nauta, eds.), pp. 136-209. Springfield, Ill.: Ch.C. Thomas 1969 Nauta, W.J.H., van Straaten, J.J.: The primary optic centers of the rat. An experimental study by the "bouton" method. J. Anat. (Lond.) 81, 127-134 (1947) Northcutt, R.G., Butler, A.B. : Retinal projections in the Northern water snake Natrix sipedon sipedon (L.). J. Morph. 142, 117-135 (1974) Oksche, A.: Retino-hypothalamic pathways in birds and mammals. In: La photor6gulation de la reproduction chez les Oiseaux et les Mammif6res (J. Benoit, I. Assenmacher, eds.), pp. 151-165. Paris: Coll. Int. C.N.R.S. No. 172, 1970 Oksche, A., Farner, D.S.: Neurohistological studies of the hypothalamo-hypophysial system of Zonotrichia leucophrys gambelii (Aves, Passeriformes). Adv. Anat. Embr. Cell Biol. 48]4, 1-136 (1974) Oksche, A., Hartwig, H.G. : Photoneuroendocrine systems and the third ventricle. Brain-endocrine interaction II. The ventricular system. Second. Int. Symp., Shizuoka 1974, pp. 40-53. Basel: Kargel 1975 Oliver, J. : Etude exp6rimentale des structures hypothalamiques impliqu6es dans le r6flexe photosexuel chez la Caille. Th6se de Sp~cialit~ Physiologie. Universit6 de Montpellier p. 68 (1972) O'Steen, W.K., Vaughn, G.M. : Radioactivity in the optic pathway and hypothalamus of the rat after intraocular injection of tritiated 5-hydroxytryptophan. Brain Res. 8, 20%212 (1968) Page, K.M.: Histological methods for peripheral nerves. J. Med. Lab. Techn. 27, 1-17 (1970) Perlia, R. : (Jber ein neues optisches Zentrum beim Huhn. Albrecht v. Graefes Arch. Ophthal. 35, 20-24 (1889) Ralph, C.L., Fraps, R.M.: Long-term effects of diencephalic lesions on the ovary of the hen. Amer. J. Physiol. 197, 1279-1283 (1959a) Ralph, C.L., Fraps, R.M. : Effects of hypothalamic lesions on progesterone induced ovulation in the hen. Endocrinology 65, 81%824 (1959b) Rep6rant, J.: Etude exp6rimentale des projections visuelles chez la Vip6re (Vipera aspis). C.R. Acad. Sci. (Paris) 275, 695-697 (1972) Rep6rant, J. : Les voies et les centres optiques primaires chez la Vip6re (Vipera aspis). Arch. Anat. micr. Morph. exp. 62, 322-352 (1973) Riss, W., Knapp, H.D., Scalia, F. : Optic pathways in Cryptobranchus allegheniensis as revealed by Nauta technique. J. comp. Neurol. 121, 31-43 (1963) Rowan, W.M. : Relation of light to bird migrations and developmental changes. Nature (Lond.) 115, 494 (1925) Stephan, F.K., Zucker, I.: Rat drinking rhythms: central visual pathways and endocrine factors mediating responsiveness to environmental illumination. Physiol. Behav. 8, 315-326 (1972) Stephan, F.K., Zucker, I.: Circadian rhythms in drinking behavior and locomotor activity of rats are eliminated by hypothalamic lesions. Proc. nat. Acad. Sci. (Wash.) 69, 1583-1586 (1972) Vullings, H.G.B., Kers, J. : The optic tracts of Rana temporaria and a possible retino-preoptic pathway. Z. Zellforsch. 139, 179-200 (1973) Westrum, L.E., Lund, R.D. : Formalin perfusion for correlative light and electron microscopical studies of the nervous system. J. Cell Sci. 1, 229-238 (1966)
Received November 27, 1975 / in final form January 10, 1976
Cell and Tissue Research 9 by Springer-Verlag 1976
Retinohypothalamic Pathway in the Duck (Anas platyrhynchos) * * * N. Bons Laboratoire de Neuroendocrinologie, ERA 85 du CNRS, PhysiologieAnimale, Universit6 de Montpellier II, Montpellier, France
Summary. Retinohypothalamic connections were studied in the duck after unilateral optic nerve transection using both light and electron microscopic techniques. Degenerated endings of optic fibers were found only in a circumscribed part o f the anterior hypothalamic area, i.e. the ventral region of the contralateral suprachiasmatic nucleus. Images o f degenerating boutons were observed in frozen sections (method according to Johnstone-Bowsher), and their presence confirmed by electron microscopic examination. These degenerating boutons make synaptic contacts with dendrites or dendritic spines o f neurons o f the suprachiasmatic nucleus. In the same material, the decussation of the optic chiasma was studied with the light microscope. Uncrossed retinal fibers were found in the marginal optic tract, the basal optic root and occasionally also in the isthmo-optic tract. Key words: Retinohypothalamic connections - Suprachiasmatic nucleus Optic chiasma - Duck.
Introduction The influence of photic (or photoperiodic) stimuli on the activity o f gonads was first demonstrated by R o w a n (1925) and Bissonnette (1930) in the starling and by Benoit (1934) in the duck. Since that time, this has been confirmed in a large variety of avian species. More recent experimental evidence has demonstrated that the anterior pituitary and the hypothalamus are essential links in the photo-gonadal response Send offprint requests to: Dr. No611e Bons, Laboratoire de Physiologie Animale, Universit6 de
Montpellier II, F-34060 Montpellier, France. * Dedicated to Professor Dr. W. Bargmann on the occasion of his 70th birthday ** Supported by the DGRST and "European Training Program Brain and Behaviour Research" I wish to express my gratitude to Professor Andreas Oksche, who repeatedly offered me the scientific facilities at the Department of Anatomy of the University of Giessen, and who provided me with valuable neuroanatomical suggestions throughout the progress of these studies.
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mechanism (for literature, see Benoit and Assenmacher, 1970; Farner, 1973). Moreover, the direct influence of light on the hypothalamus of blinded ducks was shown to stimulate the gonadotropic axis (Benoit et al., 1950). These results speak in favour o f the involvement ofextraretinal receptors (ERP) in the gonadal response. Experiments with the house sparrow have demonstrated convincingly the role o f the highly sensitive ERP in a number of physiological regulations. Therefore, the question has been raised whether or not the classical retinal photoreceptors are actually involved in the non-visual biological function of light (Menaker, 1971). There exist, however, some pertinent data which favour the concept o f a dual influence of photoperiodic information via both retinal and extraretinal photoreceptors. At low light levels, intact ducks were shown to have a significantly greater testicular response than experimentally blinded animals (Benoit et al., 1953). It is interesting to note that unoperated short-day adapted quail (6L (1 lux) 18D) responded to a four week exposure to long day conditions (18L (5 lux) - 6D) by an increase in testicular weight which was twice that o f the blinded congeners. The plasma concentration of testosterone was unchanged after five weeks of long day conditions in blinded quail, while it was doubled in intact controls (Bons et al., 1975). Therefore, the participation of the retina in the neuroendocrine regulation of birds cannot be disregarded. This physiological evidence has provided renewed interest in the problem o f the anatomical pathways between the retina and the hypothalamus (for reviews, see Nauta and Haymaker, 1969; Oksche, 1970; Oksche and Farner, 1974). On the other hand, lesion experiments have shown that, in addition to the classical infundibular gonadotropic area o f the hypothalamus (Follett, 1973), a second area located in the upper anterior hypothalamus might also be involved in the photoperiodic regulation of the gonads (Assenmacher, 1958, in duck; Ralph and Fraps, 1959, in chicken; Oliver, 1972; and Davies and Follett, 1974, in quail), thus placing emphasis on a circumscribed region of the hypothalamus near the optic chiasma. Keeping in mind physiological parameters, we initiated a series of anatomical investigations concerned with the problem of a possible retinohypothalamic nervous route in the duck. Although the primary concern was not to map the pathways of the primary and accessory optic tracts, we begun by a reinvestigation o f the optic tract (posterior to the decussation). This became important as some of our preliminary results were found to disagree with the data presented in the classical literature on the topic. The main studies were then focussed on the search for retinohypothalamic connections by exploring the optic pathways and possible terminals of degenerating nerve fibers within the hypothalamus after unilateral transection of one optic nerve.
Material and Methods
Male ducks (Anas platyrhynchos) were used in the present investigation. The right optic nerve was completelycut. Animals werethen sacrificedunder sodium pentobarbital (0.4 ml/kg) anaesthesia 4.5 h to 21 days postoperativelyby an intracarotid perfusion of 9% 0 saline followed by fixative. The material used for light microscopywas fixed 5, 8, 13, 14 and 21 days after nerve transection by perfusion with 10% formol in saline and either stored for two and a half months in fixative
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and subsequently dehydrated, cleared and embedded in paraffin, or stored for one week in fixative and then sectioned on a freezing microtome. Six and 12 gm thick paraffin sections were cut in frontal, sagittal and oblique planes. In the latter, the plane of the section was always parallel to the plane of the optic nerves in front of the optic chiasma. The course of degenerating fibers was traced in serial paraffin sections by silver impregnation (modified procedure based on the methods of Nauta and Nauta-Gygax). The general neuroanatomy and nuclear pattern of the diencephalon and the mesencephalon was analyzed in paraffin sections stained with Cresylviolet (Fernstr6m, Kltiver-Barrera), Eryochrom cyanin R (Page), and with the Bodian silver method. The brains with degenerating optic nerves were sectioned on a freezing microtome at 20 or 30 lain,and serial sections were made in a frontal or sagittal plane. In order to analyze the degenerating nerve fibers, the Fink-Heimer, Hjorth-Simonsen and Johnstone-Bowsher techniques were used. However, the latter method was superior to the others in order to demonstrate the nerve endings. Therefore, it was used for routine work. Adjacent sections to that which had been treated by the silver method were stained using the Page technique. In all cases, the degenerating optic fibers were easily identified on the basis of their increased argentophilic staining; however, the finer retinal fibers were better seen in the hypothalami of animals eight days after operation. This period of survival was then respected in further experimental work. For comparative purposes serial sections of the brains of Coturnix coturnix japonica, Columba livia and Pica pica were prepared. For electron microscope studies, the ducks were perfused 4.5, 7, 18, 23, 28, 46, 70 h and 8 days after the operation with 9%o saline followed by 4% paraformaldehyde solution. Small blocks of the anterior hypothalamus, contralateral and ipsilateral to the sectioned optic nerve, were post-fixed without washing in a 2% osmic acid solution (Westrum and Lurid, 1966) and stained with uranyl acetate during dehydratation in ethanol (Kokko and Rechardt, 1968: procedure VIII), and embedded in Epon or Araldite (Luft, 1961). The brains of one sham-operated animal (sacrificed 45.5 h after injury) and those of unoperated animals were examined using the same techniques.
Results A. Optic chiasma (cf. Bons, 1969) The optic nerves o f the d u c k are c o m p o s e d o f fascicular a r r a n g e m e n t s o f myelinated a n d u n m y e l i n a t e d fibers. E a c h optic nerve enters the chiasmatic region a n d divides into five o r six large b u n d l e s which cross the m i d l i n e a l t e r n a t i n g with the b u n d l e s o f the c o n t r a l a t e r a l nerve. The m a j o r i t y o f the optic fibers decussate in the optic chiasma. However, a small g r o u p o f d e g e n e r a t i n g fibers does n o t intersect a n d forms two postchiasmatic fascicles which project to the ipsilateral side. This is clearly seen in the obliquely sectioned b r a i n (Fig. 1). Both u n c r o s s e d optic fascicles represent 1.2% o f the d i a m e t e r o f the optic tract. O n e o f the lateral fascicles (showing a n o v a l - s h a p e d profile o f 140 p,m/40 lam) leaves the c e n t r o - d o r s a l part o f the c h i a s m a and, instead o f crossing it, unites with the dorsal division o f the c o n t r a l a t e r a l medial optic tract. Initially, the fibers o n the surface o f these ipsilateral fascicles r u n a m o n g the c o n t r a l a t e r a l fibers. Some o f t h e m reach the t e r m i n a l p o r t i o n o f the tract a n d m a k e c o n t a c t with the a n t e r i o r v e n t r a l part o f the lateral geniculate nucleus. A n o t h e r part of this ipsilateral fascicle rejoins the lateral a n d v e n t r o l a t e r a l part of the m a r g i n a l optic tract a n d projects into the tectum. T h e m o s t dorsal fibers of these lateral fascicles s u r r o u n d the external three f o u r t h s o f the lateral a n t e r i o r n u c l e u s a n d form, together with a large c o n t r a l a -
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Fig. 1. Optic system of the duck. Schematic representation of uncrossed optic fibers in the optic chiasma and the optic projections. Hatched fascicles belong to the right optic nerve, white fascicles to the left optic nerve. ROB, basal optic root; ECN, ectomammillarynucleus; EN, external nucleus; ION, isthmo-opticnucleus; lOT, isthmo-optictract (black, in order to emphasize its mainly retinopetal character); LAN, lateral anterior nucleus; LG, lateral geniculate nucleus; MOT, marginal optic tract; OC, optic chiasma; ON, optic nerve; SSN, superficial synencephalic nucleus; T, tectum; TG, tectal grey
teral fascicle, the isthmo-optic tract. The uncrossed (degenerated) fibers of this isthmo-optic tract are present only in the lateral part of the tract, because the medial part, which runs directly dorsal to the lateral geniculate nucleus, has no degenerated fibers. Both the lateral and medial parts come from the isthmo-optic nucleus which is dorsal and posterior in relation to the optic chiasma. In this context it should not be overlooked that the isthmo-optic tract o f birds is mainly formed by efferent (retinopetal) fibers. The second ipsilateral fascicle, more posterior than the first, breaks off laterally from the postero-ventral part of the chiasma, about 1 mm behind the first fascicle. This second ipsilateral fascicle splits up into the contralateral basal optic root (200 pm in diameter). This is clearly seen when the basal optic root is completely separated from the marginal optic tract (Fig. 6). All ipsi- and contralateral fibers end in the ectomammillary nucleus (Figs. 4, 5). However, most o f the ipsilateral fibers end in the ventral internal ridge of the ectomammillary nucleus. In fact, each of the three parts of the optic tract: the marginal optic tract (i.e., lateral optic tract), the isthmo-optic tract (i.e., medial optic tract) and the basal optic root are, in the duck, made up not only of contralateral optic fibers but they contain also some ipsilateral optic elements. B. Retinohypothalamic Connections (cf. Bons and Assenmacher, 1969, 1973;
Bons, 1974) I. Operated Animals 1. Nauta-stained Paraffin Sections. The great majority of the optic fibers which are posterior to the optic chiasma project into the thalamus and the mesencepha-
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Fig. 2. Series of frontal sections through the duck brain at different levels of the anterior division of the suprachiasmatic nucleus (A D). LAN, lateral anterior nucleus; LFB, lateral forebrain bundle; LGN, lateral geniculate nucleus; LOT, left optic tract (with degenerated fibers); PR, preoptic recess; P VN, paraventricular nucleus; SCN, suprachiasmatic nucleus; SO C, supraoptic commissure; SONm, medial part of the supraoptic nucleus; SONI, lateral part of the supraoptic nucleus. The highly stippled part of the SCN represents the region where degenerated endings of retinal fibers were found
lon. However, another group o f degenerated crossed fibers are directed towards the anterior part of the brain. These fibers remain lateral to the optic tract just behind the chiasma and form a short tract with an elliptic profile (600 p,m/ 200 tam in diameter). The fibers of the short tract are scattered throughout its cross-sectioned area; in a transversal plane more than 60 degenerated fibers were counted. This tract projects completely into the suprachiasmatic nucleus (Figs. 2, 3). The suprachiasmatic nucleus is limited laterally by the lateral part of the supraoptic nucleus and medially by the preoptic recess and, in its rostral region, by the medial part o f the supraoptic nucleus. After unilateral surgical interruption of the optic nerve, these degenerating retinal fibers appear to be thinner than classical degenerating optic fibers showing a caudal route (Figs. 7, 8). Black-colored, fragmented and swollen structures show a scattered pattern. N o r m a l non-degenerated fibers are light brown and regular.
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Fig. 3. Anterior hypothalamusof the duck. Diagram showing different nuclei. Arrow, projection area of retinohypothalamicfibers. LFB, lateral forebrain bundle; LO T, left optic tract; PR, preoptic recess; PVN, paraventricular nucleus; ROT, right optic nerve; SCN, suprachiasmatic nucleus; SMT, septomesencephalictract; SOC, supraopticcommissure;SONm, medialpart of the supraoptic nucleus; SONI, lateral part of the supraoptic nucleus
2. Impregnations According to Johnstone-Bowsher. This material of the duck
brain not only showed numerous degenerating fibers (Figs. 9, 10) but also degenerating endings in the left suprachiasmatic nucleus. Generally these changes were observed eight days after transection of the right optic nerve. These degenerated endings represent three different types: a) Irregularly-shaped, bulb-like boutons which are heavily silver impregnated and also show a short preterminal segment of the degenerated axon. They contact neuronal perikarya approximately 30 ~tm in diameter (Fig. 14). b) Neurofibril-containing argyrophilic ring-like boutons (not larger than 2.5 lam in diameter) which contact thin, poorly stained dendrites (Fig. 11). c) Degenerated endings forming a fine basket around the perikarya of a neuron (Fig. 12). 3. Preparations Treated According to Bodian. The results of the preparations
treated by the Bodian method are similar to the previously described results considering the site and the terminals of the retinohypothalamic fibers as well as their contact with dendrites and cell bodies. The fiber images obtained by using this method are finer than those found in preparations of the JohnstoneBowsher type (compare Fig. 11 and Fig. 13). 4. Electron Microscopic Study. The area of the anterior hypothalamus shown
by light microscopy to be the suprachiasmatic nucleus (see Figs. 2, 3) as well as the area where the degenerating boutons and the degenerated fibers were seen were observed with the electron microscope. In the suprachiasmatic nucleus, contralateral to the lesioned axons, different signs of degeneration were observed 4.5, 7, 18, 23, 28, 46, 70 h and 8 days after the operation. The ventral part of the nucleus contains numerous myelinated fibers. Eighteen hours after the interruption of the optic nerve, the axoplasm of some fibers shows shrinkage and is electron dense (Figs. 27, 29), however, the myelin
Figs. 4-8. Nauta impregnations of paraffin sections 8 days after unilateral transection of the optic nerve in the duck Fig. 4. Ipsilateral ectomammillary nucleus with several degenerated fibers (arrows) Fig. 5. Contralateral ectomammillary nucleus with numerous degenerated fibers Fig. 6. Ipsilateral basal optic root with two degenerated fibers (arrows) Figs. 7 and 8. Degenerated fibers (short arrows) leaving the contralateral optic tract (or) and running to the suprachiasmatic nucleus; n, neurons of the suprachiasmatic nucleus. Scale markers: 10 ~m
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Figs. %14. Suprachiasmatic nucleus of the duck 8 days after contralateral optic nerve transection. Johnstone-Bowsher impregnation, frozen sections Figs. 9 and 10. Degenerated fibers (arrows') Figs. 11, 12 and 14. Degenerated endings (arrows) in contact with a dendrite (d) (Fig. 11) and with neuronal perikarya (n) (Figs. 12, 14) Fig. 13. Degenerated ending (arrow) between neuronal perikarya, Bodian impregnation. Scale markers: 5 lam
Figs. 15-19. Electron micrographs o f the contralateral suprachiasmatic nucleus of the duck after unilateral optic nerve resection Figs. 16, 18 and 19. Animal sacrificed 4.5 h after the operation Figs. 15 and 17. Control animal: a, axonal arborization; b degenerated bouton; a r r o w s , synaptic contacts. Scale markers : 330 n m
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lamellae are usually still intact. Whereas in the degenerating optic nerve and optic tract (Fig. 31) numerous mitochondria are devoid of cristae and show a dark matrix, which is considered to be the first obvious sign of axonal degeneration, no mitochondria are recognized in sections of degenerating retinohypothalamic fibers. Some of the degenerating preterminal and synaptic elements of the retinal axons are observed in the ventral portion of the suprachiasmatic nucleus somewhat earlier, i.e. 4.5 h after the operation. The degeneration is characterized in the axonal arborization (i.e., preterminal portion) by a very electron dense axoplasmic matrix masking the mitochondria (Fig. 18). This opacity results from the marked increase of neurofilaments which run mainly parallel to the long axis of the cytoplasm. Degeneration in terminals is characterized by progressive darkening of the presynaptic cytoplasm and of the mitochondria, as well as by the presence of a small number of spheroidal synaptic vesicles and by numerous flattened vesicles forming clumplike arrangements (Figs. 16, 19-26). Even 4.5 h after transection of the optic nerve, a distinct increased electron density of the postsynaptic membrane thickening can be clearly seen. Seventy hours after the contralateral operation all signs of degeneration described are present, as well as considerable increase in axoplasmic density of the synaptic endings (Fig. 28). One degenerating bouton displayed a strange appearance (Figs. 22 24). It contained a concentric lamellar formation which resembles a myelin sheath in the process of degeneration. This lamellar body may indicate the decomposition of membranes rich in phospholipids. All the degenerating terminals were found to be in contact with dendritic spines and dendrites. Within the suprachiasmatic nucleus, the astrocytes show a reactive transformation during the course of degeneration of the optic fibers. Their perikarya become greatly enlarged, their processes longer and more numerous. The latter extend toward the basal lamina of the capillaries. In the distal portion, near the capillaries, the processes contain more glycogen granules than in the proximal portion which contains bundles of glial filaments. The proximal divisions of the astrocytic processes, situated close to the perikaryon, subsequently engulf the degenerating optic terminals. The phagocytic glial cells also undergo changes after optic nerve transection. Their hypertrophied somata contain numerous long cisternae of granular endoplasmic reticulum. II. Sham-operated and Control Animals No characteristic spontaneous degeneration of retinohypothalamic fibers was observed in the corresponding parts of the ipsilateral suprachiasmatic nucleus. Degenerating (or degenerated) fibers were also lacking in the suprachiasmatic nuclei of the control and the sham-operated animals sacrificed 45.5 h after the operation. This confirms the results obtained at the light microscopic level where only one spontaneous degenerated nerve ending was found in three sections of the suprachiasmatic nucleus. Furthermore, it was impossible to determine if it is belonged to a retinal axon. Glial cells of both the control and sham-operated animals showed no sign of phagocytic activity. The cell bodies of the astrocytes are inconspicuous,
Figs. 20-25. Degenerated endings in the suprachiasmatic nucleus of the duck 4.5 h (Figs. 20, 21, 25) and 70 h (Figs. 22 24) after contralateral optic nerve transection, b, Degenerating bouton; d, dendrite; thick arrows, synaptic contacts; thin arrows, concentric array of membranes. Scale markers: 330 nm
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and possess fine cell processes. Phagocytic glial cells are only occasionally seen. Their cytoplasm forms a thin rim around an irregularly-shaped nucleus.
Discussion
A. Evidence of Uncrossed Nerve Fibers in the Avian Optic Chiasma Most investigators of the avian visual system have concluded that all optic fibers decussate in the optic chiasma (Perlia, 1889; Edinger and Wallenberg, 1889; Harris, 1904; Huber and Crosby, 1929; Bltimcke, 1961; Cowan etal., 1961 ; Karten and Nauta, 1968; Hartwig, 1970). Only Cowan and his colleagues did not exclude the possibility that a few fibers may continue into the optic tract on the ipsilateral side. These authors observed a single degenerating bouton in the lateral geniculate nucleus of the pigeon, ipsilateral to the enucleated eye. However, they felt that there were no real ipsilateral fascicles. Further, Ferreira-Berrutti (1951) and Knowlton (1954) have clearly demonstrated the existence of a partial decussation of the optic fibers in the chick embryo. Our results in the adult duck are in agreement with these two earlier reports on the chick embryo. The optic fibers which remain ipsilateral terminate in the lateral geniculate nucleus, the lateral anterior thalamus, the external nucleus, the superficial and synencephalic nucleus and deep into the tectal gray matter. Another group of ipsilateral retinal fibers run into the deeper part of the marginal optic tract and end within the mesencephalon. Uncrossed optic fibers of the basal optic roots which terminate in the ipsilateral ectomammillary nucleus seem to be characteristic of the duck optic system. Nauta and Van Straaten (1947) suggested that ipsilateral optic fibers are also a component of the basal optic roots of mammals. In fact, uncrossed optic fibers are found in all groups of Tetrapods. In several mammalian species there is evidence for uncrossed retinal fibers. In the three other classes of tetrapods (amphibians, reptiles and birds) there is a lack of uncrossed fibers in certain species; however, in every class an incomplete decussation in the optic chiasma was observed. The urodele, Cryptobranchus allegeheniensis (Riss et al., 1963), possesses uncrossed optic fibers which project into the preoptic area. This finding was confirmed in Ambystoma tigrinum by Jackway and Riss (1972). These results are in contradiction to the observation of Gruberg (1969) who was not able to find ipsilateral degenerating optic fibers in the same urodele species. These negative results are probably due to the lower temperature at which the operated animals were kept and to a different staining procedure employed. Earlier results of Herrick in Ambystoma (1925) and Necturus (1949) may be interpreted as demonstrating the presence of an uncrossed retinohypothalamic and pretectal tract. In the frog, Rana pipiens, two ipsilateral optic fascicles exist, one running to the tectum, the other terminating in the pretectal area (Knapp et al., 1965). Armstrong (1950, 1951), and more recently Burns and Goodman (1967), Ebbesson (1970), Reperant (1972, 1973), Halpern and Frumin (1973), Northcutt and Butler (1973) have observed retinal fibers which fail to cross in the chiasma
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Figs. 26-31. Contralateral suprachiasmatic nucleus of the duck after unilateral transection of the optic nerve. Degenerating boutons 70h (Figs. 26, 28) and 8 days (Fig. 30) after the operation. Degenerating myelinated fibers in the same region 4.5 h (Fig. 27) and 18 h (Fig. 29) after the operation. Degenerated optic fibers in the contralateral optic tract, 18 h after the operation (Fig. 31). a, Axon; b, degenerating or degenerated bouton; d, dendrite; g, glial process; arrows, synaptic contacts. Scale markers: 330 nm
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and project to the ipsilateral diencephalon and pretectum in different species of lizards and snakes. To date, in only one species of turtles (Emys orbicularis) have uncrossed optic fibers been found (Kosareva, 1967). It appears, therefore, that uncrossed optic fibers are not only characteristic of the mammalian optic system and related to the stereoscopic vision, since they can be found in some amphibians, reptiles and birds. However, there is no evidence that uncrossed retinal fibers occur in the teleost or the elasmobranch.
B. Retinohypothalamic Pathway In our first description of retinohypothalamic connections in ducks with unilaterally transected optic nerves (Bons and Assenmacher, 1969), the Nauta-stained paraffin sections showed some very fine degenerating fibers in the anterior hypothalamus. This area which was called the "supraoptic region" was by no means restricted to the supraoptic nucleus but included a large anterior hypothalamic area with numerous scattered extensions of the supraoptic nucleus (Assenmacher, 1958). Later, Crosby and Showers (1969) and Oksche (1970) identified this circumscribed region of the anterior hypothalamus as the suprachiasmatic nucleus of birds. Using the subtle Johnstone-Bowsher impregnation method, we were able to outline the precise projection of retinal fibers and to trace their terminals within the suprachiasmatic nucleus. This nucleus is bordered on its lateral side by the lateral part of the supraoptic nucleus, and, on its medial side a) rostrally by the medial part of the supraoptic nucleus, and b) caudally by the preoptic recess and the paraventricular nucleus (Bons and Assenmacher, 1973). Finally, an electron microscopic study on degenerating fibers (Bons, 1974) within the anterior hypothalamus displayed in the contralateral suprachiasmatic nucleus not only degenerating fibers and axonal arborizations but also degenerating presynaptic profiles and postsynaptic membranes. These electron microscopic results prove that a circumscribed region of the anterior hypothalamus of the duck (suprachiasmatic nucleus) receives direct retinal input. The results obtained in the duck with silver impregnation methods (Bons and Assenmacher, 1969, 1973) are in agreement with the reports of Bliimcke (1961) and Brugi (1937) in the domestic fowl. However, in the chicken retinohypothalamic fibers seem to be more numerous than in duck. Meier (1973) employed autoradiography in a study concerned with the terminals of the retinohypothalamic fibers in birds. Labelled amino acids were incorporated into retinal neurons and subsequently transported along their axons to the terminals. Meier described a retinal projection to the anterior hypothalamic region contralateral to the labelled retina in Columba livia and Coleus monedula. On the basis of electron microscopic observations Hartwig (1974) reported the existence of degenerated optic terminals in the suprachiasmatic nucleus of unilaterally retinectomized Passer domesticus. However, in the house sparrow degenerated myelinated axons did not occur in this region. On the other hand, in recent studies on retinohypothalamic endings in several
Retinohypothalamic Pathway in the Duck (Anas pIatyrhynchos)
357
mammals and anurans, electron microscopic techniques and autoradiographic tracing have been applied (see Conrad and Stumpf, 1974). In the mammals studied, i.e. rat, hamster, guinea pig, rabbit, cat and monkey (O'Steen and Vaughn, 1968; Moore et al., 1971 ; Moore and Lenn, 1972; Hendrickson et al., 1972), these techniques revealed clearly that the retinohypothalamic tracts terminate on dendritic branches of neurons in the ventral portion of the suprachiasmatic nuclei. These results show that the only difference existing between birds and mammals is that in birds the retinal axons project only into the contralateral suprachiasmatic nucleus, whereas, in mammals the retinal axons project to both the ipsi- and contralateral suprachiasmatic nuclei. Vullings and Kers (1973) applied similar techniques in Rana temporaria. They found ipsilateral connections between the retina and the preoptic nuclei. Several recent investigations have demonstrated retinal projections to the hypothalamus of amphibians (Jackway and Riss, 1972), reptiles (Butler and Northcutt, 1971; Halpern and Frumin, 1973; Northcutt and Butler, 1974), and mammals (Karamanlidis and Magras, 1972, 1974). However, these observations were made with the light microscope using specimens stained with different silver methods. These results were partly contradictory. The body of evidence presented in recent investigations on the optic system leads us to the conclusion that retinal projections to the anterior hypothalamic region exist in all systematic groups of vertebrates. We suggest that the divergent results obtained in previous studies are due to the different silver stains applied and to the variety of surgical techniques (unilateral bulbectomy, unilateral retinectomy, uni- and/or bilateral transection of the optic nerve; cf., Nauta and Haymaker, 1969). We conclude that a direct retinohypothalamic pathway exists in birds, mammals, amphibians and presumably also in reptiles. This pathway projects to the area of the suprachiasmatic nucleus or its homologues. This area of the anterior hypothalamus plays an essential role in the photoperiodic control of the gonadotropic mechanism of birds. In mammals, the suprachiasmatic nuclei are involved in cyclic regulations leading to ovulation (Clattenburg, et al., 1972), in the control of the diurnal rhythm of the adrenal cortex (Moore and Eichler, 1972) and of several rhythmic behavioural patterns (Stephan and Zucker, 1972). Whether or not this parvocellular region of the anterior hypothalamus contains secretory neurons producing the hypothalamic adenohypophysiotropic hormones (see Barry et al., 1973) will require further investigation. In the house sparrow, Passer domesticus, it is rich in secretory perikarya of different types (Oksche and Farner, 1974; Oksche and Hartwig, 1975). In the rostral hypothalamus retinal inputs to neuroendocrine and/or other hypothalamic systems might be integrated and modulated. However, the exact physiological role of the retinohypothalamic connection is still open to discussion.
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Received November 27, 1975 / in final form January 10, 1976
