Science, Faculty of
Zoology, Department of
DSpace
UBCV
Hafeez, Mohammad Abdul
Master of Science - MSc
University of British Columbia
The pineal organ in sockeye salmon and its relation with other epithalamic components has been studied. Histological and histochemlcal methods were used and the influence of experimental photoperiods on the histology and secretion in the juvenile pineals investigated. Photosensory and supporting ependymal cells have been identified. Lipofuchsin and melanin are absent in the pineal epithelium. Besides sensory fibres, efferent end-loops are present on the sensory and supporting cells. The dorsal pineal nerve tract is probably of a mixed type. Although the blood supply is profuse an endocrine or neuroendocrine activity is not indicated. It is proposed that the pineal organ in both juveniles and adults is photosensory and secretory. The apocrine secretory activity of the sensory and some supporting cells is probably associated with either the maintenance of constant chemical composition of the cerebrospinal fluid or supply of certain substances to the nervous tissue. The subcommissural organ is metabolically more active than the pineal. The secretion consists of glycogen, mucopolysaccharides, mucoproteins, glycoproteins and aldehyde-fuchsin positive granules. Chrome-alum positive granules are abundant in the subcommissural organ. Both the pineal organ and the subcommissural organ appear independent of light's influence on secretion and histology. It seems more plausible that apocrine secretory activity is controlled by some internal factors. It is suggested that the pineal organ might be of some advantage in the light dependent behaviour of this species in terms of intensity detection. Future work is necessary in this direction.
HISTOLOGICAL AND SOME HISTOCHEMICAL STUDIES ON THE PINEAL ORGAN IN SOCK EYE SALMON ONCORHYNCHDS NERKA (WALBAUM) by ' MOHAMMAD ABDUL HAFEEZ B«Sc Aligarh Muslim University, 1 9 5 5 M.Sc Aligarh Muslim University, 1 9 5 7 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS! OF THE DEGREE OF MASTER OF SCIENCE in the Department of Zoology We accept this thesis as Conforming to the required standard The University of British Columbia August, 1 9 6 * + , In p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t of the requirements f o r an advanced degree at the U n i v e r s i t y of B r i t i s h Columbia, I agree that the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r reference and study. I f u r t h e r agree that per-mission f o r extensive copying of t h i s t h e s i s f o r s c h o l a r l y purposes may be granted by the Head of my Department or by h i s r e p r e s e n t a t i v e s . I t i s understood that copying or p u b l i -c a t i o n of t h i s t h e s i s f o r f i n a n c i a l gain s h a l l not be allowed without my w r i t t e n permission* Department of Zoology The U n i v e r s i t y of B r i t i s h Columbia, Vancouver 8, Canada August, 196^ Date ABSTRACT The pineal organ i n sockeye salmon and i t s relation with other epithalamic components has been studied. Histo-logical and histochemlcal methods were used and the influence of experimental photoperiods on the histology and secretion i n the juvenile pineals investigated. Photosensory and sup-porting ependymal cells have been identified. Lipofuchsin and melanin are absent i n the pineal epithelium. Besides sensory fibres, efferent end-loops are present on the sensory and supporting c e l l s . The dorsal pineal nerve tract i s probably of a mixed type. Although the blood supply i s pro-fuse an endocrine or neuroendocrine activity i s not indicated. It i s proposed that the pineal organ i n both juveniles and adults i s photosensory and secretory. The apocrine secretory activity of the sensory and some supporting cells i s probably associated with either the maintenance of constant chemical composition of the cerebrospinal f l u i d or supply of certain substances to the nervous tissue. . The subcommissural organ i s metabolically more active than the pineal. The secretion consists of glycogen, mucopolysaccharides, mucoproteins, glyco-proteins and aldehydefcfuchsin positive granules. Chrome-alum positive granules are abundant i n the subcommissural organ. Both the pineal organ and the subcommissural organ appear independent of light's influence on secretion and histo-logy. It seems more plausible that apocrine secretory activity i s controlled by some internal factors. I t i s suggested that the pineal organ might be of some advantage in the light dependent behaviour of this species in terms of intensity detection. Future work i s necessary in this direction. ACKNOWLEDGEMENTS The author wishes to extend his gratitude to Dr. P. Ford whose supervision and patient guidance made possible the completion of this work. Thanks are due, particularly, to Dr. W.S. Hoar for suggesting the problem, providing laboratory space and for his keen interest i n the study. The author i s also very grateful to Dr. W.N. Holmes, Dr. C.C. Lindsey and Dr. D.J. Randall for c r i t i c a l suggestions during the investi-gation and preparation of the manuscript. The help of Dr. H. Harvey i n procuring the sockeye salmon i s gratefully acknowledged. Miss A. Redlich and Dr. W. Pfeiffer assisted in translating literature. This study was supported by a grant i n aid of research from the National Research Council of Canada. TABLE OF CONTENTS. PAGE INTRODUCTION 1 LITERATURE REVIEW 3 1 . General 3 2 . Histology and photoreception ^ 3 . Experimental evidence of photosensitivity 7 k9 Secretion 1 0 MATERIALS AND METHODS 1 ^ 1 . Material, source and maintenance in ^ laboratory 1 5 2. Photoperiod experiments 1 5 3 . Histological methods ^ 7 h, Histochemical methods ^ 5 . Quantification of secretion 2 1 RESULTS 2 3 I. TOPOGRAPHY AND GROSS STRUCTURE 2 3 1 . Relation with epithalamus 2 3 2 . Relation with the skull 2 3 3 . Gross structure k. Relation with blood vessels 2 5 5 . Pigment 2 7 II. HISTOLOGY 3 0 1 . Cell types 3 0 2. Subcommissural organ and dorsal sac 3 7 epithelia 3. Nervous elements 3 8 - iv -PAGE k9 Pineal lumen, interlobular spaces and ^ their contents III. SECRETION k$ 1. Performic acid alcian blue-Periodic acid ^ 5 Schiff method 2. Bargman's chrome-alum haematoxylin- *+7 phloxin method 3 . Halmi's paraldehyde fuchsin method k-S k. Mallory-Azan method 5 0 IV. INFLUENCE OF LIGHT AND DARKNESS 5 1 DISCUSSION 5 5 1 . Sensory cells and functional significance 5 5 2. Secretion and functional significance 5 * 8 3 . Influence of light and darkness 61 SUMMARY 63 BIBLIOGRAPHY 6 6 LIST OF FIGURES FIGURE PAGE 1. Camera lueida drawing of sagittal section of head 22 of juvenile I.X120. 2. Photomicrograph of a portion of T.S. of juvenile head.Xl80. 2 6 3 . Photomicrograph of T.S. of juvenile head passing 2 6 through forebrain. X^O. h. Photomicrograph of a portion of juvenile II pineal 29 organ showing A and C type c e l l s . X 7 0 0 . 5 . Photomicrograph of a portion of juvenile I pineal 2 9 " organ showing A, B and C type c e l l s . X 7 0 0 . 6 & 7 . Photomicrograph of a portion of pineal organ in 3 2 adult sockeye salmon showing A, B and C type c e l l s . X700. 8. Photomicrograph of a portion of juvenile II pineal 3 1 * -organ (T.S.);. X700:. . 9o Photomicrograph of a portion of juvenile pineal organ showing a D type c e l l . X1260. ° 10. Photomicrograph of a portion of juvenile II pineal 3 9 organ. Ramon y Cajal's method. X 7 0 0 . 11. Photomicrograph of pineal organ in adult showing the junction of stalk and distal vesicle. X700. 12. Photomicrograph of the junction of pineal stalk ^ with subcommissural organ. X700. 1 3 . Photomicrograph of a portion of pineal organ i n adult. Romanes1 silver impregnation. X700. l^f. Photomicrograph of a portion of juvenile II pineal * * 6 vesicle showing PAS + granules. X 7 0 0 . 1 5 . Photomicrograph of a portion of juvenile II pineal organ showing AF + granules. X 7 0 0 . 1 6 . Photomicrograph of a portion of pineal organ in ^ 9 adult showing AF + granules. X 7 0 0 . LIST OF TABLES TABLE PAGE 1 . Effect of photoperiods on the secretory activity 5 2 ' in the pineal and subcommissural organs in juvenile II. A = continuous darkness, A-l = A transferred to 2^-hr. light, A-2 = A trans-ferred to 3 6 nr. ligh t . Effect of photoperiods on the secretory activity 9+ in the pineal and subcommissural organs; in juvenile II. B = continuous darkness ( 1 week), B-l = B transferred to h hr, li g h t , C = continuous light ( 1 week), C-l = C transferred to h hr, darkness. INTRODUCTION The present study was undertaken to gain some under-standing of the possible physiological role of the pineal in sockeye salmon (Oncorhynchus nerka). Earlier studies on various species of cyclostomes and fishes have suggested a photosensory role for this organ ( H i l l , l89*f; Studnicka, 1905; Holmgren, 1918; Rasquin, 1958; von Frisch, 1911; Breder and Rasquin, I9h7y 1950)o Friedrick-Freksa (1932) proposed that secretory activity in pineal sensory cells was associated with the main-tenance of cerebro-spinal f l u i d (CSF) pressure. The view that the pineal sensory cells i n f i s h and amphibia were either chemoreceptors or baroreceptors was suggested by Van De Kamer (1956). Wyman (192^) observed that larvae of Fundulus heteroclitus showed contraction of melanophores under the i n -fluence of pineal extracts and Pflugfelder (1953, 195^ and 1956) has shown that the pineal organ i n Lebistes reticulatus influen-ced the activity of the pituitary and i t s target organs. The results have been interpreted to indicate a neuroendocrine role for the pineal i n teleosts. The present study was designed to work out the morpho-logical arid histological details of this organ in sockeye salmon. Particular attention was paid to the relationship of the pineal to other epithalamic components, the existence of a sensory complex and the nerve pathway associating i t with the brain. The presence and nature of pigment, the occurrence of motor innerv-ation, and the p o s s i b i l i t y of secretory cells existing as - 2 -distinct components were also investigated. It was also pro-posed to study the chemical nature of the secretion. Sockeye salmon are anadromous species and the behaviour and downstream migration of juveniles i s light dependent (Hoar 199+). They are active and make downstream movements during low light intensity but seek cover and get less active with increasing light intensity. It was f e l t that this investigation would not only help in interpreting such light dependent re~ sponses i n terms of pineal physiology but also open up avenues for further research i n this f i e l d . - 3 -LITERATURE REVIEW General: According to Tilney and Warren (1919), Gladstone and Wakeley (19*f0) and Kelly (1962) the pineal complex in fishes consists of the pineal organ alone, the parapineal being re-duced or absent. It i s more l i k e a gland than the eye-like pineal or parietal organ i n cyclostomes and lizards respec-tively. H i l l (189*0 studied the pineal organ in representa-tives of Salmonidae, Siluridae and Percidae and noted that an anterior parapineal organ i s present in the early embryonic stages. It i s either lost or becomes reduced as development progresses. In Salmo purpuratus and S. fario this could be seen in two year old individuals,, Steyn and Webb (i960) have described the existence of a parapineal resembling the parietal eye of lizards i n adult Labeo umbratus. Complete absence of pineal and parapineal i s known for Myxinoids and Torpedo. (Tilney and Warren, 1919). A pineal foramen has been observed i n Callichthys asper (Gladstone et_al, 19^0}, Thynnus thynnus (Rivas, 1953l> Arius coelatus and Macrones gulio (Holmgren, 1959). According to Rivas although a well developed pineal window occurs i n the tuna (Thynnus thynnus). the pineal organ i s either reduced or i s absent. Holmgren (1958), however, has reported i t s presence in this species. The majority of teleosts lack a pineal foramen but a pigment free spot exists i n the pineal area of the head (Holmgren, 1959)). This spot i s very prominent i n deep-sea fishes, the skull roof lodging the anterior vesicular part of - If -the pineal (Holmgren, 1959). Histology and Photoreception: Studies on the pineal organ of various species of cyclostomes, selachians, chondrosteans and teleosts have shown that i t has photosensory c e l l s , ganglion cells and supporting cells (Dendy, 1907; Studnicka, 19P5; Holmgren, 1918; Holmgren, 1959)« Nerve fibres originating in the pineal organ form a sensory nerve tract running to the brain. (Studnicka, 1905; Dendy, 1907; Holmgren, 1918). Photosensory elements. Holmgren (1917, 1918) described i n detail the structure of the pineal and related elements of sensory cells i n selachians and amphibians. He claimed that the sensory cells are located in the periphery of the epithelium and have an outer (luminal) and an inner (peripheral.) segment. The outer segments are cytoplasmic projections into the lumen and present a variety of shapes. Quite often they are separated from the inner segments by a constriction. Osmiophilic granulations or spiral patterns form a characteristic feature. These processes range i n form from straight through spir a l , club shaped, knob-like to glo-0 bular structures. He interpreted these variations as indica-tive of a "secretion-regeneration" cycle. The inner segments contain the nuclei and the cytoplasm i s prolonged basally into axons which synapse with dendrites of more peripherally located gahglion c e l l s . Cattie (1882) also observed cells having their luminal cytoplasm i n the form of processes. The folded epithelium of the pineal in Salmo spp., Gatestomus teres and - 5 -Amia calva contains pear-shaped nerve cells which taper basaily into the fibres that are continuous with a nerve tract coursing caudad along the dorsal surface of the organ, ( H i l l , 1891*). Studnicka (1905) also observed a constriction between the outer and inner segments of the sensory cells in Acipenser sturio and Belone acus. Rasquin (1958) studied the gross morphology of pineal i n thirty species of teleosts and the histology in Atherina stipes and Astyan ax mexicanus. Steyn and Webb (I960) showed with non-specific histologic techniques, that the epithelium i n Labeo urnbratus lacked ce l l s which could be called sensory or ganglion c e l l s . Nerve elements, nerve tract and connections with brain. Sensory cells have been demonstrated i n the pineal epithelium. Their axons synapse with ganglion cells i n the basal part of the epithelium. The axons of the latter form a nerve tract running posteriad dorsal to the pineal stalk. H i l l (l89*f) did not report the presence of ganglion cells i n the pineal of Salmo spp. In Acipenser rubicundus (Johnston, 1901) and Belone acus (Studnicka, 1905) they occur near the outer limiting membrane. Few such cells occur in Osmerus mordax but i n Salmo salar they could not be seen even with Bodian's silver impregnation technique (Holmgren, 1959)• Rasquin (1958, Atherina stipes and Astyanax mexicanus) has also noted their presence. Motor end-loops have been seen on pineal support-ing and sensory cells i n Esox luclus (Van De Kamer, personal communication) and similar loops have been reported - 6 -by Kelly and Van De Kamer (I960) in these cells in adult Rana esculenta. Their existence i s interpreted as suggestive of a feed-back mechanism. The nerve tract, more precisely, enters either the habenulae or the posterior commissure or both areas of the diencephalbn. It enters the posterior commissure (P.C.) in Petromyzon planerl, Carassius auratus, Esox lucius and Belone acus (Studnicka, 1899 and 1905)» Geotria austrails (Dendy, 1907), Ami a calva (Gladstone etal, 19*f 0), Acipenser rubicundus (Johnston, 1901), Clupea harengus,Osmerus eperlanus and Acanthias vulgaris (Holmgren, 1918 b, 1918,1), Osmerus mordax and Fundulus heteroclitus (Holmgren, 1959), Polypterus senegalensis (01sson, 1958). The f i n a l destin-ation or the regions of a synapse are s t i l l not known. In Geotria austrails, Polypterus bichir and Osmerus mordax some branches from the main tract innervate the basal processes of the subcommissural organ (S.C.O.) cells lying ventral to the posterior commissure. Although connections with the habenulae are described in many species, the occurrence of an actual synapse here i s doubtful (Holmgren, 1918). H i l l (189*0 and Holmgren (1959) have denied any association of the tract with the habenulae. The latter author has seen some fibres entering the lobi o p t i c i . Supporting c e l l s . These cel l s are ependymal gl i a . In Salmo spp. they are radially arranged ovoid c e l l s with their basal processes converging on the basement membrane ( H i l l , 189*+). In Acipenser rubicundus few neuroglia are seen. Holmgren (1918) has denied the existence of both ependymal cells and neuroglia in teleost pineals. Other authors on pineal histology have reported the presence of supporting cells but have not commented on the occurrence of neuroglia c e l l s . Steyn etal (i960) state that the parietal vesicle and stalk epithelium i n Labeo umbratus consist of ependymal and astrocytic neuroglia c e l l s . Melanin. Although melanophores occur in the mesenchymal connective tissue surrounding the pineal organ, pigment i s absent i n the epithelium i t s e l f . The only reference to the presence of a brown pigment i n some pineals i s accred-ited to Studnicka (1905). In Petromyzon planeri and Geotria australis calcium phosphate and guanine deposits occur in ependymal cells alternating with the sensory c e l l s . Melanin occurs i n scanty amount. Knowles (1939) also reported that the pigment i n lamprey pineal eye i s probably guanine. The parapineal i n cyclostomes lacks pigment and more recent observations of Rasquin (1958) and Holmgren (1959) throw no light on this aspect i n f i s h pineals. Experimental Evidence of Photosensitivity; The histologic evidence for the presence of sensory cells i n the pineal of fishes and cylostomes has been substantiated by experimental studies. Young (1935) observed - 8 -that the diurnal colour rhythm displayed by ammocoetes and adults of Lampetra i s abolished in the former and disturbed i n the latter after pinealectomy. A marked shift in the position of pineal c e l l nuclei was induced when the larvae of lamprey were transferred from continuous darkness to moderate or strong light (Knowles, 1939). No changes were obtained in the position of the pigment in the epithelium which led the author to postulate that i t i s of a reflector type. Von Frisch (1911) blinded minnows, Phoxinus laevis, and subjected them to bright illumination. They s t i l l showed the capacity to undergo colour change. This response persisted to some extent even when the pineal was removed. Von Frisch proposed that besides the pineal the ventricular epithelium also has sensory c e l l s . Scharrer (1928) supported the above observation and said that i n the minnow both the epiphysis and diencephalon are concerned with light dependent responses. Breder and Rasquin (19^7) described that the pineal area in blind cave-characins Anoptichthys Jordan! and Anoptichthys antrobius i s light sensitive. Moderately pigmented individuals from the river and the cave were light negative whereas those lacking pigment were light positive as evidenced by their photo-tactic responses. They concluded that the sign of photo-taxis depends upon the degree of exposure of the pineal area. The sign of the reaction could,be reversed experimentally by - 9 -covering the normally exposed pineal area of li g h t positive f i s h inducing them to become light negative and vice-versa. Gn the basis of responses to light stimuli Breder and Rasquin (1950) classified teleosts into (1) positively phototactic species (Jenkinsla lamprotaenia, Sardinella macropthalmus. Leptocephalus larvae, Brachydanio rerio, Atherina stipes, and Carapus bermudensis) possessing a pigment free pineal area, (2) negatively phototactic species (Astyanax mexicanus, Ameiurus nebulosus, Haemulon melanurum. and Thaiassoma bifasciatum) with a covered pineal area acting as a screen against light and (.3) species with a changeable or erratic response to light where alteration in chromatophore distribution controls passage of light to the pineal area (Sphyraena barracuda, Strongylura notata, Hemirhamphus braziliensis, and Synodus synodus). Species not conforming to this pattern were also recorded. Hoar (1955) studied the responses of sockeye fry and smolts under bright illumination. Comparisons were made between normal f i s h , blinded, pinealectomized and blinded pinealec-tomized individuals. Blinded juveniles responded to light as shown by colour darkening. On pinealectomy they became completely dark simulating the dark-adapted response of normal f i s h . Both smolts and fry showed negative phototaxis after blinding, a response that did not occur on removal of the pineal. Hoar concluded that the pineal complex i s photosensory and may be involved in the negative phototactic behaviour of the juveniles during migration. More recent studies of Rasquin (1958) on Astyanax mexicanus did not reveal any significant - 10 -changes in phototaxis after pinealectomy. The author proposed that this was probably because dense pigment covering the pineal area prevents penetration of l i g h t . Grunewald-Lowenstein (1956)> observed histological and histo-chemical changes i n the pineal of Astyanax i n response to prolonged darkness and illumination. In control individ-uals the pineal cells were tightly packed whereas i n dark-adapted (8-12 weeks) nuclei became less dense and only loosely connected with neighbouring c e l l s . Blood corpuscles were scarcer in dark-adapted than in controls and light-adapted pineals and the size of the organ was larger in the former than in the l a t t e r . The observations of Dodt and Heerd (1962) on the light sensitivity of the pineal nerve in Rana temporaria are note-worthy. They recorded the afferent impulses along the incised pineal nerve and concluded that the organ i s a wave-length discriminator and i s capable of achromatic and chromatic responses. Light and short wave-length caused inhibition, whereas darkness and long wave-lengths excited the nerve. The sensitivity range l i e d between the ultraviolet and the red part of the spectrum. Infra-red rays, 'chemical and mechanical stimulations were ineffective in e l i c i t i n g a response. Secretion? Galeotti (I896) was the f i r s t to note secretion in the pineal lumen i n Leuciscus rutilus. Holmgren (1918 1 and 2) described an apocrine secretion of unknown significance i n the pineal sensory cells in Acanthias vulgaris and Rana temporaria. - 11 -According to him the cells undergo a cycle involving a gradual build up of secretion t i l l the outer segments become globular and ultimately rupture to release the secretory matter into the lumen. Following this a process of regeneration completes the cycle. He also showed the existence of mitochondria-rich ellipsoids at the junction of the inner and outer segments of the luminal processes of the sensory cells in Rana temporaria. These observations were later confirmed i n fishes by Holmgren (1959) and Rasquin (1958)«. Pineal secretion and relation with cerebro-spinal f l u i d . Friedrick-Freksa (1932) found that both sensory and supporting cells release secretion into the lumen. Only i n Dermogenys pusillus the sensory cells alone are involved in secretion. Besides an apocrine secretion he postulated also a release of secretion into the surrounding capillaries and proposed that this i s ultimately liberated into the choroid plexus by means of an increase in blood pressure. The secretion acts like a hormone controlling either the diameter of the blood vessels or the permeability of the choroidal epithelium to cause a lowering of f l u i d pressure - a process involved in the regulation of cerebro-spinal f l u i d pressure in the ventricles. Van De Kamer (1956) f i r s t suggested that the pineal i n fishes and amphibians i s primarily concerned with the regulation of cerebro-spinal pressure or acts l i k e a chemoreceptor and that a photosensory role i s of secondary consequence. He denies the existence of a secretory cycle - 12 -and emphasizes that Holmgren's theory of "Secretion-regeneration" i s based on fixation- artefacts. He says "No indications of secretory activity, were, found. It may be possible that small globules on top of the threads are secretory globules. . At present, however, there i s no con-clusive evidence to support this view". His account based ° n Esox lucius and Rana esculenta pineals did not indicate the manner i n which regulation.of C.S.F. pressure i s brought about., Holmgren (1959) does not agree with Van De Kamer's contention that the sensory cells are non^-secretory. Nature of secretion.. Grunewaid-Lowenstein (1956) using histochemical methods found that extracellular and intracellular glycogen was greater in amount in control than i n dark-adapted or light-adapted (15 week adaptation)'' pineals of As ty an ax mexicanus. Rasquin (1958) observed increase in glycogen production in dark-adapted Atherina stipes pineals. On the basis of a positive PAS reaction and careful control pre-parations (saliva digested) he and Holmgren (1959) pro-posed the release of glycogen, and mucopolysaccharides into the lumen. Rasquin postulated the existence of a glycogen cycle i n fishes having an exposed.pineal area* In Fundulus heteroclitus and,,Osmerus mordax. histochemical tests for NSS and fat gave negative results (Holmgren, 1959) whereas Van De. Kamer (1956) described aldehyde fuchsin + (basophilic) granulation in the luminal processes of Esox lucius sensory cells. , - 13 -The subcommissural organ which i s continuous with the pineal epithelium i s notable as a secretory area. Kelly and Van De Kamer (I960) reported some S.C.O. type cells arranged in f o l l i c l e s in the pineal epithelium of Rana esculent a. Bargman positive granules were seen i n Salmo salar (Holmgren, 1959) and Rana esculenta (Kelly etal I960). Olsson (1958) described that the secretion of S.C.O. i n f i s h and amphibia i s drained into the ventricle. Consequently, a positive reaction with NSS specific techniques i s not necessarily indicative of neurosecretion or a neuroendocrine role. Pharmacological experiments and neuroendocrine activity. Wyraan (192l+b, Fundulus) immersed 10 days old embryos in a solution of dessicated, powdered pineal glands and d i s t i l l e d water and noticed a concentration of a l l melanophores within an hour followed by expansion at the end of this period. In 1-2 day old larvae complete contraction occured within thirty seconds to eight minutes. Hewer (1926) did not get any such response to pineal extracts in Phoxinus laevis. Pflugfelder worked on one day old Lebistes reticulatus and observed thatpinealectomy results i n hypertrophy and hyperplasia of thyroid, hypertrophy of anterior pituitary cells and distor-tion of vertebral column. In a later work (195*+) he conducted more controlled experiments by making lesions i n different parts of the brain. Only in cases where the pineal was des-troyed did the above changes and slight acceleration of sexual - Ik -development In males occur. The administration of epiphysan or thyroxin prevented these changes. Rasquin (1958) failed to duplicate these observations i n Astyanax mexicanus. In two month old f i s h no kyphlordosis, endocrine changes or variations i n growth occurred. Implantation of Atherina stipes pineals in Cyprinodon baconi did not cause any alter-ation i n pigmentation of the host., Schonherr (1955) pointed out that destruction of pineal in Gasterosteus aculeatus has no effect on nuptial colouration or reproductive cycle. There are many references to a relation between pineal secretions and their influence on melanophores in amphibians. Bagnara's studies (1960b, 1961 and 1963) on larval Xenopus laevis are very convincing. According to him pinealectomy and transfer to darkness prevented the usual dark induced blanching. Administration of melatonin restored the blanching response. These effects persisted and did not show reversal to opposite conditions after any lapse of time. Bagnara (1963) postulated the elaboration of melatonin or a similar factor i n the •stirnorgan 1 and/or pineal organ of Xenopus under the stimulus of darkness. In the illuminated condition a simultaneous inactivation of circulating pineal factor and stimulated secretion of MSH by the pituitary induces an expansion of melanophores. He also suggested the possibil-i t y of such a mechanism in fishes. - 15 -MATERIALS AND METHODS Material, source and maintenance In lap»; Sockeye salmon juveniles and adults were used for the study. The juveniles were obtained from Cultus lake hatchery in May and July 1962, They comprised newly hatched fry and approximately one year old juveniles. They were stocked in fibre glass f i s h tanks containing running deehlor-inated water. The fi s h were fed daily on commercial dried f i s h food (JJR. Clark, Salt Lake City, Utah), The photo-period in the f i s h room was adjusted to outdoor conditions by means of an electric switch (Protect-O-lite, Geneva, I l l i n o i s ) , Whenever required the juveniles were obtained from this stock. Adult f i s h were made available from two sources namely the Technological Station of the B.C. Fisheries Research Board of Canada i n August and from the Adam's river spawning run 1962, Their size ranged from 50-56 cm (total length), Photoperiod experiments? Photoperiod experiments were planned to study the influence of light on secretory activity and histology of the organ. Three sets of experiments were arranged. Juveniles measuring 9-11 cm (standard length) were used,. The f i s h were maintained in light proof fibre glass tanks. The tanks measured 6 3 x k-6 x 36 cm , and were covered with wooden l i d s - 16 -each fi t t e d with one 8 watt mercury lamp (W, Freeman & Sons Ltd., Vancouver) placed in the centre of the roof approxi-mately 15 cm, above the water surface. The lamps were protected from below by a fibre glass screen. The intensity of light measured 27 ft c at the water surface. During the experimental period the f i s h were fed daily on commercial f i s h food. The temperature of water was maintained between h = 5°C by regulating water flow. Experiment A was started in early March, 1963. Twenty four f i s h were kept in continuous darkness for one month. At the end of this period half of them were k i l l e d i n darkness and the brains dissected out in dim light (red l i g h t ) . The remaining f i s h were exposed to continuous light by turning the lamps on. They were k i l l e d twenty-four hours (A-l) and thirty-six hours (A-2) after i n i t i a l exposure time. Experiments B and C were set up i n May 1963. In each set ten f i s h were maintained under continuous darkness and continuous light respectively for one week. At the termin-ation of this period 50% i n each were sacrificed. The other 50$ in each were immediately subjected to reversed conditions of l i g h t . Those formerly in darkness were now i n continuous light (B-l) and those formerly i n continuous light were i n darkness (C-l). Fish in each set were k i l l e d after four hours from the i n i t i a l time of exposure to these reversed conditions. Control f i s h (daily photoperiod conditions); were also k i l l e d at the time when experimental f i s h were so treated. - 17 -For histological and histochemical preparations intact heads of juveniles O-^cm, standard length) were used. For convenience these are designated as juveniles 1. The brains of juveniles II (7-9 cm or more); were dissected out to avoid d i f f i c u l t i e s in sectioning due to bone. Decalcific-ation was not considered appropriate for histochemical treat-ment. The heads and brains were quickly immersed in suitable fixatives. The fixatives were changed once. Adult f i s h were used for both histological and histo-chemical studies. Owing to d i f f i c u l t i e s in maintaining them i n the laboratory they could not be subjected to experimental photoperiods. The f i s h were k i l l e d at the sites of collection. Their heads were removed and the bone overlying the pineal region was scraped to expose the pineal. After immediate immersion i n appropriate fixatives they were brought to the laboratory. Histological methodss: 1. Heads and brains of juveniles I and II respect-ively were fixed in aqueous Bouin*s solution. Also juveniles II heads were decalcified In Perenyi's solution (Galigher, 193l+5 which i s recommended for tissues containing small deposits of calcium, Section were cut at 5/J, and 10 JJL after paraffin embedding and stained with haematoxylin-eosin. 2. Romanes' ammonlated silver nitrate-sodium chloride method (1950J was used for impregnation of nervous elements. Juvenile II and adult brains were fixed in 10$ neutral formalin - 18 -and sections were cut at 15 to kOJUU after paraffin embedding* The sections were coated with If nitrocellulose to avoid any loss of sections in the impregnating solution (pH 9.0-9o6) kept at 58°C for 20-2*f hours. 3. Ramon y Cajal"s method for astrocytes and microglia was used on juvenile I I brains fixed i n formalin, embedded in gelatin, frozen and cut at 16 JU/with a freezing microtome. *f. Some brains of experimentally treated juveniles were fixed i n Flemming's dilute solution (Humason, 1962) which has been used as a very satisfactory fixative for retinal elements. Sections were cut at 5 JJL, and 10p> and stained with Ehrlich's haematoxylin-eosin and also with Heidenhain's Iron haematoxylin methods. Histochemical methodss These methods were applied to brains of experimentally treated and control juveniles and brains of adult f i s h . 5JLL, and lO^ UU sagittal sections of paraffin embedded material were made. 1. Bargman"s chrome-alum haematoxylin-phloxin method was used for neurosecretory substance. This method i s not specific but i s s t i l l considered most satisfactory for the study of NSS: (Pearse, I960). Brains of juveniles and adults were fixed in 10% neutral buffered formalin. After deparaf-finization the sections were mordanted i n aqueous Bouin's containing 3-U % chrome-alum for 17-2K hours at 3 7°C 2. Halmi*s paraldehyde fuchsin light green orange G method (1952). The technique was applied for neurosecretory - 19 -matter and identification of basophilic and acidophilic granules. Halmi could distinguish two types of basophils selectively stained with aldehyde fuchsin i n rat adenohypophysis. The counterstain was used for acidophil granules. The material was fixed i n aqueous Bouin's containing 0.1% trichloroacetic acid instead of glacial acetic acid. According to Halmi best results are obtained with this fixation as trichloroacetic acid effectively precipitates proteins. The sections were stained with 3-10 days old paraldehyde fuchsin since after this period i t looses i t s potency as a selective stain. 3. Performic acid alcian blue and Periodic acid-Schiff technique (PFAAB & PAS; Adams, 1956). These were employed for neurosecretory substance, polysaccharides, mucopolysaccharides, mucoproteins and glycoproteins. PFA oxidation followed by staining with acid alcian blue gives a reaction for cystine, an important component of NSS i n the hypothalamus, neurohypo-physis and secretion of some adenohypophyseal c e l l s . A posi-tive reaction with PAS indicates the presence of 1-2 glycol groups occuring i n polysaccharides (glycogen) and some muco-polysaccharides, mucoproteins and glycoproteins. Juvenile and adult brains were fixed in 10% neutral buffered formalin. Control sections were prepared with saliva digestion for approx-imately 6 hours. Salivary amylase removes glycogen from the tissues. Sections were also subjected to acetylation in a mixture of acetic anhydride and dry pyridine and l e f t over-night at room temperature. Acetylation blocks 1-2 glycol groups from forming reactive aldehyde groups after oxidation - 20 -with periodic acid. After thorough washing sections were stained together with normal sections according to the usual procedure. The use of orange G was omitted to avoid masking of Schiff's reaction. k, Mallory-Azan technique (Koneff, 1938). The method was used for identification of acidophilic and" basophilic granules. Brains were fixed in 10$ neutral formalin and the sections were postfixed i n 3$ potassium dichromate for 2k hours before actual staining. 5. Some series of sections were selected and stained using alternate slides, with Bargman's, PFAAB and PAS and Mallory-Azan techniques to substantiate the reactions of one method with that of the other. 6, Adult and juvenile brains were fixed i n 10$ neutral formalin. 5 and 10 u thick sections were tested for melanin and lipofuchsin in the pineal organ. Two methods were employed (a) Nile-blue sulphate ( L i l l i e , 1956) for differentiation of lipofuchsin from melanin and (b) ferrous sulphate-ferricyanide ( L i l l i e , 1957) for melanin. The f i r s t method depends on stain-ing of melanin a dark green colour when sections are subsequently treated with 1% H^ SO^ and dehydrated in 100$ acetone. Lipo-fuchsin does not stain i n this method. It retains green or dark green stain when sections are stained with nile-blue and the step involving the use of B^SO^ and acetone i s omitted. The reaction i s pH dependent. Method "b" i s considered speci-f i c for melanin and depends on melanin entering into a complex ++ formation with Fe. - 21 -Quantification of Secretion: In tables 1 and 2 a positive sign (.+) indicates the presence and a negative sign (-) shows the absence of a certain secretory substance. For evaluating the effect of light on the secretory activity the amount of secretion has been arbi-t r a r i l y quantified by noting differences apparent to the eye under the microscope. The differences i n the relative amount of secretion under various photoperiod conditions are repre-sented by differences i n the number of positive signs. A difference of two positive signs has been considered s i g n i f i -cant . - 22 -Fig. 1. Camera lucida drawing of sagittal section of head of juvenile I sockeye salmon. X120. B. V. blood vessel CR.R. cranial roof C. T. connective tissue D. S. dorsal sac F.B. fore-brain H.C. habenular commissure M.B. mid-brain OP.T. optic, tectum PAR. paraphysis PG. pigment (melanin) P.D.V. pineal d i s t a l vesicle P.O. pineal opening P.S. pineal stalk P.C. posterior commissure SK. skin covering head S.C.O. subcommissural organ III VEN. third ventricle - 23 -RESULTS Topography and Gross Structure: Relation with epithalamus. The pineal organ, the most posterior diverticulum of the epithalamus, l i e s immediately in front of the mid-brain and dorso-medial to the fore-brain. As seen in median sagittal section ( f i g . 1) i t consists of an anterior, broad, vesicular part, the di s t a l vesicle (P.D.V.), and a narrow posterior por-tion, the stalk (P.S.). The stalk establishes a connection with the brain. The entire organ l i e s obliquely to the longi-tudinal axis of the brain; dorsally the di s t a l vesicle and part of the stalk shield the posterior half of the fore-brain. Interposed between the fore-brain and the ventral side of the pineal l i e s the dorsal sac (D.S.) which i s a spacious hollow outpouching of the epithalamus. Except a small anterior seg-ment and lateral expansions the entire dorsal sac i s covered by the pineal organ. In the region of the stalk the lateral extensions of the dorsal sac are turned dorsad meeting with each other above the stalk ( f i g . 2 ) . Posteriorly and medially the stalk connects with the subcommissural (S.C.O.) organ and the posterior commissure (P.C.). In the same plane but ven-t r a l l y i t joins the dorsal sac epithelium. Immediately in front of and i n contact with this junction l i e s the habenular commissure (H.C.). Relation with the skull. The di s t a l vesicle l i e s i n contact with the overlying - 2h -cartilaginous roof, of the cranium in juveniles. A foramen. in this region of the skull i s absent but the cartilage i s thinner here and forms a fossa ( f i g . 1 and 3) to lodge the distal vesicle. , Probably the pressure exerted by the growing, , pineal i s responsible for this formation. In the juveniles the skull roof i s transluscent and the distal vesicle i s clearly visible through i t in intact animals. With advancing age, increasing opaqueness obscures i t . In adults a change from cartilage to bone makes i t entirely invi s i b l e . However, the pineal fossa persists as seen during dissection. ........ Gross structure. The pineal organ consists of a pseudostratified epithelium enclosing a central lumen i n the juveniles. The distal vesicle has thicker epithelium with pseudostratification more distinct here than i n the stalk. . In juveniles 1 the epithelium i s thrown into ridges and furrows ( f i g . 1) in the. vesicle. The ridges contain clusters of cells alternating with furrows where the epithelium i s single layered or has fewer c e l l s . It appears that a differential growth of cells forms these ridges which represent areas of most rapid growth. With increasing age these ridges start infolding forming lobules as i s evident i n juvenile 11 pineals. In contrast to the parietal eyes in lampreys and lizards and the frontal organ i n amphibians the dorsal and ventral epithelia do not • differ i n thickness and an eye-like appearance i s lacking. - 25 -The central lumen i s continued into the stalk where the epithelium i s thinner and devoid of infolding. At the level of the junction of stalk with subcommissural organ dorsally and with the dorsal sac ventrally, the lumen communi-cates with the third ventricle by a narrow aperture (P.OP., f i g . 1). Although the topography and morphological features remain basically the same, certain noteworthy changes are en-countered with increase in age and are particularly evident in the adult pineals. The d i s t a l vesicle increases in size and the stalk becomes proportionately longer. Gradual infold-ing of the epithelium results in the formation of folds or lobes hanging into the lumen. Thus, the lumen divides into 2>-h intercommunicating chambers. In adults the process Is carried to the extent that the lumen of the distal vesicle i s reduced to narrow sinuous interlobular spaces ( i l . sp.). Some infold-ing also occurs i n the stalk in adults but the central lumen i s never obliterated. Relation with blood vessels. The pineal organ i s bounded dorsally by meningeal con-nective tissue (C.T., f i g . 1) particularly on i t s proximal part (stalk). This tissue also Invades the space between the pineal and the dorsal sac. Many blood vessels course through this tissue anteriorly, dorsally and ventrally ( f i g . 1, B..V.). They run in immediate contact with the pineal outer limiting membrane. One dorsal longitudinal vessel can be seen even - 26 -Pig. 2. Photomicrograph of a portion of T.S. of juvenile head, cr., cranium; d.s., dorsolateral expansions of dorsal sac; hab., habenula; p.s., pineal stalk. ' H and E. X130. Fig. 3» Photomicrograph of T.S. of juvenile head passing through forebrain. d.s., dorsal sac; p.f., pineal fossa; p.og., pineal organ. H and E. XkO. - 27 -with the naked eye (during dissection)) which gives rise to a right and a l e f t branch about half way along the longitudinal pineal axis. These flow on either side of the forebrain and then dip ventrad. A longitudinally running vessel i s seen ventral to the pineal organ ( f i g . 1). The precise course and destination of the various vessels requires further investi-gation. However, an important change occurs in the relation-ship of blood vessels to the pineal epithelium during growth of the organ. The inpushing of the outer limiting membrane carries with i t the overlying blood vessels which get trapped in the resulting intralobular septa ( i l . s.), thus associating a greater number of cells with the blood vessels. Such a change i s quite evident in Juvenile 11 and i s more so on the dorsal than on the ventral aspect. Although blood vessels are rarely carried into the ventral intralobular septa, they are closely applied to the epithelium. In adults this process i s very pronounced. Pi gment. Sections of juveniles show melanin below the skin i n the pineal and adjacent anterior and posterior regions (PG., f i g . 1). It i s notable that the amount of pigment immediately dorsal to the pineal area i s scantier than anterior or posterior to i t . Between the roof of the cranium and the pineal organ also, pigment i s generally absent. Occasionally one or two clumps of black granules or scattered granules are seen. Anterior and posterior to the pineal such granules are often encountered. There i s no indication of presence of black or - 28 -dark brown pigment in the pineal epithelium-afact which i s consistent with past observations i n other f i s h species (Gladstone fetal, 19*f0). - 29 -Fig. h. Photomicrograph of a portion of juvenile II pineal organ (sagittal section) showing A (s.c.) and C type (sp^c) c e l l s , b.pr., basal process; i . s . , inner segment; o.s., outer segment. Note the coagulum, granules and fragments of outer segments in the lumen ( 1 ) . Osmiophilic granule i s indicated by arrow. / H and E. X700. Fig. 5. Photomicrograph of a portion of juvenile I pineal organ showing A (s.c.) B (sp.c.) and C type (sp*iC) c e l l s . cr.r., cranial roof; d.s.ep., dorsal sac epithelium; 1 . , lumen of pineal; l.pr., luminal process. Bargmans Chrome-alum. X700. Histology; On the basis of their nuclear features, cytoplasmic characteristics, the position of cells in the epithelium, their relationship with the basement membrane, the following types of cells have been identified. Type A, These are large cells with round to oval nuclei con-taining generally one and rarely two nucleoli. They occur throughout the thickness of the epithelium but are most abun-dant toward the luminal aspect. The cytoplasm i s pulled out into processes at both the basal and luminal aspects ( f i g . *0. The latter are termed here luminal processes as they occur to-ward the lumen of the organ, ending on or near the inner boun-dary of the epithelium and often extending into the lumen. They are either upright or recline on neighbouring epithelial c e l l s . Often these processes are divided into two segments by a constriction forming an inner segment proximal to the nucleus and a distal outer segment ( i . s . , o,s., f i g . *f). The terminal outer segments vary i n shape ranging from straight pointed forms to clubs, knobs or globules. In rare instances bright haema-toxyphilic f i b r i l s are seen in the apex. Eosinophilic spiral or straight threads often stream into the lumen either from the c e l l bodies, apparently devoid of luminal processes, or from the apices of the luminal processes. They appear to result from a disintegration of the outer segments. Detachment of the latter can be seen in many regions. Their fragments are present in the lumen as well as in the interlobular spaces ( f i g . h)„ - 31 -The inner segments of few of these cells exhibit an osmiophilic body immediately in front of the nucleus ( f i g . *f). Similar globules are characteristically present in the e l l i p -soid of the retinal photoreceptors. The occurrence of an ellipsoid has been described in pineal sensory cells (Holmgren, 1918, 2, Rana temporaria) and in l a c e r t i l i a n parietal sensory cells (Steyn, I960; Eakin and Westfall, 1961). On the basal aspect the cytoplasmic basal processes run obliquely or straight toward the basement membrane but do not end on i t ( f i g . h). Instead they turn either to the l e f t or the right and run parallel to the basement membrane. Silver impregnation revealed that they are neuraxes. On the basis of their structural details and the presence of neuraxes the A type cells are identified as sensory cells ( f i g . s . c ) . The nuclear chromatin shows considerable variation in amount and distribution. It i s uniformly distributed in some but in others i t shows a shift and crowding near the nuclear periphery. Similar granules can even be seen lying across the nuclear membrane. Also i n rare instances the nucleolus l i e s near the nuclear wall. Such observations have also been made by Holmgren (1959) in the pineal sensory cells in various f i s h species. In juvenile 1 these c e l l s , as characterized above, are not identifiable in H and E stained sections. The lumen appears largely clear with few projections from the luminal aspect of the epithelium. Whether they are protoplasmic pro-jections or some kind of coagulate i s d i f f i c u l t to say. In - 32 -Fig. 6. Photomicrograph of a portion of pineal organ in adult sockeye salmon (sagittal section) showing A ( s . c ) , B (sp.e.) and C type (sp'.c.) ce l l s . b,pr,, basal process; b.v., blood vessel; f . c , free c e l l s ; i l . s p . , interlobular space; i l . s . , intralobular septum; i . s . , inner segment; o.s., outer segment. Note the coagulum and granules near the inner boundary of epithelium. Bargmans chrome-alum. X 7 0 0 . Fig, 7« Photomicrograph of a portion of pineal organ in adult (sagittal section); showing A ( s , c ) , B (sp,c.) and C (sp* , c ) type c e l l s . Note the basal processes of B and C type cells ending on the basement membrane. Bargmans chrome-alum. X700. - 33 -sections stained with Halmi's method few luminal processes can be identified belonging to cells with nuclei similar to A type cells ( f i g . 5)» In adult pineals these cells appear fewer in number than in the juvenile 1 1 . Often the outer and inner segments can be discerned in the luminal projections ( f i g . . 6 and 7 ) . They present a variety of shapes and sometimes the club-like outer segments are haematoxyphilic and contain granules. Detached fragments of these processes l i e freely i n the inter-lobular spaces. In Bargman stained sections phloxinophilic caps or concentric bodies are frequently encountered either at the base or the side of the nuclei. They are probably sections of basal processes of neighbouring cells cut in a transverse plane. Type B. These are also large cells but show variations both i n size and shape of nuclei. They range in shape from round through oval to elongated types. The nuclear chromatin i s similar in distribution to that i n A type c e l l s . The elongated nuclei are usually hyperchromatic. A central or eccentric nucleolus i s present i n each. They possess a basal cytoplas-mic process which varies in length depending on the position of c e l l bodies i n the epithelium. They are predominantly located between the middle and the luminal extent of the epithelium. In the distal vesicle they are arranged in bunches and their basal processes run toward the periphery where they are seen to end on the basement membrane ( f i g . 8 ) . Usually - 3^ -Fig. 8. Photomicrograph of a portion of juvenile 11 pineal organ (T.S. )1 b.pr., basal processes of B type cells ending on basement membrane; b.v., blood vessel; d.s.ep., dorsal sac epithelium. C type cells can also be seen but are not very clear. Lumen i s obliterated due to compression. H and E. X 7 0 0 . Fig. 9. Photomicrograph of a portion of juvenile pineal organ showing a D type c e l l . A type B (sp.c.) c e l l i s clear with i t s basal process (b.pr.) running toward basement membrane. H and E. X1260. - 35 -blood vessels are present i n these areas*. At the point of contact with the membrane the processes form feet-like structures which are faintly v i s i b l e . Although the nuclear characters of these cells are similar to those of A type, the direction of the basal pro-cesses and their contact with the basement membrane as well as= the absence of luminal processes provide dependable evidence for distinguishing the two types, Dendy (1910} also noted the similarities between the ce l l s which he designated as supporting and sensory cells: i n the parietal eye of Sphenodon punctatus. This may be possible as the pineal cells are derived from the ependymal cells of the embryonic neural tube (Gladstone etal. l$kQ% Kelly, 1962). In juvenile I these cells are very prominent in the distal vesicle as clusters with generally pear-shaped nuclei with their processes converging, very often i n bundles, on the basement membrane. Their number decreases i n the stalk. In the pineals of adult f i s h they are greater in number than A type c e l l s . Fig, 6 and 7 show their basal processes, their relation with the basement membrane and the neighbouring blood vessels i n the intralobular septa, Type E, These are the smallest cells in the epithelium,. The nuclei are distinct from A type cells i n being hyper-chromatic ( f i g , *f,6 and 7 ) . The cytoplasm is; very scanty* The nuclei show a variety of shapes*. Round, oval, spindle - 36 -shaped, squarish and irregular forms are commonly encountered. A nucleolus i s visible particularly in round and oval nuclei.. At their basal aspect the cyto-plasm in each i s formed into a short process which attaches to the basement membrane. As they are predominantly abund-ant i n the basal part of the epithelium many cells l i e jutting against the outer limiting membrane or the intra-lobular septa. Their number i s greater i n the stalk than in the distal vesicle, the variety of shapes more clearly observed in the former, i Type D, Rarely cells are seen with large, round nuclei surrounded by abundant cytoplasm and characterised by lack of luminal or basal processes. These are present both i n juveniles and adults. Fig, 9 shows a c e l l situated directly on the intralobular septum. They appear very different from the above categories. As such cells did not show up i n silver impregnated sections their identity as nerve cells i s subject to doubt. It i s quite possible that repeated and more controlled silver impregnation treatment i s required before they can be clas s i f i e d . Neuroglia, The above described types of cells do not show structural details characteristic of either astrocytic or oligodendrocytic neuroglia ( f i g . 10), In the substance of the brain astrocytes and oligodendrocytes are often identifiable i n sections impregnated with neuroglia stains. - . 3 7 -The presence of f a i n t l y impregnated processes and the r e l a t i o n s h i p of these c e l l s w i t h blood v e s s e l s and. nerve c e l l s a i d considerably i n t h e i r I d e n t i f i c a t i o n , , As examined i n j u v e n i l e I I p i n e a l s none of the c e l l types show n e u r o g l i a type processes. A s i n g l e b a s a l process i n each c e l l ending on the basement membrane i s a f e a t u r e more l i k e that of ordinary ependymal g l i a as seen i n the e p i t h e l i a l i n i n g the v e n t r i c u l a r w a l l and the subcommissural organ. Thus i t seems appropriate t o c a l l B and C types as supporting c e l l s (sp.c. and sp'c. r e s p e c t i v e l y , f i g . 6 and 7 ) ' i n accordance w i t h other workers i n . t h i s , f i e l d . This i s a r a t h e r l o o s e d e s i g n a t i o n but serves w e l l t o d i s t i n g u i s h them, from .the, sensory cells,.. They could be c a l l e d ependymal... c e l l s but t h i s gives r i s e t o confusion as a l l the p i n e a l c e l l s are of ependymal o r i g i n and such a terminology needs care. Subcommissural and d o r s a l sac e p i t h e l i a . As already i n d i c a t e d , the p i n e a l e p i t h e l i u m grades i n t o the subcommissural and d o r s a l sac e p i t h e l i a i n the median.plane. This t r a n s i t i o n i s marked by a change i n the s t r u c t u r e ; of the e p i t h e l i a l c e l l s . i n the l a t t e r two organs. In the subcommissural organ the n u c l e i are predominantly o v a l or elongated., Their s i z e and shape i s subject t o v a r i a t i o n s as these c e l l s appear m e t a b o l i c a l l y very a c t i v e - an obser-v a t i o n described elsewhere. The c e l l s are, arranged i n a p s e u d o s t r a t i f i e d s t r i p , l y i n g , v e n t r a l to the p o s t e r i o r comm-i s s u r e ( f i g . 12). They are densely packed and each possesses - 38 -a basal process directed toward the posterior commissure and an apical process on the ventricular aspect. The latter either end on the wall forming the ventricular roof or pro-trude into the ventricle as hair-like projections. The ventral limb of the pineal epithelium undergoes a reduction in cellular components at the level where i t turns around the habenular commissure. Beyond this point round to oval nuclei, often b i f i d , become prominent. The cells have abundant granular cytoplasm and are arranged in a single layer. On the ventricular aspect c i l i a - l i k e pro-jections form a striated border. Secretory granules are usually present along the epithelium covering the striated border as well as i n the cavity of the dorsal sac (d.s, ep», f i g , 8 ) . Nervous elements, A large number of nerve fibres i s present i n the pineal epithelium of juveniles and adults. Their origin and details are better seen under high magnification. The fibres arise singly from the base of pineal c e l l s . They run toward the basement membrane obliquely or perpendicularly,, In the vicinity of the membrane they appear to end as stumps. However, their relation with the membrane indicates that their peripheral continuation i s cut as the fibres would take an irregular course. Peripherally many nerve fibres run parallel to the intralobular septa and the limiting membrane, and often they are seen running across the neighbouring epithelial cells as long fibres ( f i g , 11 and 13 , n,f,)}. These are actually b - 39 -Fig., 10 Photomicrograph of a portion of juvenile II pineal organ (sagittal section)). Note lack of astrocytic or oligodendrocytic processes. Ramon y Gajal's method. X 700. - IfO -continuations of neuraxes (nx.) arising from the pineal c e l l s . This i s particularly clearly seen in the region of the stalk ( f i g , 11): where c e l l neuraxes curve to continue parallel to the basement membrane. In few instances they enter the basal region of the epithelium where they f i r s t curve frontalward for a short distance but eventually move back to run posteriad accompanying other parallel fibres,. The lat t e r are frequently seen to run along the wall of blood vessels. The number and frequency of these pineal nerve fibres increases proximally. Dorsally between the outer limiting membrane and the base of the cells the fibres form a thick bundle forming the pineal nerve tract ( f i g . 11 and 12, n,,tr,.) which i s traceable further proximally into the posterior commissure. A nerve tract i s not seen ventrally but many neuraxes course into the epithelium both parallel and perpendicular! to the basement membrane. These fibres; do not seem to enter the habenular commissure although they can be traced as far as the point where the pineal epithelium turns ventrad to meet the dorsal sac epithelium. The direction of the fibres ventrally suggests that they may turn around laterally and move toward the dorsal surface to join the nerve tract. In both juveniles and adults during i t s course through the posterior commissure no fibres; are seen to branch out from the nerve tract i n the region of the optic tectum^ Also, the tract fibres dip down to run across the subcom-missural epithelium but they do not end on the c e l l s . Further - kl -Fig. 11. Photomicrograph of pineal organ i n adult at the junction of stalk and distal vesicle, n.f,, nerve fibres; n.tr,, nerve tract5 nx., neuraxis entering nerve fibre layer. Romanessilver impregnation. X 700. Fig. 12, Photomicrograph of the junction of pineal stalk with subcommissural organ (s,col), in adult• c.t., connective tissue^ n.tr., pineal nerve tract entering posterior commissure ( p . c ) ; III ven., third ventricle. Romanes' silver impregnation. X ?00t - 1+2 -caudadi the tract fibres end abruptly and cannot be traced in serial sections. Their terminal ends appear curved suggesting that they turn sideways in the posterior commissure and run along with fibres of the latter and in the corresponding direction. There occurs a reduction in the number of pineal tract fibres as they move caudad.. This observation coupled with the topography of the commissure supports the view that a change occurs in the direction of the pineal nerve tract. Efferent innervation. Besides the above described sensory innervation loop-like nerve endings; are observed on both the A and B type pineal cells (f*ig. 13» e.lp.)). These end-loops are very prominent in the pineal of adults. Their fibres are directed toward the basement membrane. Their direction i s variable and they criss-cross frequently. It i s clearer in the stalk where they can be traced running along the fibres of the pineal nerve tract. The existence of the loops indicates an efferent innervation and that the pineal nerve tract i s probably of a mixed type consisting of both sensory and motor fibres. The end-loops are very prominent in the distal vesicle c e l l s . In the stalk few cells show them but whenever seen they are distinct., They are lacking i n C type c e l l s . Nerve c e l l s or ganglia.. Contrary to the observations of Holmgren (1918, 1920,-; Rasquin (1958)• etc. i n various f i s h pineals, nerve cellsi are not seen i n either the Juveniles or adults. Their absence has been noted i n Salvelinus spp. ( H i l l , 1891) and i n - ^ 3 -Fig. 13- Photomicrograph of a portion of pineal organ in adult, e.lp., efferent end-loops; n.f., nerve fibres;; nx., neuraxis. Note complete absence of end-loops on C type (sp».c.) c e l l s . Romanes" silver impregnation. X 700. - kh -Salmo salar (Holmgren, 1959). Steyn (1959c) has described direct continuation of some sensory c e l l neuraxes into the nerve tract in the parietal eye of Cordylus polyzonus. Pineal lumen, interlobular spaces and their contents. The lumen and adjacent interlobular spaces i n juveniles and adults contain a coagulum. Its amount i s vari-able i n different regions and also shows variations from pineal to pineal. In juvenile I i t i s scant and increases with age as can be seen i n juveniles II and adults. It occurs in patches and frequently l i e s close to the epithelial boundary,. Granular clumps of the coagulum are located near the margin of type A and B cells ( f i g * *+,6 and 7 ) . Fragments of luminal segments of A type cells are often observed lying free i n the lumen ( f i g . *f),. Some A, type cells even show them in the process of breaking off from the c e l l bodies. Granular threads can be seen streaming out into the lumen. Similar granules and threads occur within the epithelium and usually their continuity with those i n the lumen i s discernible suggesting that the luminal contents are derived from the pineal epithelium. Many free cells, are also scattered i n the lumen either singly or i n groups. They resemble the epithelial types ( f i g , 6, f,.c>. Strikingly, the cytoplasm in these cells i s very clearly seen. In some cel l s i t i s formed into a pro-cess at both ends of the nucleus. In this regard they resemble the subcommissural type c e l l s . The occurrence of free cells seems to result from an extrusion of these cells from the pineal and/or subcommissural epithelium. Such an expulsion has been described as a common feature in the pineal and parietal eye epithelium in f i s h and reptiles respectively (Holmgren, 1959; Kelly etal. I960; Steyn, 1957, 1959 c). Secretions The relationship of pineal cells with the granular coagulum in the lumen and interlobular spaces indicates an apocrine secretory activity. The A type cells are clearly involved i n this process and i t appears probable that the B type cells also take part in i t . The C type cells which are predominant peripherally do not show granules i n their v i c i n i t y and seem non-secretory. The basal cytoplasmic processes of the cells as well as the nerve fibres are devoid of granules. The chemical composition of the secretory matter i s described as follows. PFAAB-PAS -method., Pineal cells i n both juveniles and adults give negative result with PFAAB.. On the other hand a positive reaction i s obtained i n the neurohypophysis and the adeno-hypophysls. Thus cystine and acid mucopolysaccharides are absent i n the pineal or the subcommissural organ. PAS positive granules are seen in a l l the pineals tested but the reactivity i s far less than in the pituitary,. It i s thus apparent that the reactive substances are limited in amount. The granules occur generally near the inner boundary of the pineal cells- as well as in the lumen ( f i g , I 1*). - 1 + 6 -Fig. Ik, Photomicrograph of a portion of juvenile II pineal vesicle showing PAS * granules i n the luminal pro-cesses (l.p r . ) , within the' epithelium (arrow) and the lumen (1) . PFAAB-PAS. X 700. - h7 -They are most abundant in the extreme anterior end of the distal vesicle, i n juveniles.. The cytoplasmic; basal pro-cesses of the cells show few granules, but very rarely. Within the epithelium they form rosettes which occur probably within the c e l l cytoplasm. Such granules are. not associated with C type cells. A positive reaction, with PAS. treatment, i s not obtained after acetylation of the. sections indicating, the presence of 1:2 glycol groups. Saliva, digestion-greatly reduced the intensity of the reaction. These tests show that ..glycogen and probably mucopolysaccharides are present. These substances have been reported in f i s h pineals (Rasquin, 1958;; Holmgren, 1959). The subcommissural.organ i s very active as a secretory area and the amount ,of glycogen, and mucopolysaccharides i s greater here than i n the pineal organ. The granules are intensely reactive and often occur in columns in the epithelium, particularly in the apical processes. Few are also present in the basal processes. The crowding of granules i n the apical region points a release into the ventricular cavity. Bergman's chrome-alum haematoxylin^phloxin method. The cytoplasm of the cells i s generally devoid of Bargman positive granules. The luminal processes, however, show in few instances only some positive,reaction. Maximum reactivity i s obtained in .the luminal coaguiunu In these areas the reactive substance, usually , appears amorphous , although, granules ..can also be seen rarely. - he -Unli&e the pineal organ the subcommissural organ contains numerous chrome-alum positive granules. They stain a dark-blue or black and range i n size from very fine to large granules arranged in columns or even clumped in some parts of the epithelium. They are most abundant i n the apical processes and are few in the basal processes. The pineal nerve tract shows no such granules. However, they occur abundantly on the apical striated-border lining the ventricular roof. The coagulate in the third ventricle when seen stains similarly indicating that the secretion i s released into i t . Except rarely in the pineal cells of the adults the basal processes lack chrome-alum positive granules. Consequently the release of these granules into blood vessels occuring in the pineal or the subcommissural organ seems doubtful. Halmi's paraldehyde fuchsin counter stain method. The luminal processes of sensory cells contain baso-philic granules (aldehyde fuchsin + =; AF+). Fig. 15 and 16 show knob-like outer segments f i l l e d with these granules. The basophilic reaction i s more intense in the distal vesicle than in other areas. The basal processes of cells give a negative AF reaction but in rare instances few light green positive (LG) granules are present. They are seen i n the distal vesicle cells in Juveniles as well as i n adults. In the latter they are present near the basement membrane. The luminal coagulate contains a large number of AF positive granules and seems to be drived from the epithelial c e l l s . - 1+9 -Fig. 1%, Photomicrograph of a portion of juvenile II pineal organ showing AF * granules i n the luminal processes (l.pr..) and the lumen (l.X d.s.ep.,dorsal sac epithelium. Halmi,1s paraldehyde - fuchsin* X 700* Fig. 16. Photomicrograph of a portion of pineal organ in adult showing AF + granules. Abbreviations same as above. Halmi's paraldehyde - fuchsin. X 700,. ~ 50 -The subcommissural cells as, well as the dorsal sac cells are also reactive to AF. The granules in the former are crowded in the apical processes and are absent or scanty i n the basal processes. They are also absent i n the pineal nerve tract layer, whereas: the third ventricle contains subcommissural type granules. Again these observation suggest that they are released into the ventricle. Uh the dorsal sac the granules are far more reactive to AF than anywhere else £fig. 15, d..s.ep.). The cytoplasm of the cells i s studded with them. They are liberated into the cavity of the sac as i s evident from a similar reaction of the coagulum in the latt e r . Et i s d i f f i c u l t to say whether cystine i s responsible for AF positive reaction i n the pineal and subcommissural organs, PFAAB i s considered a more specific test for cystine CPearse, 1961);. Negative results with this method in these organs in contrast to positive reaction in the pituitary with PFAAB and AF show that cystine i s probably absent in the former. It i s quite possible that the reactivity with AF i s due to some other amino acid radical found commonly in the pineal, the subcommissural organ and the dorsal sac. Mallory°Azan method. In both juveniles and adults the c e l l cytoplasm and the luminal coagulum show acidiphilia. Distinct granules are rarely seen in the luminal processes i n juvenile pineals. In the adult pineal A type cells acidophilic droplets or granules - 51 -are sometimes seen. The coagulum in the lumen i s generally amorphous. Some acidophilic granules are observed near the basement membrane and also in a space surrounded by cells arranged i n a f o l l i c l e - l i k e pattern. Secretory - granules, usually acidophilic, are very distinct in the subcommissural organ. Sometimes they also show basophilia. The granules are abundant in the apical processes and the ventricular coagulum whenever seen i s similarly stained. Influence of Light and Darknesss: None of the c e l l types in the pineal show differences in either cytological detail or position in the epithelium under the influence of darkness or l i g h t . Melanin and lipofuchsin are absent as revealed by histochemical tests used for their identification. Consequently no changes i n their distribution and amount could be expected. The effect of experimental photoperiods on the secretory activity i s presented in tables I and 2. Table 1 shows that there are no differences in the amount of secretion in the dark•= adapted and light-adapted pineal organ and subcommissural organ. The amount i s \maffected even i n tissues transferred from 2k hr light to 36 hr, l i gh ts The quantity i s comparable in these to that in the controls •; • In experiments B, B-l and C, C.-l (table 2) the secretory activity i s same in light-adapted (one week) and dark-adapted (k hr.) pineals and subcommissural organ. Reversal - 52 -T a b l e 1. E f f e c t o f p h o t o p e r i o d s on t h e s e c r e t o r y a c t i v i t y i n t h e _ p i n e a l and s u b c o m m i s s u r a l o r g a n s i n j u v e n i l e I I . A = c o n t i n u o u s d a r k n e s s , A - l = A t r a n s f e r r e d t o 2h h r l i g h t , A-2 = A t r a n s f e r r e d t o 36 h r . l i g h t . ? = i n d i c a t e s a b s e n c e o f o r g a n i n s e c t i o n s e x a m i n e d . F o r e x p l a n a t i o n o f s y m b o l s see page 21. E x p e r i m e n t A, C o n t r o l s Methods P i n e a l o r g a n Subcommis-s u r a l o r g a n P i n e a l o r g a n Subcommis-s u r a l o r g a n PFAAB - - -PAS .(.+ ++++ +++ ++++ Chrome-a lum h a e m a t o x y l i n -p h l o x i n ++ ++++ ++ ++-!- + M a l l o r y - A z a n ++ ++•+ ++ - * - • ! - - » • H a l m i • s p a r a l d e h y d e - • f u c h s i n ++++ +++ ++++ 0 m E x p e r i m e n t A - l E x p e r i m e n t A-2 PFAAB - - - -PAS ++ ++++ +++ ++++ Chrome-alum h a e m a t o x y l i n -p h l o x i n ++ ++++ + +++ M a l l o r y - A z a n ++ +++ +++ +++ Halmi»s p a r a l d e h y d e -fuchslnv +++ +++ +++ ? - 53 -of the above photoperiods (C, C-l) also does not seem to ef-fect the amount of secretion. No differences are noted be-tween the above and the control sections, A comparison of results between sets "A, A-l, A-2" and "B, B-l; C, C-l" i n d i -cates that the length of exposure to darkness or light i s not significant in bringing about an effect. This observation refers only to the periods of time used i n these experiments. It i s however, possible that a much longer period of conti-nuous light or darkness may be necessary to i n i t i a t e an effect. Table 2, Effect of photoperiods on the secretory activity in the pineal and subcommissural organs in juvenile II. B = continuous darkness (1 we^k), B-l = B transferred to k hr. lig h t , C = continuous light (1 week), C-l = C transferred to h hr. dark-ness. Symbols same as i n Table 1. Experiment B Experiment B-l Methods Pineal organ Subcommis-sural organ Pineal organ Subcommis-sural organ PFAAB - • - ' -PAS ++ ++++ ++ ++++ Chrome-alum haematoxylin-phloxin ++ +++ ++ ++++ Mallory-Azan +++ ++++ ++ Experiment C Experiment C-l PFAAB - - - -PAS ++ ++++ • M r ++++ Chrome-alum haematoxylin-phloxin ++ ++++ ++ ? Mallory-Azan +++ +++ +++ ++++ - 5 5 -DISCUSSION Sensory Cells and Functional Significance;; The pineal organ i n lower vertebrates has long been implicated in light perception. The present observations in sockeye salmon also strengthen this view. Electron microscope studies substantiate the light microscope obser-vations and confirm that the sensory cells i n the frontal and pineal organs of amphibians and the parietal and pineal organs of lizards are remarkably similar in histological and cyto-logical detail with rods and cones in the vertebrate eye retina (Kelly, 1962 ) o The presence of outer and inner segments con-taining lamellar folds and mitochondria-rich ellipsoids res-pectively, a c i l i a r y connection of the former with the latter and sensory neuraxes at the base of cells are the main features common to the pineal or parietal eye and retinal photoreceptors (Steyn, i960; Eakin and Westfall, 1961)). The unpublished electron microscope studies of Horstmann have demonstrated a similarity between the sensory cells of the f i s h pineal organ with those of the lateral eye retina indicating a photosensory function of these c e l l s . Besides the histological similarity of sensory cells i n sockeye to those i n other f i s h , experimen-t a l evidence (Hoar, 1955) has been put forth that the juvenile sockeye pineal i s photosensory. Whether these cells are chemosensory or pressure receptors can not be decided from the present observations. These pos s i b i l i t i e s should not be ruled out u n t i l methods are developed for such a study. - % -The observations of Dodt and Heerd (1962) that the pineal nerve in frog i s insensitive to chemical and mechanical s t i -muli and the general histological similarity of sensory cells in sockeye to those in other lower vertebrates favour a photo-sensory role. In juveniles of sockeye the pineal area i s trans-luscent and would probably allow enough penetration of lig h t . Although the retention of a photosensory pineal in addition to eyes does not seem indispensable, i t provides a useful sensory mechanism operable under certain conditions. The dorsal location of the organ i s ideal and may be of s i g n i f i -cance specially during the early part of l i f e when juveniles l i e buried in gravel or at times when they seek cover during their downstream migration. Apart from such situations the pineal organ would have no special significance unless d i f f e r -ences i n the intensity threshold are observed in the pineal sensory cells and the retinal c e l l s . Measurement of nervous activity from incised pineal nerve tract would prove valuable in this regard. In adult sockeye owing to thick bone overlying the pineal area the situation becomes complicated with respect to penetration of light. This i s often an argument against the theory of a photosensory role for the pineal i n such species. The possibility of penetration, however, should npt be entirely disregarded i n the absence of direct obser-vations. Sufficient penetration of light has been observed (Ganong, Shepherd, Wall and Brunt, 1963} using photoelectric - 57 -cells within the brain of rats 0 Also, in sockeye adults a pineal fossa i s retained and the organ does not l i e as deep as would appear from outside. Such fossae are particularly prominent i n deep-sea fishes (Holmgren, 1959))* Thus a cer-tain degree of photosensitivity in adult pineals seems pos-sible. A decrease in photoreceptor c e l l number in the adults is suggestive of decreased photosensitivity. The organization of the pineal i s such that the sensory cells are distributed a l l around the epithelium. In contrast to the parietal eye of lizards and the cup-shaped retina i n the lateral eye photomechanical shift in their posi-tion would be of l i t t l e advantage against excessive amounts of light, particularly as melanin is also lacking i n this species. It seems quite l i k e l y that the skin and meningeal pigment act as a compensatory mechanism. The sockeye salmon has been grouped with fishes where the amount of light reaching the pineal i s controlled by changes in the distribution of pigment overlying the pineal area (Breder et a l . 1950; Hoar, 1955). This mechanism would provide the f i s h with a movable screen against excess lig h t . The presence of nerve cells and the destination of the pineal nerve i n the brain seems to be intimately related to a photoreceptor function. The present observations have failed to establish conclusively the f i n a l destination of the nerve tract. The optic tectum as well as the subcommissural organ does not receive branches from the tract. It i s possible i1 that the tract fibres follow the posterior commissure fibres - 58 -and synapse in either the lateral geniculate nuclei or the thalamus., The posterior commissure fibres have connections in these areas (Ariens Kappers etal, 1 9 3 6 ) . Synapse forma-tion in these areas would not be unusual as optic nerve fibres are also known to synapse here. Efferent fibres whose endings have been observed on sensory and supporting cells may have their origin in the same or neighbouring regions. Secretion and Functional Significance; The ever accumulating evidence of a neuroendocrine role of the pineal in mammals has inspired studies along simi-lar lines in lower vertebrates (reviews by Kitay and Altschule, 195^; Kelly, 1 9 6 2 ; Ariens Kappers, 196*0. In the lower "'vertebrates, however, a neuroendocrine role has only been indicated by the experimental studies of Bagnara ( I 9 6 3 ) on the control of chromatophores in Xenopus laevis larvae. The general absence of secretory granules i n the vicini t y of the blood vessels, except in rare instances in the adults, and along the nerve fibres shows that a neuroendocrine activity i s absent in sockeye. The possibility of such a role should be kept in mind since detection of certain specific substances depends on the specificity of the methods as well as the amount of reactive substances present (Eearse, i 9 6 0 ) . A histochemical detection of tryptophan derivatives i n the amphibian pineals has not been possible although some success has been achieved with spectrofluorometric methods (Eakin etal, I 9 6 3 ) . The subcommissural organ, anatomically associated with the pineal organ, i s also not neuroendocrine i n function. - 59 -Although i t contains abundant chrome-alum positive, AF positive and PAS positive granules these are released into the third ventricle. These substances seem to be produced in the epithelium i t s e l f and except a positive reactivity to N S 8 specific techniques there i s nothing else to help in cl a s s i -fying them with neuroendocrine or endocrine substances. The similarity in the chemical nature of the apo-crine secretion i n the pineal and the subcommissural organ and the common direction of release might suggest that i t i s i n some way related with the cerebro-spinal f l u i d (CSF). It may be involved either i n maintaining a constant chemical compo-sition of the latter or i n supplying the nervous tissues with certain chemical substances. This view also takes into account the continuity of the pineal lumen with the third ventricle. The chemical composition of the CSF has been studied to greatest extent in the mammals. It consists mainly of inorganic salts some dextrose and l i t t l e protein (CMaximow & Bloom, 19575* Glycogen i s present i n large amounts i n the epen-dymal cells of a l l classes of vertebrates. Oksche (1958) found that anuran epiphyseal cells are specially rich i n glyco-gen during the winter season and that these may supply glycogen to nervous tissue. Possibly some absorption of glycogen from the CSF occurs in sockeye also. Besides, the occurrence of glycogen i n the sensory c e l l processes may be u t i l i z e d either in the formation of some yet unknown substance essential for their functioning as has been suggested by Eakin etal (1962, Ryla regilla) or i n their normal metabolic activity. In the - 60 -lateral eye retins of vertebrates glycogen i s sometimes found in the photoreceptor cells (Carasso, I960; Yamada, i960). Its presence as a metabolically active substance i n the sensory cells i s quite probable but a predominant role appears to be related with the CSF. This view i s particularly emphasized because the glycogen^rich subcommissural organ lacking i n sensory cells also drains i t into the ventricle. The fate of the other secretory substances such as mucopolysaccharides, AF positive and Chrome-alum positive (subcommissural) substances is subject to speculation only. These may be incorporated as part of the protein component of the CSF or may even be a waste. The latter explanation does not seem probable spe-c i a l l y i f they are considered actively secreted substances and not mere artifacts as suggested by Van De Kamer (1956, fi s h and amphibia). Also the secretion of these substances in the subcommissural organ i s hardly doubtful. The presence of a motor innervation on both the photo-sensory and supporting cells can only be accounted for in terms of a pineal-CSF relation. Kelly and Van De Kamer (i960, Rana esculenta) have suggested that these efferent loops are either autonomic fibres or motor endings involved i n a feed-back mechanism. The1 regulatbn of photosensitive pigment syn-thesis or release i s not known to occur under a nervous con-t r o l in the vertebrate eye retina. The most li k e l y stimu-latory role seems to be control of the apocrine secretion into the pineal lumen particularly as neuroendocrine activity i s improbable i n this species so far as the present evidence i s concerned. - 61 -Influence of photoperiods. In view of improbability of an endocrine or neuro-endocrine activity the importance of light or darkness as a factor controlling i t s secretory activity does not seem pos-sible. The photosensitivity of the pineal organ does not necessarily warrant that i t i s subject to an influence of this factor with regard to secretion. It has been reported by Grunewald-Lowenstein (1956) that the pineal organ i n fishes is very resistant to the effect of light both in regard to i t s histology and secretion. She obtained some histological changes i n the pineal of Astyanax mexicanus after long expo-sures to light and darkness. In sockeye salmon where an apocrine secretion is tied up with the cerebro-spinal f l u i d an internal control seems more plausible than a control by light. Changes in the amount of CSF or i t s chemical compo-sition are not known to be induced by l i g h t . The independence of pineal from light i s also shown by Eakin etal (1963) in Hyla r e g i l l a where no significant differences occurred i n the amount of glycogen i n light-adapted and dark-adapted pineals. The subcommissural organ also gave similar results although differences between light-adapted and dark-adapted 1stirnorgan' were s t a t i s t i c a l l y significant. The subcommissural organ in sockeye i s not seen to receive nerve fibres from the pineal tract. Consequently any changes, secretory or histological, induceable by light have to be brought about by the agency of the eyes. However, a complete independence from light i s seen for this organ in this species. - 62 ~ The. view that the pineal organ i s photosensitive in sockeye i s handicapped by the results of photoperiod experi-ments. If the pineal i s indeed photosensitive then the asso-ciated pigment, i f present, must show differences under light and darkness. This aspect requires more investigations speci-f i c a l l y directed toward detecting a photosensitive pigment, before an analysis of the influence of light on the photosen-sory cells i s made. - 63 -SUMMARY 1. A parapineal i s absent i n sockeye. The pineal i s lodged distally i n a pineal fossa i n the cranial roof. Proxi-mally i t i s anatomically continuous with the subcommis-sural organ. 2. Sensory cells i n the pineal are considered photoreceptor in function. The pineal nerve fibres form a dorsal nerve tract which enters the posterior commissure and does not give branches to either the optic tectum or the subcommissural organ. It i s suggested that i t probably forms synapses in the thalamus or the late r a l geniculate nuclei. 3. Efferent nerve endings occur on both sensory and sup-porting c e l l s . Probably the dorsal nerve tract i s of a mixed type. l+. Supporting cells are ependymal and other neuroglia i s absent. Nerve cells could hot be detected i n silver impregnated sections. 5. Melanin and lipofuchsin are absent in the pineal epithe-lium. 6. The sensory cells and some supporting cells show an apocrine secretory activity, 7. An endocrine or neuroendocrine role of the pineal or the subcommissural organ i s improbable because of the general lack of association of secretory granules with the blood vessels or the nerve fibres. - 6*+ -8. The secretion of the pineal and subcommissural organ contains glycogen, mucopolysaccharides, mucoproteins and glycoproteins and basophilic (AF positive) granules. The subcommissural organ has abundant chrome-alum posi-tive granules whereas such positivity i s very limited i n the pineal organ. The subcommissural organ seems metabolically more active than the pineal. It i s pro-posed that the secretion of both the organs i s related with maintenance of constant chemical composition of the cerebro-spinal f l u i d and/or supply of some chemical sub-stances to the nervous tissue. 9. The pineal i s considered to be a useful photosensory organ in sockeye salmon only under eonditions when the efficiency of the eyes may be reduced. However, i t might be of some advantage i f future studies revealed differences i n the intensity threshold of the pineal sensory cells and the retinal photoreceptor c e l l s . 10. Both the pineal organ and the subcommissural organs are found to be independent of influence of lig h t or darkness. An internal stimulatory control of secretion seems more plausible i n view of these observations. 11. Suggestions are made that further investigations be car-ried with regard to (a); measurement of penetration of light through the skull in juveniles and adults, (b) measurement of nervous activity in the pineal nerve which would throw light on the intensity threshold, (c) develop-ment of methods to study whether the sensory cells have - 65 -any ehemoreceptor or baroreceptor function and (d) the presence of photosensitive pigment. — 66 — BIBLIOGRAPHY^ Ariens: Kappers, C.U., Huber, G.C. and Croshy, E.C. 1936. The Comparative Anatomy of the Nervous; System of Verte-brates including Man. Vol. 2, Ni,Y. Ariens Kappers, JJ. I960. The development, topographical relations and innervation of epiphysis cerebri in the albino rat. Z. Zellforsch., 52s 163-215. Ariens Kappers, J . 196V. Survey of innervation of the pineal organ in vertebrates. Am. Zool., hi *+7-5lo Bagnara, JT.T.. 1960b. Pineal regulation of the body lightening reaction i n amphibian larvae. Science, l l +8l-l l f83. Ibid. 1961. Onset of pineal and hypophyseal regulation of melanophores in Xenopus. Amer. Zool., Is 339-3M). Ibid. 19630 The pineal and the body lightening reaction of larval amphibians. Gen. Comp. Endocrinol. 3, No. I, pp.-86-100. Bentheld, F.V. and Moll. JJ. 195h* The vascular system of the mouse epiphysis with remarks on the comparative anatomy of the venous trunks i n the epiphyseal area. Acta Anat, ([Basel)), 22 s 227=235. Bern, H.A. 1962. The properties of neurosecretory c e l l s . Gen.. Comp. Endocrinol, Suppl. Is 117=132. Bern, H.A., Nishioka, R.S. and Hagadorn, I.R. 1962. Neuro-secretory granules and the organelles of neuro-= secretory cells.. Neurosecretion, Memoirs of the Society for Endocrinology, 12s 21-3*+. Ibid, I 9 6 L Association of elementary neurosecretory granules with Golgi complex. J . Ultrastruct. Research, 5s 311-320. Breder, CM. and Rasquin, Pi 19*+7» Comparative studies in the light sensitivity of blind characins from a series of Mexican caves. Bull. Amer. Mus. Nat. Hist., 89(5)s 325-351* Ibid, 1950. A preliminary report on the role of pineal organ in the control of pigment cells and light receptor reactions in recent teleost fishes. Science, I l l s 10-12, '^Not all references cited. - 67 -Carasso, N. I960, Role de. 1 "ergastoplasme dans 1' elaboration du glycog&he au cours de l a formation du "paraboloide" de cellules visuelles. CR, Acad. Sci., Paris, 250, 600-602. Cattle, J.T. 1882. Recherches sur l a gland pineale. Arch, de Biol. 101-19 1*. Cintron, C, and Kelly, E. 1961. An analysis of pineal regeneration in developing newts. Amer. Zool. 1 (4) . ; Clausen, H.J. and Poris, E.G. 1937. The effect of light on sex activity in the li z a r d , Anolls carolinensis, with special reference to the pineal body. Ahat. Rec, 69 i 39-53. Combescot, ch. et Demaret, JJ. 1963. Histophsiologie de l'epiphyse chez l a tortue d'eau Emys leprosa (Schw.). Annales D'Endocrinologie, tome 24-, No. 2. Dendy, A. 1907. On the parietal sense orgen and associated structures in the New Zealand lamprey, Geotria austral!s. Q.I.. Micr, Sci., 51? 1. Dendy, A. 1910. On the structure, development and morphological interpretation of pineal organ and adjacent parts of the brain in the tuatura (Sphenodon punctatus). Phil. Trans. Roy, Soc, Ser. B, 201, 227. De Robert!s, E. and Pellegrino De I r a l d i , A. I96I. Plurivesicular secretory processes and nerve endings i n the pineal gland of the rat, J. Biophys. Biochem. Cytol., 10; 361-372. De Robert!s, E. 1962. Ultrastructure and function in some neuro-secretory systems. Neurosecretion, Memoirs of the Society for Endocrinology, No. 12s 3-20. Dodt, E, and Heerd, E. 1962. Mode of action of pineal nerve fibres in frogs. J. Neurophysiol., 25s ^05-^29. Eakin, R.M. 196la. Photoreceptors i n the amphibian frontal organ. Proc. Natl. Acad. Sci,, 4-7; 108*+-1088. Ibid. 1961b. Fine structure of some l i t t l e known photoreceptors;. Amer, Zool., IsM+6, Ibid, 1962. Lines of evolution of photoreceptors. I, Gen. Physiol., 1*6 s:359-360. Eakin, R.M. and Westfall, J.A. 1959. Fine structure of the retina in the r e p t i l l i a n third eye. If, Biophys. Biochem, Cytol., 6sl33-13 l+. - 68 ~-Ibid. 1 9 6 1 . Further observations on.the,fine structure of the parietal eye of lizards. Ji. Biophys. Biochem. Cytol., 8s*f83°V99. •Ibid. 1 9 6 1 . The development of photoreceptors i n the stirnorgan of the tree-frog Hyla r e g i l l a . Embryologia (Ngoya), 6 : 8*f - 9 8 . Ibid. 1 9 6 2 . Effect of vitamin deficiency on photoreceptors in the lateral, and median eyes of the l i z a r d Sceloporus Occidentalls. Amer. Zool., Eakiny • R.M.y •'Quay-,.vE..-B.-<-,and'.i-We,stf.-al-L,. vJ<, A. . 1 9 6 1 . ' Cytochemical,; and cytological studies of the parietal eye of the l i z a r d . Sceloporus occidentalis. Z. Zellforsch., 53s: Mf 9=4-70. Eakin, R.M., Stehbins,* R.C.-and Wilhoft, - D.C. 1 9 5 9 . Effeet,,of pinealectomy and sustained temperature on the thyroid of the lizard,.Sceloporus Occidentalls. Proc. Soc. Exp. Biol. .(N.Y.)),. 1015162-.: Edinger, T. 1 9 5 6 . Paired pineal organs. In "Progress in Neurobiology!1 p.. 1 2 1 . Farrel, G. i 9 6 0 . Epiphysis cerebri i n the control of steroid secretion. Federation Proc. , 1 9 s 6 0 1 - 6 0 4 - . Ibid. i 9 6 0 . Adrenoglomerulotropin. Circulation suppl. (No. 5> part 2 ) , 1 0 0 9 - 1 0 1 5 . Friedriek-Freksa, H. 1932. . Entwicklung,. bau. und bedentung der parietalgegend bei teleostiern. Zeitschrift ftir Wissenschaftliche Zoologie, 14-1: 52-14-2. Galambos, R. 1958 A glia-neural theory of brain function. Proc. Natl. Acad. Sci. U.S., 4 - 7 M 2 9 - 1 3 6 . Ganong, W.F., Shepherd_. M.D., Wall, J.R., van. Brunt, E.E. and Clegg, M.T. 1963. Penetration of light into the brain of mammals. Endocrinology»72, 9 6 2 . , Gilbert, G.J. 1 9 5 8 . Subcommissural organ secretion i n dehydrated rats. Anat. Rec, 132s:563-567. 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The epiphsis of teleosts and Ami a. J. Mbrph. Hoar, W.S., 195*+. The behavior of juvenile Pacific salmon with particular reference to the sockeye. Ji. Fish. Res.. Bd. Canada, lit 69-97. Ibid. 1955. Phototactic and pigmentary responses of sockeye salmon smolts following injury to the pineal organ. J. Fish. Res. Bd. Canada. 12(1)s 178-185. Holmgren, N. 1918(1). Zum bau der epiphyse von Squalus acanthias. Arkiv for Zoologi, 11(24-)}. Ibid. 1918(2). Zue kentnnis der parietalorgane von Rana temporaria. Bd. 11(24-). Arkiv for Zoologi. Ibid. 1918 b. Uber die epiphysen nerven von glupea sprattus und harengus. Arkiv for Zoologi, Bd. II, No. 25, S.I. Holmgren, U. 1959. On the structure of the pineal area of teleost fishes with special references to a few deep sea fishes. Goteborgs Kungl. Vetensk. Vitterhets-samh. Handl., B. 8, 1-66. Ibid. 1958. Secretory material in the pineal body as shown by aldehyde-fuchsin following performi© acid oxidation. Stain Tech. 33 sl4-8-14-9. Humason, G.L. 1962. Animal tissue techniques. Freeman co., U.S. Hungerford, G.F. and Panagiotis, N.M. 1962. Response of pineal l i p i d to hormone imbalances. Endocrinology, 71? 936-91+2, ~ 70 -Katzenelbogen, S. 1 9 3 5 . The cerebro-spinal f l u i d and i t s relation to the blood. Baltimore, The Johns Hopkins Press., Keene, L. and Hewer, E.E. 1935. . The subcommissural organ and the mesocoelic recess i n the human brain and the Kelly, D.E. 1 9 5 8 . Embryonic and larval epiphsectomy in the salamander Taricha toroso and observations on scoliosis. Ibid. 1 9 6 2 . Developmental patterns i n the newt pineal organ, Amer. Zool., Vol. 2 , No. 3 . Kelly, D.E. 1 9 6 3 . The pineal organ of the newt, a developmental study. Z. Zellforsch., 58* 6 9 3 - 7 1 3 . Ibid, (to be published)}, infrastructure and development of amphibian pineal organs. Ibid. 1 9 6 2 . Pineal organsz Phot©reception, secretion and development. American Scientist, Vol. 5 0 , No. 4-, 5 9 7 - 6 2 5 . Kelly, D..E. and Van De Kamer, J..C. i 9 6 0 ..Cytological and cytochemical investigations on the pineal organ of the adult frog (Rana esculenta) Z. Zellforsch., 52s; 6 1 8 - 6 3 9 . Ibid. i 9 6 0 . Some cytological and histochemical considerations on the amphibian pineal organ. Anat. Rec, Vol. 1 3 8 , No. 3 , PP. 3 5 9 - 3 6 0 . Kit ay, JJ.I. 1 9 6 3 . Relation entre l a glande pineale et l a fonction hypophyso-corticosurrenalienne.. Ann ales D'Endocrinolgie, tome 24, No. 2 . Knoche, H. 1 9 5 6 . Retina, Zwischenhirnes und hypophyse. Z. Zellforsch, 4-5s 2 0 1 . Knowles, F.G.W, 1 9 3 9 . Photomechanical changes in the pineal of lampreys, jr.. Exp. Biol., 16a 524—529. Koneff,A.A» 1 9 3 8 , Adaptation of the Mallory-Azan staining method to the anterior pituitary of the rat. Stain Techno!,, 13*4-9-52. Laurens, H. 1 9 1 6 * The reactions of the melanophores of Ambystoma larvae.. The supposed influence of the pineal organ, jr. Exp. Zool., 20s; 2 3 7 - 2 6 1 . Reissner^s; fibre. J J . Anat 69s 5 0 1 - 5 0 7 . » 71 *= Ibid. 1917o The reaction of the melanophores of Ambystoma tigrinum larvae to light and darkness. J. Exp., Zool., 23s: 195-205. Le Gros Clark, W.E. 194-0. The nervous and vascular relations of the pineal gland. J. Anat. (Lond.), 74-s; 4-70-4-92. Lerner, A.B., Case, J.D.. Takahashi, Y., Lee, T.H., and Mori, W. 1 9 5 8 . Isolation of melatonin, the factor that lightens melanocytes. J. Am. Chem. Soc, 80s 2 5 8 7 . Maximow, A.A. and Bloom, W. 1 9 5 7 . A text Book of Histology. W..B. Saunders Co. McCord, CP; and Allen, E.P. 1 9 1 7 . Evidences associating pineal gland function with alteration i n pigmentation. JJ. Exp. Zool., 23s 207-224-. Mikami, S. 1951. Cytological and histological studies on the pineal gland of domestic animals. Tohuko J. Agric. Res., 2s 4-1-4-8. Miline, R., Deceverski, V. et krst i c , R. I 9 6 3 . Influence d'excita-tion olfactives sur l'systeme habenulo-epiphysaire, Ann ales D'Endocrinologie, tome 24-, No. 2 . Tbid. 1 9 6 3 . Contribution a 1 ' etude de 1 ' influence de l 1 i r r a d i a t i o n s sur le ganglion de 1'habenulae et glande pineale. Annales D'Endocrinologie, tome 24-, No. 2 . Miline, R. 1 9 6 3 . La part du noyau paraventriculaire dans l'histo-physiologie correlative de l a gland pineale.. Annales D'Endocrinologie.. tome 24-, No. 2 . Milcou, S., Milcou, I. and Nanu, L. I 9 6 3 . Le role de l a glande pineale dans le metabolisme glucides. Annales D' Endocrinologie. tome 24-, No. 2 . Mori, W. and Lerner, A.B. i 9 6 0 . A microscopic bioassay for melatonin. Endocrinology, No. 4-, 67s 4-4-3-4-50. Moszkowska, A. 1 9 6 3 . L1antagonisme epiphyso-hypophysaire. Annales D'Endocrinology, tome 24^ , No. 2 . Olsson, R. 1 9 5 8 . Studies on the Subcommissural organ. Acta Zool. (Stock.)., 3 9 - 7 1 - 1 0 2 . Ibid. 1 9 5 8 . The subcommissural organ. Stockholm. Ibid. 1 9 6 2 . Reissner's fibre apparatus i n i t s most primitive condition. Gen. Comp. Endocrinol, Vol. 2 , No. 6. - 7 2 -Palay, S,L, 19M-3, Neurosecretion, 5. JD« Comp. Neurology. 79:24-2. Ibid. 194-5. Neurosecretion, 7. J. Comp. Neurology. 82:128-14-3. Palkovits, M. and Foldvari, P.I. 1963. Effect of the subcommissural organ and the pineal glandon the adrenal cortex. Endocrinology, 72s 28, Pearse, A.G. I960. Histochemistry, theoretical and applied. L i t t l e Brown & Co. Pellegrino de I r a l d i , A. Zieher, L.M. and De Robertis, E. 1963. The 5-bydroxytryptamine and synthesis of normal and denervated pineal gland. Life Sciences, 9s 69I-696. Pflugfelder, 0. 1953. WIrkungen der epiphysectomie auf die post-embryonal entwicklung von Lebistes reticulatus. Roux* Arch. Entw. Mech. Org. I h b : 11^-136^ * Ibid., 1954. Wirkungen partieller zerstorungen der parietal region von Lebistes reticulatus. Peters' Arch. Entwick. Mech. Vol. 14-7, pp. 4-2-60., " Ibid. 1956. Physiologie der epiphyse. Verh.. dtsch. Zool. Ges., 53-75. Polyak, S. 1957.- Efferent or exogenous fibres of the retina. In "The vertebrate visual system", pp.. 250-251, The Univ. Chicago press. Quay, W..B. 1956. Volumetric and cytologic variations: in the pineal body of Peromyscus leucopus with regard to sex, captivity and day length. Jl. Morph., 98 s 4-71-4-95. Ibid. 1956. The demonstration of secretory material and cycle in the parenchyma of the mammalian pineal organ. Experimental Cell Research, 10s: 5^1-568, Quay, W.B. and Kahn, H. I963. Pineal histology and cytochemistry in organ culture. La Cellule, tome 63, fascicule 2, 24-7-257. Quay, W,B,, and Renzoni. A 61963. Comparative and experimental studies of pineal structure and cytology in passeriform birds,. Rivista, di Biologia, Vol. 56, fascicule 4, pp* 39^+28 „ Rasquin, P. 1958. Studies i n the control of pigment cel l s and light responses i n recent teleost fishes. I. morphology of the pineal organ. 2. reactions of the pigmentary system to hormonal stimulation. Bull.. Amer. Mus. Natl* Hist., 115t 1-68, - 73 -Rivas;, L.R. 1953. The pineal apparatus of tuna and related fishes as a possible light receptor controlling phototactic movements. Bull. Mar. Sci. Gulf & Carribean, 3sl68-l80. Romanes, G.J.. 1950. The staining of nerve fibres i n paraffin sections with si l v e r . J. Anat., 104—115. Roth, W.D. 1964-. Comments on Ariens Kappers review and observations on pineal activity. Amer Zool., 4-t 53°-57» Roth, W.D., Wurtman, R . JJ . and Altschule, MI.D. 1962. Morphologic changes in the pineal parenchymal cells of rats exposed to continuous light and darkness. Endocr. 71888-892. Scharrer, E. 1928. Die Llchtemfindlichkeit erlitzen (unter-suchungen); uber das zwischenhirn der fische. Z. verg. Physiol., 7, 1-38. Scharrer, E. and Scharrer. B. 194-5. Neurosecretion. Physiol. Rev., 25? 171-181. Scheer, B.T., Matsumoto, K,. and Shane, G. 1962. Abstract submitted to A.I.B.S. Mtgs. 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Thesis/Dissertation
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Zoology
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Histological and some histochemical studies on the pineal organ in sockeye salmon oncorhynchus nerka (Walbaum).
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