Structure and Alleged Functions of Avian Pineals. Department of Biology, University of Pittsburgh,, Pittsburgh, Pennsylvania 15213

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1 AM. ZOOLOCIST, 10: (1970). Structure and Alleged Functions of Avian Pineals CHARLES L. RALPH Department of Biology, University of Pittsburgh,, Pittsburgh, Pennsylvania SYNOPSIS. The pineal is structurally very diverse among the avian species that have been examined. In some birds, especially owls, the pineal is virtually absent. Sympathetic innervation is provided by projections from the superior cervical ganglia. Several cell types are present in the pineal body, among which are large cells, associated with lamellar bodies, that are commonly considered to be abortive or vestigial photoreceptors. Diurnal cycles of serotonin and melatonin content of the pineal are responsive to photoperiod, and there is a small amount of evidence that the avian pineal may serve as a pacemaker for diurnal rhythms of activity. Pinealectomy indicates a role for this body in controlling gonadal function, but the evidence is not consistent. Indole amines may be the mediators of this presumed endocrine role, but the supporting evidence is not very convincing. Literature relating to the avian pineal has been cited in reviews by Tilney and Warren (1919), Gladstone and Wakely (1940), and Kitay and Altschule (1954). I have attempted here to provide a current and somewhat comprehensive review of what is known about the pineal body of birds. 1 I hope this is a critical work, although space is inadequate to do a thorough evaluation. It is inevitable that birds will be compared with mammals when interpreting pineal structure and function, since so much more work has been done with the latter. However, for lack of space I have largely avoided drawing such parellels, and also have considered the possibility that such an approach could be misleading, because the pineal complex seems to have assumed different functions in different classes, and birds and mammals are not very closely related organisms. EMBRYOLOGY The development of the epiphysis cerebri of birds has been examined in only a The original work of the author cited herein was supported by N.S.F. Grant GB-5605 and N.I.H. Grant NB i If the reader is aware of a significant publication that is not included in the topics discussed, I would appreciate having it brought to my attention. Abstracts are cited only when they contain information not published in a more definitive form known to me. few species, the most studied being the domestic chicken (Gallus domesticus) (Chiodi, 1940; Spiroff, 1958). Detailed descriptions of development of the pineal also have been published for Phalacrocorax carbo (cormorant) (Charvat, 1954) and Larus canus (gull) (Wetzig, 1961). These, plus a few partial descriptions for other species, are not adequate for drawing sweeping conclusions about the embryology of bird epiphyses. Enough variation has been discovered, however, to suggest that this is a fertile field for study. The anlage of the pineal of birds forms from a thickening of the epithelium in the roof of the third ventricle. It is considered to be an epiphyseal structure, as it develops caudal to the velum transversum. Although definitively it appears to be a median, single structure, there is some evidence from early chick embryos that the pineal has a bilateral origin (Saint-Remy, 1897; Hill, 1900; Livini, 1904), and that the left epiphyseal evagination develops faster than the right (Cameron, 1903). (There also are indications of a bilateral origin of the pineal structures in other classes of vertebrates.) Others have failed to find any evidence for a dual origin in the chick and consider it a rare exception for more than a single primordium to be involved in forming the pineal (Spiroff, 1958). However, in several species of birds the primary pineal organ appears to be 217

2 218 CHARLES L. RALPH attached at its base on the right-hand side of the intercommissural region, and in a few species an accessory or secondary pineal parenchymal proliferation has been seen, usually anterior and to the left of the the primary pineal stalk (Quay and Renzoni, 1963, 1967). Evaginations rostral to the velum transversum have been reported in Larus (Klinckowstrom, 1892), and Gallus (Dexter, 1902). These would seem to correspond to the anlage of the paraphysis of other vertebrate classes. In the chicken, the paraphysis is reported to persist in the adult as a portion of the epithelial roof of the forebrain (Dexter, 1902). In the swan (Cygnus olor), Krabbe (1955) describes the development of a prolongation from the distal end of the epiphysis, which eventually becomes detached and forms a "parietal corpuscle", bearing a resemblance to the parietal eye of saurians. He also noted an eye-shaped enlargement that remains attached to the epiphyseal anlage in Melopsittacus undulatus (parakeet) embryos. However, Renzoni (1965a) failed to find in the parakeet, or in any other of the several species examined (Quay and Renzoni, 1967), any structure that could be interpreted as a rudimentary eye and suggests that Krabbe made an error in interpretation. The presence of pigment in or around the developing pineal is sometimes noted (Klinkowstrom, 1892; Studnicka, 1905; Wetzig, 1961), suggesting, perhaps, the potential for light absorption. The wall of the epiphyseal evagination at first consists of an undifferentiated ependymal layer which is continuous with the ependyma forming the roof of the diencephalon. In the 5-day chick embryo, the evagination has budded off from its central lumen several hollow cell clusters, lined by ependyma and separated from each other by a loose mesenchyme (Mihalkovicz, 1874; Tilney and Warren, 1919; Gladstone and Wakely, 1940; Moszkowska, 1958). By the sixteenth day of incubation there are numerous well-defined.follicles separated by connective tissue. The pineal body is now a rather compact organ surrounded by a capsule (Spiroff, 1958). Further morphological changes arc relatively minor. Spiroff (1958) has recorded the volume of the pineal body of the chicken, relative to its volume in a 5-day embryo, between five days of incubation and sexual maturity. Between five and 12 days of incubation the relative volume increases 4282%; between 12 days of incubation and five days post-hatching there is little change in volume; between five days and two months post-hatching the relative volume increases 272%; from two to four months posthatching there is almost no change in relative volume. Spiroff comments that several endocrine organs show rapid increases in size between 10 and 21 days of incubation, a period during which the pineal body is not noticeably growing. These results are partly at variance with those of Moszkowska (1956, 1958) who states that the growth of the embryonic chick pineal is relatively slow during the first 14 days of incubation, accelerates during the sixteenth day, and reaches a peak between days The latter period corresponds to the time during which the hypophysis grows most slowly, according to Moszkowska. She suggests that this is an indication of an epiphysis-hypophysis antagonism, and presents evidence from the in vitro culturing of these organs that such an antagonism exists (Moszkowska, 1956, 1963). Vidmar (1953) explanted the pineals from chick embryos and grew them in tissue culture. Epithelial buds developed follicles that differentiated into two cell types, somewhat like in vivo follicles. MORPHOLOGY The pineal body in birds lies between the cerebral hemispheres and the cerebellum and extends dorsally toward the skull. The overall shape and size vary greatly among species and taxonomic groups (Funkquist, 1912; Chiodi, 1940; Renzoni, 1965&; Quay and Renzoni, 1963, 1967). Furthermore, pineal size is not related to

3 AVIAN PINEAL 219 body size. To cite an instance, the pineals of the pelagic cormorant (Phalacrocorax pelagicus) and the willet (Caloptrophorus semipahnatus), birds of comparable size, are quite different in volume, the cormorant's being several times larger (Quay, 1965a). The largest avian pineal, in proportion to body size, has been found in the parakeet (Melopsitlacus undulatus) (Renzoni, 1965a). The largest pineal in terms of absolute size is probably that of the emu (Dromareus), which weighs, after fixation in formalin, 0.1 g (Cobb and Edinger, 1962). In some species the pineal is almost absent (Quay, 1968). Renzoni (1968a) has established that in five species of Strigiformes (owls) the pineal body is rudimentary, or more often, absent. This confirms the reports of Krabbe (1955) and Breucker (1967) that the pineal of Strix aluco, one of the owls examined by Renzoni, is a rudimentary structure. Most curiously, the pineal of a closely related species, Strix flammed, is reported to be very large (Studnicka, 1905). This possible discrepancy should be investigated further. Studnicka (1905) has classified avian pineals into three basic structural patterns: (1) saccular with thick walls, (2) follicular and tubular, or (3) solid (see Oksche and Vaupel-von Harnack, 1966). There are, however, intermediate and mixed structural types and some of the simplified diagrams in the literature used to support this classification should probably be suspect. In the house sparrow (Passer domesticus), for example, the pineal is figured in the earlier literature as a simple, saccular type, but as Quay (1965a) points out, the multiple foldings and tubular formations of the walls actually present a much more complex picture. Another complication is that the pineal in the post-embryonic animal may undergo considerable modification. In Passer domesticus the pineal of the young animal is larger and more follicular than in the older bird where it appears to be more solid 1969). Romieu and Jullien (Ralph and Lane, (1942c), Jullien (1942a, 1942b) and Spiroff (1958) have observed in Callus a progressive change from vesicular to compact condition occurring during development. Shellabarger (1953) notes that the pineal of White Leghorn males has a follicular arrangement in embryonic stages which is lost by approximately the ninetieth day post-hatching, the time at which sexual maturation is complete. However, Desogus (1926) claims that the pineal of Gallus shows an inactive compact state when the hypophysis is active and the bird is forming eggs and an active follicular condition when it is reproductively quiescent. Unfortunately, at present none of the variations between or within species can be implicated with certainty in any adaptive role or physiological function of the pineal. How much continuity is maintained between the canals and follicles of the pineal body and the third ventricle is an unsettled matter. In the gull (Lams canus), Wetzig (1961) suggests a permanent communication exists between the pineal and the ventricle. A similar situation prevails in the cormorant (Phalacrocorax carbo), according to Charvat (1954), where the pineal stalk is hollow and connected with the diencephalon. Romieu and Jullien (1942c) believe that for the first two or three weeks in Gallus the lumina of the follicles are in communication with the recessus pinealis of the ventricle and that secretory products are passed into the cerebrospinal fluid. Spiroff (1958) found the connection persisting as long as three months post-hatching in the same species. However, in other species the stalk appears solid with no evident channels between the pineal body and the ventricle (Studnicka, 1905). Quay and Renzoni (1967) argue that the frequent closure of the lumen of the pineal stalk, especially in higher groups of birds, suggests that this is not a pathway for release of secretion products. The rich vasculature of the pineal body is probably a more likely route for any secretory efflux.

4 220 CHARLES L. RALPH VASCULARIZAT1ON Definitive studies of vascularization of the pineal body remain to be done. Only for gallinaceous birds is there any substantial information. Spiroff (1958) notes that in the chick embryo the epiphyseal mass becomes surrounded by an intricate vascular network by the fifth day. Capillaries filled with blood cells are present among the follicles by the sixth day. By day 12, capillaries encircle the follicles, but never penetrate them. The pineal body of the adult pigeon (Columba livia) is supplied by the posterior meningeal artery, which usually arises from a posterior cerebral artery and then ascends along the length of the pineal body, enclosed in the leptomeninges encapsulating the latter (Baumel, 1962). In Gallus, according to Beattie and Glenny (1966), a similar situation prevails. The pineal is supplied by "the posterior meningeal artery, which arises as a branch of the cranial ramus of the left internal carotid.... It... ascends the anterolateral face of the pineal stalk where it ramifies al or near the base of the gland proper to form a circus vasculosus or the rele pincalis." What appeared to be a pair of efferent vessels was seen emerging from the antero-dorsal aspect of the jjineal body and then after curving along the posteromedial surface of the cerebral hemispheres, they joined the internal jugular vein. The passerine pineal has been described as highly vascularized, with large meningeal venous blood sinuses (Quay and Renzoni, 1963). There is little known about possible lymphatic vessels within or adjacent to the avian pineal. Lymphoid tissue is found in the pineal stroma of some species (Quay, 1965a). In Gallus, lymphoid tissue is found in or near the walls of superficial pineal blood vessels as early as four days posthatching and reaches a maximum at three months (Spiroff, 1958). By the sixth month, lymphatic nodules have generally disappeared from the pineal body, according to Romieu and Jullien (1942a) and Spiroff (1958), but Quay (1965a) found them in year-old chickens. Romieu and Jullien (1942a) hypothesize that the lymphocytes of the meninges and cerebrospinal fluid have their origin in the pineal lymphoid nodules, but there is no adequate evidence to support such a contention. INNERVAT1ON Although earlier reports denied the presence of nerve fibers in the pineal (see Studnicka, 1905), it is now commonly agreed that the avian pineal is a richly innervated structure. A brief but critical review of Uie small volume of literature on this subject has been provided by Kappers (1965). Tubahara (1955), Stammer (1961), and Quay and Renzoni (1963) describe thin, non-myelinated, branching fibers surrounding and penetrating follicles and coursing along blood vessels in a dense plexus. According to Stammer the great majority of the fibers arise from the cranial part of the sympathetic nervous system, and Quay and Renzoni state that "the parenchyma of the distal part of the pineal in some species receives an innervation anteriorly from a (sympathetic?) trunk accompanying a venous sinus." In chickens, jungle fowl (Gallus gallus), and Japanese quail (Coturnix colurnix japanica), Hedlund and Nalbandov (1969) have confirmed, by fluorescent histochemical techniques, that bilateral superior cervical ganglionectomy results in complete sympathetic denervation of the pineal. In the pineal stalk there are fewer and much thicker fibers. Stammer (1961) attributes their origin to the vagus or trigeminal nerves. Quay and Renzoni (1963), on the other hand, describe large neurons in the stalks of passeriform pineals which receive small fibers from smaller and more distal ones and send thick axons into a layer of nerve fibers which forms the beginning of the tractus pinealis. The fibers of this tract are described as extending down the pineal stalk in distinct bundles. In some species the fibers of the tractus

5 AVIAN PINEAL 221 pinealis can be followed into the anterior end of the posterior commissure, and in others they disappear into the habenular commissure (Quay and Renzoni, 1963). There is a bit of inconsistency in the literature on the matter of the presence or absence of neurons in the pineal. It has been stated that nerve cell bodies are absent in species of Ciconiiformes, Anseriformes, Falconiformes, Galliformes, and Charadriiformes (Stammer, 1961), and in Larus canns (Wetzig, 1961), but they are said to be present in Melopsiltacus undulatus (Renzoni, 1965a) and in species of Passeriformes (Quay and Renzoni, 1963). In the latter case, they are considered to be confined largely or exclusively to the stalk region. This matter warrants continued investigation. (See Kappers, 1965, p. 121, for skeptical comments). In the basal part of the pineal stalk in Passer domcsticus, Quay and Renzoni (1963, 1966) have observed what they consider to be a small neurosecretory nucleus and tract (commissuro-pineal). These were found especially in the vicinity of the habenular commissure. The belief that the cells are neurosecretory is mainly based on their staining characteristics and the authors concede that further studies will have to be done before anything conclusive about them can be said. Oksche (1961) describes aldehyde-fuchsin-stainable ependymal loops that extend into the pineal stalk. CYTOLOGY There is no standardized terminology for cell types. However, there are a few commonly used names, based largely on cellular geography as visualized by light microscopy. The large, columnar cells lining the pineal lumen (or lumina of follicles) are usually called ependymocytes (Studnicka, 1905), although they are structurally diverse. In the lumina of embryonic and adult pineals there frequently is seen fibrous, globular, or amorphous materials, and it has been suggested that they are secreted by the ependymocytes (Romieu and Jullien, 1942b; Wetzig, 1961; Stammer, 1961). Among and peripheral to the ependymocytes (at least in gallinaceous birds and in those with thick-walled tubules or follicles) are round, smaller cells, the hypendymocytes (Romieu and Jullien, 19426; Quay and Renzoni, 1963). A third type, sometimes characterized, are the pinealocytes, which have large nuclei and resemble neurons in having processes. They are at the borders of the follicles, according to Romieu and Jullien (1942b). However, what is seen in the way of cell types depends upon the species examined, its age (as some evidence of cell replacement exists), physiological state, and the staining technique used. Electron microscopy offers additional distinguishing features for differentiation of cell types, but again in the few studies reported there is little agreement on what is being visualized. Oksche (1968) describes in sparrow, pigeon, duck, and chicken, sensory-like cells with variable features. Some are elongate cells with clubshaped, mitochondria-laden apical ends protruding into the follicles. Others are described as associated with a complex of vacuoles and lamellar bodies extending into the lumen. In addition, there are smaller, supporting cells which sometimes bear microvilli and cilia on their apical surfaces. (See also Oksche and Vaupel-von Harnack, 1965«, 1966). Fujie (1968) describes for the chicken three types of cells: (1) pinealocytes; the most numerous parenchymal cell; flask or pear-shaped, with a large, spherical nucleus in the basal half; at the apical end a cilium projects into the lumen; in some cases myelin-like membranes are formed by superimposed or partially flattened cilia; (2) supporting cells inserted among the pinealocytes and extending microvilli into the lumen; (3) glial cells, small in size, uncommon, and containing many filaments. Quay, et al., (1968) designate two primary kinds of parenchymal cells in the pineal of the parakeet: (1) Type I, with cilia and laminated membranes and whorls

6 222 CHARLES L. RALPH extending into the lumen and abundant dense-cored vesicles; a possible sensory capacity is suggested for this cell; (2) Type If, with niicrovilli projecting into the lumen and abundant microfibrils; a secretory function for this cell is suggested. Bischoff (1969) characterizes three types of cells in the pineal of Japanese quail: (1) epenclymal cells; numerous cilia and a few microvilli projecting into the follicular lumen; (2) secretory cells lacking cilia and containing numerous membranelimited granules; frequently the same granules are seen in the lumen of the follicle; (3) photoreceptor cells with synaptic contacts along their basal border and a modified cilium at the apical end projecting into the lumen (in some cases as a membrane-bound, cytoplasmic extension containing a granular matrix, and in others as a concentric, membranous complex lying within the luminal space). Whether or not there actually are photoreceptor cells in the pineal of any bird is quite unsettled. With the light microscope some investigators have failed to find any evidence for them (Wetzig, 1961), whereas others believe they have seen what appear to be photoreceptors (Quay and Renzoni, 1963; Renzoni, 1965b). The electron microscope, as noted above, reveals cells witli concentric, irregular whorls of lamellae and modified ciliary structures which some investigators suggest might be photosensory (Quay, et al, 1968; Bischoff, 1969), while other consider them to be abortive or largely vestigial and doubt they are sensory (Oksche and Vaupel-von Harnack, 1965", 19656, 1966; Gonzalez-Gonzalez and Garcia-Hidalgo, 1966; Collin, 19660,6/, 1967, 1968; Oksche, 1968). These cells lack the orderly arrangement of lamellae that characterize the outer segments of the photoreceptor cells of amphibian and reptilian pineals. If the avian pineal is a photosensory organ, its receptor units are not a conventional kind. BIOCHEMISTRY The avian pineal contains biologicallyactive indole derivatives. Serotonin (5-hydroxytrypfamine) has been detected by fluorometry (Quay, 1966), by bioassay (Hedlund and Ralph, 1967), and by lluorescence histochemistry (Owman, 1964; Fuxe and Ljunggren, 1965). The amounts extractable vary among species; the pigeon is reported to have a maximum of about 250 ng in its pineal (Quay, 1966), whereas the Japanese quail's pineal has at most only about 1.4 ng (Hedlund and Ralph, 1967). 5-Hydroxyindole- 3-acetic acid has been detected by fluorometry in the pigeon's pineal (Quay, 1966). Melatonin, measured by bioassay (Ralph, el al, 1967), has been found in the pineals of four species of birds. An enzyme involved in synthesis of melatonin, hydroxyindolc-o-methyl transferase (HIOMT), is very active in the pineals of birds (Axelrod, et al, 1964; Quay, ; Lauber, et al., 1968; Axelrod and Lauber, 1968). HIOMT from Japanese quail lacks the high substrate specificity of the comparable enzyme from cow pineal (Axelrod and Lauber, 1968). Only one other tissue, in addition to the pineal, has been shown to have HIOMT activity, and that is the retina, but HIOMT is not found there in all avian species examined, and when present is in low activity (Quay, 19656). 5-Hydroxytryptophan decarboxylase, an enzyme which forms serotonin from 5-hydroxytryptophan, also has been detected in the pineal of quail (Snyder and Axelrod, 1964). EFFECTS OF LICHT ON PINEAL CHEMISTRY AND STRUCTURE The amount of indole amines present in the pineal varies diurnally. Serotonin is at a maximum near the beginning of the light phase, in a conventional 24-hour photoperiod, and at a minimum near the middle of the dark phase, in both the pigeon (Quay, 1966) and Japanese quail (Hedlund and Ralph, 1967). This cycle can be phase-shifted by altering the phasing of the imposed diurnal photoperiod

7 AVIAN PINEAL 223 (Quay, 1966; Hedlund and Ralph, 1968). A different and much lower-amplitude cycle of extractable 5-hydroxyindole-3-acetic acid has been described for the pigeon's pineal (Quay, 1966). In Japanese quail and three species of African weaver birds, pineal content of melatonin is highest in the dark phase of their photocycle and lowest in the light (Ralph, et al., 1967). Pineal HIOMT activity of young Japanese quail undergoing sexual maturation (but not older quail) varies diurnally, being more active in the dark phase and less in the light; this cycle appears to be sustained for three days in continuous light (Sayler and Wolfson, 1969). The phasing of HIOMT activity in the quail is consistent with its observed melatonin cycle (Ralph, et al., 1967). Strangely, unlike the response in quail, light enhances rather than diminishes HIOMT activity in chickens (Axelrod, et al., 1964; Winget, et al., 1967; Lauber, et al., 1968). It now becomes of great interest to determine whether the chicken has a melatonin cycle that is the inverse of the quail's. However, melatonin content may not be predicted by HIOMT activity of the pineal, and furthermore, melatonin content does not necessarily reflect synthesis or secretion rate of this indole. Chickens exposed to long light periods have larger pineals than those kept in darkness (Axelrod, et al., 1964; Winget, 1966; McFarland, et al., 1969). In contrast, prolonged illumination of the cluck is said to cause its pineal to diminish in weight, but the follicular cells are taller and have more mitochondria, although no data are given (Milcou and Postelnicou, 1964). In contrast to chickens kept in 14 hours of light per day, those in continuous darkness are reported to have substantially less parenchymal cell volume and reduced lipid content (McFarland, et al., 1969). In female (but not male) Passer domesticus subjected to increasing daily photoperiods, the parenchymal nuclei were slightly larger (Quay and Renzoni, 1963). Parenchymal cells of chickens reared in long photoperiods (20L:4D) are stated to be larger, contain more small granules, lysosomes, and lipid droplets; nerve fibers of these animals have fewer cored synaptic vesicles than in chickens on short photoperiods (4L:20D) (Fujie, 1968). Lane, et al., (1969) failed to find any evidence of morphological or cytological differences between pineals from Japanese quail reared under either 10 hours or 18 hours of light per day and Ralph and Lane (1969) did not observe any structural changes in the pineals of wild P. domesticus that could be correlated with changes of natural photoperiods. EFFECTS OF SUPERIOR CERVICAL GANCLIONEO TOMY Both McFarland, et al. (1968) and Sayler and Wolfson (19686) report that removing the superior cervical ganglia retards the rate of oviposition and the time of onset of oviposition in Japanese quail. Ganglionectomy did not affect testicular or cloacal gland activity in males, however, and the observed effects on the females are quite transitory. In assessing these results it should be kept in mind that ganglionectomy alters innervation of several organs in addition to the pineal, including eye structures, and that the effects on egg-laying may result from denervation of one or more of these. After denervation of the pineal by ganglionectomy, HIOMT activity in 51^-weekold chicks is almost twice as high in light as in darkness (like normal animals); blinding by enucleation also fails to diminish enzymatic activity (Lauber, et ah, 1968). This suggests that chicks might perceive light through non-retinal receptors. The thin skull does allow a considerable amount of light to penetrate to the brain, and the pineal itself is properly positioned to be illuminated by light impinging on the dorsum. However, sexually mature Japanese quail fail to sustain a pineal serotonin cycle following bilateral ganglionectomy (Hedlund and Ralph, 1968). This suggests that either young birds have responses to non-retinal illumi-

8 224 CHARLES L. RALPH nation that are not found in adults or the serotonin cycle and the melatonin cycle are not driven by light in an analogous manner. POSSIBLE ROLE IN EXTRA-RETINAL EFFECTS OF LIGHT Because enucleated ducks respond to illumination by accelerating sexual development, it has been proposed that part of the brain itself, may be sensitive to light (Benoit, 1964). There is no question that photoreception occurs in the epiphyseal organs of reptiles and amphibians, and therefore, the possibility that the avian pineal is also a photoreceptor is a very attractive idea, despite the lack of convincing support from structural studies. Morita (1966) attempted to record electrical responses to changes in illumination in the pineal body of adult pigeons (Columba livia) and failed to find any activity that could be interpreted as photoreception. Ralph and Dawson (1968) obtained similar negative results with Japanese quail (ranging in age from 7 days to 6 months) and sparrows {Passer domesticus) (about 30 to 120 days of age). All failed to show electrical activity related to changes of illumination. A unique approach to the possibility of photoreception by the pineal was employed by Oishi and Kato (1968). Radioluminous paint one which emits orange light and another which emits green light was painted on the skull overlying the pineal region in sexually-mature Japanese quail that were maintained in continuous light. Other quail were pinealectomized or sham-operated before the paint was applied. When transferred from continuous light to 8L:16D, intact quail, with luminous paint emitting orange light, maintained their testicular size for two weeks, whereas the testes of those painted with the green emitter had regressed. Thus, despite a short-day treatment, gonadal activity was maintained in the intact birds with the orange emitter. However, pinealectomized birds painted with either color regressed, suggesting that the pineal was mediating the effect of the supplemental illumination. These results are coherent with the finding of Woodard, et al. (1968) that in Japanese quail red light accelerates sexual maturity much more than green light. Nevertheless, Oishi and Kato's evidence that the pineal is the site of reception can be challenged on the following grounds. When the pineal is removed, thick scar tissue forms along the skin incision, transparency of the skull is reduced around the aperture for pineal removal, dense blood clots form, and the injured tissues become more opaque at and around the site of extirpation, all of which diminish penetration of light through the dorsal surface of the head. Thus, assuming the site of photoreception for extra-retinal stimulation of gonads is the diencephalon, as it seems to be from the results of Benoit (1964) and Lisk (personal communication), 2 then the effect of pinealectomy may result from opaquing a normal light path to the diencephalon, and not from the absence of pineal photoreception. That the bird's pineal is not a photoreceptor, has been indicated by the highly significant discoveries of Menaker. He has presented evidence that a function of the pineal in sparrows {Passer domeslicus) is to serve as a pacemaker for circadian locomotor rhythms (Gaston and Menaker, 1968). Perching patterns in normal sparrows remain rhythmic in continuous darkness, but pinealectomy abolishes this circadian activity. In contrast to its effect on free-running activity rhythms, pinealectomy does not abolish the entrainment response to light cycles. Furthermore, bilaterally enucleated sparrows show entrainment to 24-hour light cycles in a manner very similar to normal birds (Menaker, 1968a). Thus, neither the eyes nor the pineal are necessary for entrainment, but the pineal is essential for continuance of 2 Supplementary light delivered by optic fiber to the diencephalon was as effective as illumination of the retina in causing gonadal hypertrophy in Mexican house finches.

9 AVIAN PINEAL 225 the circadian rhythm in continuous darkness. To test the hypothesis that the extraretinal receptor is localized in the sparrow's brain, Menaker (19686) enucleated birds and then presented them with light cycles or such low intensity that they could not entrain to them. Feathers were then plucked from the top of the head; within a few days all birds became synchronized to the light cycle. India ink was then injected beneath the scalp to increase opaqueness of the featherless area; all birds resumed a circadian, free-running rhythm. When the ink deposit was removed, the birds once again became synchronized to the light cycle. The pineal was then removed from four of the birds; there was no effect on the entrainment by light of the activity rhythm. Menaker's finding that enucleated, pinealectomized birds entrain to light cycles, strongly suggests that the brain is photoreceptive. In this case, the opaquing which would have resulted from pinealectomy, did not interfere with their response to light cycles. This appears to be in conflict with the results of Oishi and Kato, and with my explanation of their results. However, Menaker tested the effects of extraretinal light on entrainment of rhythms, but Oishi and Kato looked for testicular maintenance. Perhaps more light penetration is required for the latter. 3 Also, it is probable that the intensity of the light source, and the amount received by the sparrows, was greater in Menaker's experiments than that from the radioluminescent paint on Oishi and Kato's quail. Further evidence that neither the eyes nor the pineal is necessary for entrainment of circadian rhythms comes from the study of Harrison and Becker (1969) on time of oviposition in chickens. Enucleated, 3. Note added in proof: Underwood and Menaker (Science, 167: ) have demonstrated recently this is not true in P. domesticus. At intensities down to 20 lux the testicular response of blind birds to stimulatory photoperiods was indistinguishable from the response of normal birds. An extra-retinal photoreceptor capable of mediating the gonadal response to photoperiodic stimuli is indicated. pinealectomized birds shifted time of laying in accord with shifted light-dark cycles. In this case, however, the pineal does not appear to be necessary even for maintaining a free-running circadian pattern of oviposilion. EFFECTS OF PINEALECTOMY The study commonly cited as the first to demonstrate that pinealectomy in birds causes gonadal hypertrophy is that of Foa (1912). The experiment, in which pinealectomy was attempted in 63 chicks, had 15 survivors, of which only three were male. Because females showed no effects of pinealectomy, it is from the data on the three males that conclusions are drawn. When sacrificed at about nine months of age their testes were heavier than those of control birds of the same age. (The data were published twice see reference; a photograph of the comb of the same animal is identified as bird no. 1 in the French version and as no. 2 in the Italian one). Another study by Foa (1914) employed seven pinealectomized chicks, out of 10 pinealectomies attempted. Five of these were females, and when sacrificed five to nine months later no differences could be found between operated and control birds. Differences were found in the two males (pinealectomized when 32 days old and sacrificed six and 10 months later). As in the previous study, the testes were heavier and the comb was larger than in unoperated controls. A third study by Foa (1928) (also published in 1929; see reference) involved two pinealectomized male chicks and one control. The weight of the testes was presumably greater in the pinealectomized birds than in the control, but there are discrepancies in the data reported in the two publications (as shown in his tables of data), and even within the French paper the weights of body and testis and the ages are reported differently (pp. 153 and 158), making it difficult to accept these results as valid.

10 226 CHARLES L. RALPH Izawa (1922, 1923«) attempted pinealectomy in 36 chickens, four to five weeks of age. Of the four animals surviving the operation, two males and one female had been successfully pinealectomized. The cocks grew more rapidly, began to crow earlier, and showed more rapid development of the comb and testes than controls. The female, in contrast to Foa's negative finding, likewise showed accelerated sexual development. The endocrine glands, other than the gonads, were normal. In a second study, Izawa (1923b) pinealectomized 80 chickens, 25 to 42 days of age. Eleven lived to the time of autopsy between 197 and 300 days. Gonads of pinealectomized males and females were heavier than those of controls, as was body weight and several other organs. Clemente (1923) pinealectomized 30 chicks, and the 21 surviving showed a retarded development at first, but then rapidly overtook and surpassed the controls. Some operated males exhibited precocious singing, large combs and wattles, greater aggression, and larger testes than controls, but since the report contains only descriptions of some of the animals and no numerical data it is of little value. Yokoh (1927) pinealectomized a number of young cocks (31 to 40 days old) and found that they had significantly more rapid body growth, larger testes, and more prominent combs than unoperated cocks. Patay and Chalard (1952), reporting briefly on a study then in progress, noted the precocious appearance of singing and sexual behavior in two young pinealectomized cocks, and the enlargement of the comb and testes and a heavier body weight in several others. One pinealectomized female had a larger comb and heavier ovary than its control. Shcllabarger and Breneman (1950) successfully pinealectomized 77 White Leghorn cocks at four days post-hatching. No significant differences were found between control and experimental birds in body weight, or weights of adrenal, thyroid, or pituitary glands at any age. Pinealectomized cocks had significantly smaller testicular weights at 19 days of age, but were not different from the controls at 28 and 40 days. Thereafter, the pinealectomized birds had larger testes than the controls up to 94 days of age when again there was no difference. Comb weights of pinealectomized chicks were significantly larger than those of controls only at 70 days of age. In a subsequent study, Shellabarger (1952) again observed the inhibitory effect of pinealectomy on the testes of chicks about 20 days old and concluded that the pineal is progonadotropic in young chicks. In a third report, Shellabarger (1953) presents further evidence that pinealectomized chicks had hypertrophied testes at 40, 54, and 60 days of age, suggesting that at these times the pineal is antigonadotropic. Also, the gonadotropic potency of the adenohypophysis was increased in pinealectomized chicks. Additional evidence that the pineal is progonadotropic in young chicks is contained in the abstract by Harrison (1968), reporting that pinealectomized chicks had depressed comb size at six and 10 weeks, and the age at first egg was delayed in pinealectomized pullets. On the other hand, pinealectomized birds had more advanced spermatogenesis than controls when in reduced light (6 hours/day). Other accounts of pinealectomy in chickens which follow, report either inhibition of sexual development following pinealectomy, suggesting that the pineal normally stimulates sexual maturation, or no significant effects. Christea (1912) obtained 12 survivors out of 30 attempted pinealectomies in young cocks. He found a marked retardation in the appearance of secondary sexual characters (comb, spurs, voice) and growth of feathers. In a brief report of Urechia and Grigoriu (1922) two cocks, out of a large number of attempted pinealectomies, survived for eight months (age at operation not given). They showed regression of secondary sexual characteristics for two months and then later regained them. At sacrifice they appeared not to be different from controls,

11 AVIAN PINEAL 227 except that the combs may have been slightly larger in the operated birds. The testicular weights were the same as controls, but the pituitary of the operated birds was at least three times larger than the normal gland. Badertscher (1924) extirpated the pineal from 101 newly hatched chicks but only 25 reached maturity. Pinealectomy was complete in five cocks and six hens; in 14 birds the pineal was partially removed. The results from the experiment were quite variable and the author concludes that there were no significant differences among the birds in body weight, testicular weight, or sexual behavior. Traina (1933) destroyed the pineal by electrocautery in young cocks and reported that one bird, pinealectomized when 83 days old and sacrificed 5i/ 2 months later, had a greater body weight and testicular weight and a more developed syrinx than the control. When he repeated the experiment (operation at 113 days and sacrifice 6 months later), three birds survived and the opposite result was obtained; namely, the operated animals had smaller testes; body weights and comb size and syrinx were about the same in operated and nonoperated birds. Reporting on a series of small experiments, Parhon and Cahana (1939a, >,c, 1940a, >,c) could find no significant effect of cauterizing the pineal o chickens and ducks on blood glucose, calcium, cholesterol, phosphorus, or liver glycogen or of injection of pineal extract on muscle glycogen or phosphorus. Mikami (1950) pinealectomized 42 newly hatched chicks and observed the development of endocrine glands between 45 and 255 days of age. No significant differences were found between operated and control birds in body weight, pituitary, thyroid, adrenal, or testicular size. However, no numerical data are given. Zadura, et al. (1969) performed pinealectomy in 18 one-month-old cockerels and examined the testes and adrenals of nine of them at 80 days and the remaining nine at seven months after the operation. Pinealectomized birds had smaller testes than normal controls and retarded spermatogenesis. No morphologic changes were noted in the adrenal cortex. There are five reports on pinealectomy in Japanese quail. Again the observed effects are inconsistent. Homma, et al. (1967) pinealectomized 1-week-old quail and then exposed them to different photoperiods. Under three types of light schedules-24 LL, 12L:12D, and 8L:16D-there was no discernible difference, as compared to controls, but under 14L:10D, pinealectomized females showed an increase in oviducal weight (p<0.05), but no significant difference in ovarian weights. Renzoni (1967a) found that pinealectomy of 15- and 16-day-old quail by thermocautery did not cause significant variation in body or testicular weight in males, or significant change in onset of ovulation or the number of ova produced in females. Sayler and Wolfson (1967) pinealectomized quail at 7-9 days after hatching. Males did not show any differences in body weight, testicular weight, or adenohypophyseal weight, but females autopsied at 43 and 47 days of age had, on the average, smaller ovaries than normal or shamoperated birds. Oviducts also were subnormal in pinealectomized birds (Sayler and Wolfson, 1968a). At the next sampling time, 55 days, pinealectomized and shamoperated females had identical ovarian weights. (Homma, et al., 1967, autopsied quail at 49 days of age that had been pinealectomized at four weeks and kept in an 8L:16D photoperiod; they found no significant difference in ovarian weights, but did note retarded oviducts in the operated birds, which they attribute to surgical stress and not to removal of the pineal.) Pinealectomized quail exposed to continuous light, according to Sayler and Wolfson (1968a), were delayed in onset of egglaying, and enucleated, pinealectomized quail exposed to 16L:8D had reduced ovarian weights (Sayler and Wolfson, 1968b). Arrington, et al. (1969) pinealectomized newly-hatched quail and reared them

12 228 CHARLES L. RALPH under non-stimulatory photoperiods (2L: 22D) from three weeks of age until sacrificed at four, five, six and seven weeks. Pinealectomy was ineffective in preventing the gonadal inhibition expected in exposure to short light periods. Another group was raised under similar conditions until eight weeks of age, and then exposed to stimulatory photoperiods (14L:10D) for four weeks, at which time all had become sexually mature. The rate of sexual maturation of pinealectomized birds was not significantly affected. They were then exposed to a non-stimulatory photoperiod (4L:20D). Pinealectomy was not effective in preventing the gonadal atrophy expected under such conditions. The experiments of Stalsberg (1965) are a unique and important contribution to this confused subject. He surgically removed the epiphysis from day White Leghorn chick embryos, of which 85 survived until autopsy at 18 and 63 days of age. Graded amounts of epiphyseal tissue were present when examined, ranging from 0.01% of normal volume to nearly normal size. However, no correlation could be made between the amount of pineal tissue present and any of the test parameters. There were no indications that the chicken's pineal is in any way related to survival, body size, adenohypophyseal weight, function of any endocrine gland, weight of testis or ovary, weight of comb, or sex ratio. These results make the significance of the curious growth patterns of embryonic pineals, as described above (Spiroff, 1958; Moszkowska, 1958) even more puzzling. Pinealectomy in Harris' sparrow (Zonotrichia querula) failed to prevent the gonadosuppressive effect of short daily photoperiods (Donham and Wilson, 1969). This is the only published report on pinealectomy in a seasonally breeding bird. Since pinealectomy is a traumatic operation in birds (mortalities of 50% or more are common), one cannot escape the suspicion that some of the effects reported are due to side effects of the surgery and not specifically to the loss of the pineal. It has been our experience in pinealectomy of quail that when the pineal is pulled out (a common method of extirpation), a large amount of choroid plexus also is removed. Therefore, the possibility of damage to the roof of the diencephalon must be taken into consideration. In fact, an anomaly has been described wherein 60% of pinealectomized chicks show as adults a spinal deformation, but different surgical methods revealed that lesions of the roof of the diencephalon were responsble for this condition and not the absence of the pineal (Thillard, 1966). EFFECTS OF FEEDING, IMPLANTATION, AND INJECTION OF PINEAL SUBSTANCES On the assumption that the pineal body is a secretory organ, a number of investigators have looked for effects of feeding, implanting, and injecting pineal preparations and pineal amines. McCord (1914), in a most interesting experiment, fed pineal bodies of cattle to young mammals and chickens. Included in the study were 16 chicks which were fed beef pineal powder (administered in pellet form with lactose as an adjuvant). Birds receiving pineal powder grew in size at a much faster rate than controls. Especially interesting is the finding that the pineals of young cattle (veal) greatly accelerated maturation, whereas pineals of older cattle did not. The precocious animals did not grow beyond normal size, they merely grew more rapidly. Kozelka (1933) did two series of implantation experiments. In the first, two male chicks received pineal implants from adult birds: one received 14 implants over 28 days and die other 25 over a period of 44 days. In the second series, two females and two males ("halfgrown birds") were implanted 18 to 36 times with pineals from young chicks from about the 40th to the 114th day post-hatching. The increase in body weight and growth of comb of the birds receiving implants differed in no significant way from those of their control brothers and sisters.

13 AVIAN PINEAL 229 Two young cocks given a pineal extract ("Epiphysan"), one orally and the other by injection, had retarded growth of comb and wattles, according to Trautmann (1937), and at autopsy three months after the treatment began, the testes were smaller than in the control. Parhon and Postelnicu (1945), in badly executed experiments, tested the effect on plumage development of feeding pineal powder (source not indicated) to recently hatched ducklings. At the end of the first month, of the four fed pineal powder, two appeared to have more advanced feather development than the controls. (Disappearance of the hens during the night and disease among the young truncated the experiment.) Injections of pineal extract into ducklings caused no noteworthy effects on plumage. Moszkowska (1947) tested the effects of the hen's pineal on a female guinea pig's sexual maturation and estrus cycle. The first vaginal opening of this animal, which had received under its skin every 2 or 3 days a pineal of a hen (2 to 3 years old) for 40 days, occurred at the same time as its control. The same guinea pig received additional implants of hens' pineals during two subsequent estrus cycles; there were no effects observed. On the contrary, pineals of chick embryos implanted at the time of the first vaginal opening caused, in three guinea pigs, permanent estrus (cited in Moszkowska, 1958; original source not given). White Leghorn male chicks, either pinealectomized when two days old or nonoperated, were injected by Shellabarger (1952) with a solution of beef pineal powder (5 mg per day from the fifth to the nineteenth day) and then autopsied on the twentieth day. The injections caused a small increase in testicular growth in nonoperated birds and prevented testicular atrophy in pinealectomized birds. Shellabarger (1953) injected intact chicks daily with a lyophilized beef pineal powder and found a depression of testicular weight (at 46 and 60 days). Injection of this preparation into pinealectomized males, autopsied 60 days later, prevented testicular hypertrophy. Pineals of gonadectomized males (capons) were slightly larger than those of intact males, and capons receiving injections of the preparation had slightly smaller pineal weights than non-injected capons. However, several other pineal preparations prepared by various methods and given on a similar schedule were without effect. Also, there was no effect on pineal weight or histology from injections of sex hormones, thyroxin, thiouracil, or from thyroidectomy. Renzoni (1967&) daily injected Japanese quail (males for 25 days and females for 75 days) with pineal extracts (animal source not given) and found no significant difference in testicular or body weights of males, or in time of onset of lay or number of eggs produced in females. Homma, et al. (1967) implanted pellets containing melatonin (1, 10, or 100 ^g) under the skin of 1-week-old Japanese quail. They were autopsied at four weeks of age. Those implanted with 1 ^g of melatonin showed retardation of gonadal and oviducal growth (p<0.05). However, 10 ^g pellets caused less inhibition and 100 ^g pellets were without effect. Ten laying quail injected for four days in succession with a large amount (1 mg) of melatonin laid, on the average, two hours earlier, but the number of ova was not affected. Renzoni (1968&) injected Japanese quail with melatonin and serotonin solutions and found that melatonin did not affect testicular, body, or pineal weights in males or influence the body weight, onset of ovulation, or number of eggs in females. Serotonin (30 and 500 jug daily from the fifteenth to the forty-fifth day), however, in one out of three experimental series, caused males to have lower body weights and females (given 500 ^g serotonin through the hundredth day) to have slightly lower body weight and to start lay a few days earlier. Sayler and Wolfson (1968a) gave adult, sexually-mature, male and female Japanese quail large doses of melatonin (1, 10, or 20 mg) subcutaneously in oil for 26

14 230 CHARLES L. RALPH days. There was no effect on body, gonadal, oviducal, or hypophyseal weights. Oviposition also was not influenced. Pinealectomized birds injected daily with 1 mg o nielatonin were not different in organ or body weights from controls. Melatonin (1 and 10 mg for 16 days) injected into juvenile quail had no effect on body weight or organ weight, except that the adenohypophyseal weights of melatonininjected birds under non-stimulatory light (8L:16D) were significantly higher than controls. Ishibashi, ct al. (1967) injected three varieties of chickens with estrogenic compounds and found that they tended to have increased pineal weight and cell size, while testicular weight was greatly depressed. An inverse relationship between gonad and pineal is suggested. The amounts of materials administered in some of these studies could be considered massive doses, physiologically speaking, but since nothing is known about the rate of uptake from depots or the half-life of circulating melatonin in birds, one cannot presently make a valid argument to support such an opinin. However, one can at least entertain the idea that in such amounts, nonspecific effects of the pineal substances could produce the observed "inhibitions" or "stimulations." Even pathological effects cannot be ruled out, because the pineal monoamines have very marked effects on the central nervous system. Injections of melatonin (0.03 to 0.06 mg/g) into young chicks induce sleep, usually lasting 30 to 60 minutes (Spooner and Winters, 1965; Hishikawa, et al., 1969). Serotonin injected intraperitoneally into newly-hatched chicks produces a syndrome characterized by ataxia, decreased muscle tone and motor activity, and stupor. In sufficiently high doses (0.1 mg/g) it causes convulsions and death (Hehman, et al, 1961). CONCLUSIONS The avian pineal, although examined in relatively few species, is remarkably varied in its morphology. In some owls it is virtually absent. During development the pineal shows curious spurts of growth and undergoes considerable cytological modifications as the bird matures; some of the changes seem to coincide with the beginning of sexual function, but others cannot be correleated with any physiological events. A common trend seems to be from a follicular, glandular appearance in the young to a more compact, inactiveappearing body in the mature animal. The effects of pinealectomy in gallinaceous birds suggest, overall, that the pineal in the very young bird may be progonadotropic and in the older bird (sexually immature and mature) it may be antigonadotropic, but the evidence is equivocal. Many of the effects of pinealectomy, which are commonly very slight and transitory, perhaps can be accounted for by the trauma of the operation or by brain injury. Certainly, birds tend to do very well in most cases without their pineal. The effects of administering pineal substances also are very mixed and marginal. Forty-seven years ago, a reviewer of pineal literature stated: "In the studies on the pineal gland, one has very often the impression that the authors' desire to consider the organ as incretory in function is greater than their critical judgement regarding published results" (Krabbe, 1923, Endocrinology, 7:379). Even now, the statement has considerable validity and better progress might be made in solving the riddle of pineal function if investigators would entertain the possibility that pineal secretions may not have specific targets, such as reproductive structures, but rather have broad actions or perhaps phase rhythms in the central nervous system. The pineal body is innervated by sympathetic projections from the superior cervical ganglia. The content of indole amines and HIOMT activity in the pineal vary diurnally and the phasing of their cycles is responsive to photoperiod. In the house sparrow the pineal may serve as a "biological clock", the timephasing of which can be "set" by a non-

15 AVIAN PINEAL 231 retinal photoreceptor. The avian pineal probably is not a photoreceptor, at least not in the conventional sense. It has occurred to this reviewer that the pineal in most birds appears to be in an appropriate position to serve as a "light funnel", conducting light from its expanded distal end just beneath the venous sinus and vault of the skull through its elongated stalk between the cerebrum and cerebellum to its insertion on the roof of the diencephalon. That is, photic information about the bird's environment (overhead illumination primarily) may be preferentially (although not exclusively) conducted by the relatively transparent pineal to the diencephalic receptors postulated by Benoit. This hypothesis, highly speculative though it is, may at least encourage re-interpretation of some of the data in an area that is replete with confusion, oversimplification, and biased conclusions. REFERENCES Arlington, L. C, R. K. Ringer, and J. H. Wolford Effect of pinealectomy of Coturnix quail reared under non-stimulatory photoperiods. Poultry Sci. 48: Axelrod, J., and J. K. Lauber Hydroxyindole- O-methyltransferase in several avian species. Biochetn. Pharmacol. 17: Axelrod, J., R. J. Wurtman, and C. M. Winget Melatonin synthesis in the hen pineal gland and its control by light. Nature 201:1134. Hadertscher, J. A Results following the extirpation of: the pineal gland in newly hatched chicks. Anat. Rec. 28: B.'iiimel, J. J Asymmetry o encephalic arteries in the pigeon (Columba livia). Anat. Anz. 111: llcattie, C. W., and F. H. Glenny Some aspects of the vascularization and chemical histology of the pineal gland in Galhis. Anat. Anz. 118: Benoit, J The role of the eye and of the hypothalamus in the photostimulation of gonads in the duck. Ann. N. Y. Acad. Sci. 117: Bischoff, M. B Photoreceptoral and secretory structures in the avian pineal organ. J. Ultrastructure Res. 28: Nreucker, H Vergleichende histologische Studien an der Zirbel der Vogel. Verh. Anat. Ges (Anat. Anz. Suppl. 120: ). Cameron, J On the origin of the epiphysis cerebri as a bilateral structure in the chick. 1'roc. Rojal Soc. Edinburgh 25: Charvat, Z Vyvoj epifysy u kormorana. Ceskosl. Morfol. 2: Chiodi, V Sviluppo, struttura e topografia deu'epifisi. Riv. Biol. 29: Christea, Genital organs and pineal gland. (In German). Rev. Spec. Med. (Rumania). (Cited by Krabbe, Endocrinology, 7:395). Clemente, G Contribute* allo studio della ghiandola pineale nell' uomo e in alcuni animali. Endocr. Patol. Cost. 2: Cobb, S., and T. Edinger The brain of the emu (Dromareus novaehollandiae, Lath) I. Gross anatomy of the brain and pineal body. Breviora (Cambridge) 170:1-18. Collin, J. P. 1966a. Contribution a l'etude des follicules de l'epiphyse embryonnaire d'oiseau. C. R. Acad. Sci., Paris, D, 262: Collin, J. P. 1966b. fitude preliminaire des photorecepteurs rudimentaires de l'epiphyse de Pica pica L. pendant la vie embryonnaire et postembryonnaire. C. R. Acad. Sci., Paris, D, 263: Collin, J. P. 1966c. Sur revolution des photor cepteurs rudimentaires epiphysaires chez la Pie (Pica pica L.). C. R. Soc. Biol. 160: Collin, J. P Le photorecepteur rudimentaire de l'epiphyse d'oiseau: le prolongement basal chez le Passereau Pica pica L. C. R. Acad. Sci. Paris, D, 265: Collin, J. P Rubans circonscrits par des vesicules dans les photorecepteurs rudimentaires epiphysaires de l'oiseau: Vanellus vanellus (L.) et nouvelles considerations phylogenetiques relatives aux pinealocytes (ou cellules principales) des Mammiferes. C. R. Acad. Sci., D, Paris. 267: Desogus, V Contributo allo studio della pineale e dell'ipofisi degli uccelli in stato di maternita. Monitore Zool. Ital. 37: Dexter, F The development of the paraphysis in the common fowl. Amer. J. Anat. 2: Donham, R. S., and F. E. Wilson Pinealectomy in Harris' sparrow. Auk 86: Foa, C Ipertrofia dei testicoli e della cresta dopo l'asportazione della glandola pineale nel gallo. Pathologica 4: (Also published as: Hypertrophie des testicules et de la Crete apres l'extirpation de la gland pineale chez le coq. Arch. Ital. Biol. 57: ) Foa, C Nouvelles recherches sur la fonction de la glande pineale. Arch. Ital. Biol. 61: Foa, C Nuovi esperimenti sulla fisiologia della ghiandole pineale. Arch. Sci. Biol. Bologna 12: (Abstracted as: Nuovi esperimenti sulla fisiologia della ghiandola pineale. Boll. Soc. Ital. Biol. Sper., 3: ; also published as: Nouvelles experiences sur la physiologie de la glande pineale. Arch. Ital. Biol. 81: ). Fujie, E Ultrastructure of the pineal body of the domestic chicken, with special reference to the changes induced by altered photoperiods.

16 232 CHARLES L. RALPH Arch. Histol. Jap. 29: Funkquist, L. H Zur Morphogenie und Histogencse des Pinealorgans bei den Vogeln und Saugetieren. Anat. Anz. 42: Fuxe, K., and L. Ljunggren Cellular localization of monoamines in the upper brain stem of the pigeon. J. Comp. Neurol. 125: Gaston, S., and M. Menaker Pineal function: The biological clock in the sparrow? Science 160: Gladstone, R. J., and C. P. G. Wakely The pineal gland. Williams and Wilkins, Baltimore. Gonzalez-Gonzalez, G., and F. Garcia-Hidalgo Ultraestructura de la glandula pineal des las aves. Trab. Inst. Cajal Invest. Biol. 58: Harrison, P. C Reproductive development of pinealectomized chicks. Poultry Sci. 47:1679. Harrison, P. C., and W. C. Becker Extraretinal photocontrol of oviposition in pinealectomized domestic fowl. Proc. Soc. Exp. Biol. Med. 132: Hedlund, L., and A. V. Nalbandov Innervation of the avian pineal body. Amer. Zoologist 9:1090. Hedlund, L., and C. L. Ralph Daily variation of pineal serotonin in Japanese quail and Sprague-Dawley rats. Amer. Zoologist 7:712. Hedlund, L., and C. L. Ralph Effect of photoperiod phase and ganglionectomy on pineal serotonin in Japanese quail. Amer. Zoologist 8:756. Hehman, K. N., A. R. Vonderahe, and J. J. Peters Effect o serotonin on behavior, electrical activity of the brain, and seizure threshhold of the newly hatched chick. Neurology 11: Hill, C Two epiphyses in four-day chick. Bull. Northwestern Univ. Med. Sch. 2: Hishikawa, Y., H. Cramer, and W. Kuhlo Natural and melatonin-induced sleep in young chickens A behavioral and electrographic study. Exp. Brain Res. 7: Homma, K., L. Z. McFarland, and W. O. Wilson Response of the reproductive organs of the Japanese quail to pinealectomy and melatonin injections. Poultry Sci. 46: Tshibashi, T., K. Sugihara, and Y. Shimoda Morphological changes of the pineal gland in estrogen-treated chickens. Med. Biol. 74: (In Japanese) Izawa, Y On the experimental removal of the pineal body in chickens. Trans. Soc. Pathol. jap. 12: Izawa, Y. 1923a. A contribution to the physiology of the pineal body. Amer. J. Med. Sci. 166: Izawa, Y. 1923b. Further experiments of removal of the pineal body in chickens. Trans. Soc. Pathol. Jap. 13: Jullien, G. 1942a. Sur l'origine et la formation des v sicules closes dans l'epiphyse des Gallinaces. C. R. Soc. Biol. 136: Jullien, G. 1942fc. Sur les formations ve'siculaires de la glande pineale des oiseaux. Bull. Mus. Hist. Natur. Marseille 2: Kappers, J. A Survey of the innervation of the epiphysis cerebri and the accessory pineal organs of vertebrates, p In J. A. Kappers and J. P. Schade, [ed.], Progress in brain research. Vol. 10. Elsevier, Amsterdam, The Netherlands. Kitay, J. I., and M. D. Altschule The pineal gland. Harvard Univ. Press. Klinckowstrom, A Untersuchungen iiber den Scheitelfleck bei Embryonen einiger Schwimmvogel. Zool. Jahrb., Abt. Anat. Ontog. 5: Kozelka, A. W Implantation of pineal glands in the Leghorn fowl. Proc. Soc. Exp. Biol. Med. 30: Krabbe, K. H Development of the pineal organ and a rudimentary parietal eye in some birds. J. Comp. Neurol. 103: Lane, K. B., C. L. Ralph, and S. Gilbert Lack of correlation between sexual maturation and pineal cytology in Japanese quail. Anat. Rec. 163:215. Lauber, J. E., J. E. Boyd, and J. Axelrod Enzymatic synthesis of melatonin in avian pineal body: Extraretinal response to light. Science 161: Livini, F Abbozzo dell'occhio parietale in embrioni di uccelli (Columba livia dom., Callus dom.). Monit. Zool. Ital. 16: McCord, C. P The pineal gland in relation to somatic, sexual and mental development. J. Amer. Med. Assoc. 63: McFarland, L. Z., K. Homma, and W. O. Wilson Superior cervical ganglionectomy in the Japanese quail. Experientia 24: McFarland, L. Z., W. O. Wilson, and C. M. Winget Response of the chicken pineal gland, blood and reproductive organs to darkness. Poultry Sci. 48: Menaker, M. 1968a. Extraretinal light perception in the sparrow, I. Entrainment of the biological clock. Proc. Nat. Arad. Sci. 59: Menaker, M. 1968b. Light perception by extraretinal receptors in the brain of the sparrow. Proc. 76th Annual Convention, APA, p Mihalkovicz, V Entwicklung der Zirbeldriise. Zentralbl. Med. Wiss. 16: Mikami, S The effect of pinealectomy on body growth and on the other endocrine organs in the domestic fowl. Nippon Juigaku Zasshi, Gap. J. Vet. Sci.) 12: (In Japanese). Milcou, S. M., and D. Postelnicou L'influence de l'illumination prolonged sur la structure de l'epiphyse chez canard. Rev. Roumaine Endocrinol. 1: Morita, Y Absence of electrical activity of the pigeon's pineal organ in response to light. Experientia 22:402. Moszkowska, A Difference d'activit entre l'^piphyse de quelques mammiferes et l'epiphyse de poule. Ann. Endocrinol. 8: Moszkowska, A L'antagonisme ^piphyso-

17 AVIAN PINEAL 233 hypophysaire. tude in vitro par la methode de E. Wolff. C. R. Acad. Sci. 243: Moszkowska, A L'antagonisme epiphysohypophysaire. fitude in vivo et in vitro chez l'embryon de poulet Sussex. Ann. Endocrinol. (Paris) 19: Moszkowska, A L'antagonisme epiphysohypophysaire. Ann. Endocrinol. (Paris), 24: Oishi, T., and M. Kato Pineal organ as a possible photoreceptor in photoperiodic testicular response in Japanese quail. Mem. Fac. Sci., Kyoto Univ., Ser. Biol. 2: Oksche, A The fine structure, neurosecretory, and glial structure of the median eminence in the white-crowned sparrow. Mem. Soc. Endocrinol. 12: Oksche, A Zur Frage extraretinaler Photorezeptoren im Pinealorgan der Vogel. Arch. Anat. Histol. Embryol. 51: Oksche, A., and M. Vaupel-von Harnack. 1965a. Vergleichende electronenmikroskopische Studien am Pinealorgan. Progr. Brain Res. 10: Oksche, A., and M. Vaupel-von Harnack Ober rudimentare Sinneszellstrukturen im Pinealorgan des Hiihnchens. Naturwiss. 52: Oksche, A., and M. Vaupel-von Harnack Electronenmikroskopische Untersuchungen zur Frage der Sinneszellen im Pinealorgan der Vogel. Z. Zellforsch. 69: Owman, C New aspects of the mammalian pineal gland. I. Functional significance of fetal pineal gland of rat. II. Monoamine stores in mammalian pineal gland. Acta Physiol. Scand. 63: Suppl. 240,40 p. Parhon, C. I., and M. G. Cahana. 1939a. Modifications biochemiques apres la cauterisation de l'epiphyse chez les oiseaux. Bull. Soc. Roumaine Endocrinol. 5: Parhon, C. I., and M. G. Cahana Sur la cholesterolemie des oiseaux ayant subit la cauterisation de l'epiphyse. Bull. Soc. Roumaine Endocrinol. 5: Parhon, C. I., and M. G. Cahana. 1939c. Recherches sur le glycogene musculaire apres l'injection d'extrait epiphysaire. Bull. Soc. Roumaine Endocrinol. 5: Parhon, C. I., and M. G. Cahana. 1940a. Recherches sur la hydration et le phosphore des muscles apres l'administration d'extrait epiphysaire. Bull. Soc. Roumaine Endocrinol. 6: Parhon, C. I., and M. G. Cahana Recherches sur le glycogene hepatique chez les oiseaux, apres la cauterisation de l'epiphyse. Bull. Soc. Roumaine Endocrinol. 6: Parhon, C. I., and M. G. Cahana. 1940c. Recherches sur le phosphore sanguin chez les oiseaux apres la cauterisation de l'epiphyse. Bull. Soc. Roumaine Endocrinol. 6: Parhon, C. I., and D. Postelnicu L'extrait epiphysaire exerce-t-il une influence inhibitrice sur le developpement <lu plumage definitif chez les oiseaux? Acta Endocrinol., Bucarest. 11: Patay, R., and A. Chalard A propos de l'epiphysectomie chez Gallus domesticus L. C. R. Acad. Sci. 235: Quay, W. B. 1965a. Histological structure and cytology of the pineal organ in birds and mammals, p In J. A. Kappers and J. P. Schade, [ed.], Structure and function of the epiphysis cerebri. Elsevier, Amsterdam. Progress in brain research, vol. 10. Quay, \V. B Retinal and pineal hydroxyindole-o-methyl transferasc activity in vertebrates. Life Sci. 4: Quay, W. B Rhythmic and light-inducetl changes in levels of pineal 5-hydroxyindoles in the pigeon (Columba livia). Gen. Comp. Endocrinol. 6: Quay, W. B Occurrences and biological significance of pineal atrophy among families of birds. Amer. Zoologist 8: Quay, W. B., and A. Renzoni Comparative and experimental studies of pineal structure and cytology in passeriform birds. Riv. Biol. 56: (In Italian and English) Quay, W. B., and A. Renzoni Studies on the "commissuro-pineal neurosecretory cells" of birds. Riv. Biol. 59: (In Italian and English) Quay, W. B., and A. Renzoni The diencephalic relations and variably bipartite structure of the avian pineal complex. Riv. Biol. 60:9-75. (In Italian and English) Quay, W. B., A. Renzoni, and R. M. Eakin Pineal ultrastructure in Melopsittacus undulatus with particular regard to cell types and functions. Riv. Biol. 61: (In Italian and English) Ralph, C. L., and D. C. Dawson Failure oc the pineal body of two species of birds (Cohirnix coturnix japonica and Passer domesticus) to show electrical responses to illumination. Experientia 24:147. Ralph, C. L., L. Hedlund, and W. A. Murphy Diurnal cycles of melatonin in bird pineal bodies. Comp. Biochem. Physiol. 22: Ralph, C. L., and K. B. Lane Morphology of the pineal body of wild house sp: rrows (Passer domeslicus) in relation to leprodiu tion and age. Can. J. Zool. 47: Renzoni, A. 1965a. L'epifisi nel Melopsittacus undulatus. Riv. Biol. 48: (In Italian and English) Renzoni, A Ancora sull'epifisi degli uccelli. Boll. Zool. 32: Renzoni, A. 1967a. La fisiologia dell'epifisi negli uccelli. I. Pinealectomia in Coturnix coturnix japonica. Soc. Ital. Biol. Sper. 43: Renzoni, A La fisiologia dell'epifisi negli uccelli. II. L'azione di estratti epifisari sullo sviluppo sessuale di Coturnix cohirnix japonica. Soc. Ital. Biol. Sper. 43: Renzoni, A. 1968a. Osserva/ioni comparative sull'epifisi degli Strigiformi ed Ordini affini. Arch. Ital.

18 234 CHARLES L. RALPH Anat. Embriol. 73: Renzoni, A. 1968i>. La fisiologia dell'epifisi negli uccelli. III. L'azione della melatonina sullo sviluppo sessuale di Coturnix coturnix japonica. Soc. leal. Biol. Sper. 44: Roinieu, M., and C. Jullien. 1942«. Sur l'existence d'unc formation lymphoidc dans l'cpiphyse des Callinaccs. C. R. Soc. Biol. 136: Romicu, M., and G. Jullien, Caracteres his- K)logi( iics ct hislopliysiologit ucs des vcmcules cpiphysaires des Gallinaccs. C. R. Soc. Biol. 136: Romieu, M., and G. Jullien. 1942c. Involution et valcur rnorpliologi( iie des vesicules closes de la glande pineale des Oiscaux. C. R. Soc. Biol. 136: Saint-Remy, G Notes I. fibauches epipliysaires et paraphysaires paires che/. mi embryon de poulet monstrueux. Bibliogr. Anat. 5: Sayler, A., and A. Wolf son Avian pineal gland: progonadotropic response in the Japanese quail. Science 158: Sayler, A., and A. Wolfson. 1968a. Influence of the pineal gland on gonadal maturation in the Japanese quail. Endocrinol. 83: Sayler, A., and A. Wolfson. 1968/;. Role of the eyes and superior cervical ganglia on the effects of light on the pineal and gonads of the Japanese quail. Arch. Anat. Histol. Embryol. 51: Sayler, A., and A. Wolfson Hydroxyindole-Omethyl transferase (HIOMT) activity in the Japanese quail in relation to sexual maturation and light. Neuroendocrinology 5: Shellabarger, C. J Pinealectomy vs. pineal injection in young cockerel. Endocrinology 51: Shellabarger, C. J Observations of the pineal in the While Leghorn capon and cockerel. Poultry Sci. 32: Shellabarger, C. J., and W. R. Breneman The effects of pinealectomy on young White Leghorn cockerels. Indiana Acad. Sci. 59: Snyder, S. H., and J. Axelrod A sensitive assay for 5-hydroxytryptophan decarboxylase. Biochem. Pharmacol. 13: Spiroff, B. E. N Embryonic and post-hatching development of the pineal body of the domestic fowl. Amer. J. Anat. 103: Spooner, C. E., and W. D. Winters Evidence for a direct action of monoamines on the chick central nervous system. Experientia 21: Stalsberg, H Effects of extirpation of the epiphysis cerebri in 6-day chick embryos. Acta Endocrinol., Suppl. 97, 119 p. Stammer, A Untersuchungen iiber die Struktur und die Innvervation der Epiphyse bei Vogeln. Acta Biol. (Szeged) 7: Studnika, F. K Die l'arietalorgane. Avcs. In, A. Oppel, Lehrbuch der vergleichenden mikroskopischen Anatomie der Wirbeltiere, vol. 5. Fischer, Jena. Thillard, M. J Lesions diencephaliques provatrices de scolioses chez le poulet. Bull. Assoc. Anat. 51: Tilney, F., and L. F. Warren The morphology and evolutionary significance of the pineal body. Amer. Anat. Mem., Wistar Institute of Anatomy and Biology, Philadelphia. Traina, S Influenza della pineale sulla laringc e sulla siringe. Ann. Laringol. Otol. 33: Trautmann, A Die Wirkung der Zirbeldiiisenpraparates "Epiphysan" auf die germinalen und akzidenlellen Geschlechtsmerkmale juveniler Tiere. Deut. Tieriirztl. Wochenschr. 45: Tubahara, M Ueber die Innervation der Epiphyse. Kurume Med. J. 2: Urechia, C. E., and C. Grigoriu L'extirpation de la glande pineale et son influence sur l'hypophyse. C. R. Soc. Biol. 87: Vidmar, B The development in vitro of the embryonic pineal body of the fowl. J. Embryol. Exp. Morphol. 1: Wetzig, H Die Entwicklung der Organe des Zwischenhirndaches (Epiphyse und Plexus choroideus anterior) bei der Sturmmowe Larus canus L. Gegenbaur's Morphol. Jahrb. 101: Winget, C. M Morphological and biochemical changes associated with a change in photoperiod (Galliis domesticus). Amer. Zoologist 6:506. Winget, C. M., C. A. Warren, and C. W. DeRoshia Interrelationships of the pineal gland, the diencephalon and the pituitary (Gallus domesliens). Amer. Zoologist 7:732. Woodard, A. E., J. A. Moore, and W. O. Wilson Effect of wavelength of light on growth and reproduction in Japanese quail (Coturnix coturnix japonica). Poultry Sci. 47: Yokoh, A Experimentale Untersuchungen iiber die Doppelexstirpation der Epiphyse und der Diemdriise. Z. Gesamte Exp. Med. 55: Zadura, J., J. Roxzkowski, and S. Cakala Effect of pinealectomy on the weight and histological changes in the testes and adrenals of cockerels. Acta Physiol. Pol. 20: [Also in Polish; Acta Physiol. Pol. 20: ] Note added in proof: The following publications have appeared subsequent to the presentation of this review: Collin, J. P Distinction et rapports entre les pedicules basaux des photorccepteurs rudimentaires secretaires et les afferences nerveuses monoaminergiques de l'epiphyse d'oiseau. Recherches chez le poussin de Passereau (Pica pica L.). C. R. Soc. Biol. 163: Donham, R. S., and F. E. Wilson Photorefractoriness in pinealectomized Harris' sparrows. Condor 72: Hamner, W. M., and R. J. Barfield Ineffectiveness of pineal lesions on the testis cycle of a finch. Condor 72:

19 AVIAN PINEAL 235 Kannankeril, J. V Effect of pinealectomy on the hypertrophy of the rudimentary right gonad following sinistral oxariectomy in the Japanese quail (Coturnix coluniix japonica). Anat. Rec. 166:328. Munns, T. W Effect of different photoperiods on melatonin synthesis in the pineal gland of the canary (Serbius canarius) and testicular activity. Anat. Rec. 166:352. Oksche, A., Y. Morita, and M. Vaupel-von Hainack Znr Feinstruktur und Funktion des Pinealorgans der Taube (Columba livia). Z. Zellforsch. 102:1-30. Oksche, A., and H. Kirschstein Electronmikroskopische Untcrsuchungen am Pinealorgan von Passer domeuicus. Z. Zellforsch. 102: