Cell Tiss. Res. 182, 139-143 (1977)
Cell and Tissue Research 9 by Springer-Verlag 1977
Short Communication
Photoreceptor-like Outer Segments in the Pineal Organ of the Lovebird, Uroloncha domestica (Ayes: Passeriformes) A Scanning Electron Microscopic Study * ** Manfred Ueck, Reiko Ohnishi, and Kenjiro Wake Department of Anatomy, Osaka City University Medical School, Abenoku, Osaka, Japan
Summary. In the pineal organ of the lovebird, Uroloncha domestica, bulbous, cup-shaped and elongated outer segments of photoreceptor-like pinealocytes are demonstrated by scanning electron microscopy. These scarce outer segments, 4-11 Ixm in length, extend into the pineal lumen. The present structural observations speak in favor of photosensitive pinealocytes in the pineal organ of Uroloncha domestica. The relation of the photoreceptor-like pinealocytes to acetylcholinesterase-positive nerve cells and a nervous connection between the pineal and the brain indicate that the pineal organ of this passeriform species may be the site of neuroendocrine and photoreceptive functions. Key words: Pineal organ - Uroloncha domestica (Aves, Passeriformes) Photoreceptor-like cells - Scanning electron microscopy. Introduction The pinealocytes of lower vertebrates display a sensory (for literature, see Dodt et al., 1971) and a secretory function (for literature, see Ueck, 1974). One of the morphological characteristics of sensory pinealocytes is the presence of cone-like outer segments with regularly arranged membrane disks. These cone-like outer segments are absent from the pinealocytes of mammals. Sensory-like secreting pinealocytes with reduced outer segments were shown in the pineal organs of lizards (cf. Oksche and Kirschstein, 1968; Collin, 1969) and birds (cf. Collin, 1969; Ueck, 1972; Menaker and Oksche, 1974). Two different types of outer segments were observed in avian pineals by use of the transmission electron microscope: (1) a membrane whorl originating from a modified cilium (Oksche and Send offprint requests to: Prof. M. Ueck, Zentrum fiJr Anatomic und Cytobiologie, Aulweg 123, D-6300 Giessen, Federal Republic of Germany, or Prof. K. Wake, Department of Anatomy, Faculty of Medicine, Tokyo Medical and Dental University, Yushima, Bunkyoku, Tokyo, 113 Japan
* Supported by a fellowship from the Japan Society for the Promotion of Science to M. Ueck ** Supported by a grant from the Ministry of Education of Japan to K. Wake and by a grant of the Deutsche Forschungsgemeinschaft to M. Ueck
140
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Kirschstein, 1969), and (2) a bulbous cilium with homogeneous content (Ueck, 1969). The membrane whorls are variable in shape and display an irregular arrangement of their lamellae. Therefore, it is difficult to reconstruct the shape of these complexes from transmission electron micrographs. It has not yet been shown with certainty whether these irregular membrane whorls possess a photoreceptive function. Ueck (1973), using a modified osmification according to Eakin and Brandenburger (1970), demonstrated particles 50A in diameter on the whorl membranes which is in agreement with the observations of Eakin and Brandenburger (1970), who had shown similar particles on the outer segment membrane disks in the retina of the lateral eye. It has therefore been suggested that the osmiophilic particles are somehow correlated to a photopigment. In passeriform birds Ueck and Kobayashi (1972) observed acetylcholinesterase-positive nerve cells in the pineal parenchyma and a tract running through the pineal stalk toward the brain. From these findings they concluded that the pineal organ of passeriform birds displays, in addition to a well developed secretory function, also a sensory function. Ralph and Dawson (1967) in Passer domesticus and Morita (1966) in the pigeon failed to record direct light-dependent impulses from the pineal stalk. Herbut6 and Bayl6 (1974) in the pineal gland of the Japanese quail, however, observed not only spontaneous firing but also responses to photic stimulation. Therefore, there is some indication that the pineal organ of some avian species may have a sensory function in addition to its secretory activity. In the present study using the scanning electron microscope different types of outer segment structures were demonstrated in the pineal organ of Uroloneha domestica.
Material and Methods Four lovebirds, Uroloncha domestica (Aves: Passeriformes), were decapitated; their pineal organs were isolated and fixed in 5 ~ glutaraldehyde in 0.1 M phosphate buffer, pH 7.4, for 90min at 4~ After washing in 0.1 M phosphate buffer containing 0.34 M sucrose for 4 h, the pineals were transferred to a 2~o solution of tannic acid for one day at 4~ followed by washing in 0.22 M phosphate buffer for 2h. The material was postfixed in 1 ~ OsO 4 at 4~ for 4h, then dehydrated. After fracturing in liquid nitrogen and critical point drying, the pineals were coated with gold and observed in a Jeol JSM - 50 A scanning electron microscope.
Results and Discussion
In comparison with the scanning electron microscopic observations on the pineal organ of the teleost fish, Salmo gairdneri (Hartwig and Pfautsch, 1973) and the lamprey, Lampetra japonica (Morita, 1975), only very few outer segments were found to occur in the pineal of the bird, Uroloncha domestica. Scattered outer segments of varying form and size extend beyond the surface of the pineal wall into the pineal lumen. Figure 1 a shows a bulbous outer segment, 4.2 Ixm in length and 2.01xm in diameter. In Figure 1b a structural complex formed by an inner and a corresponding outer segment of a pinealocyte is seen within the pineal lumen. The inner segment is 3.6 ~tm long and 3.3 ~tm thick. The cup-shaped outer segment displays a longitudinal axis of 3.6 ~tm, a transversal axis of approximately 2.0 Ixm
Outer Segments in the Pineal Organ of the Lovebird
141
Fig. 1 a - d . Different types of outer segments in the pineal organ of Uroloncha domestica, a Bulbous outer segment, x 15,300. b Cup-shaped outer segment; i invagination of cilium into the outer segment; t thickening o f the border of the outer segment; m microvillus-like evagination; is inner segment; x 11,700. e Elongated outer segment showing a bifurcation (bl; b2), a raphe (r) and a finger-like process (p); the distal part of the outer segment is bent down toward the base. x 12,600. d The same outer segment as in e after rotation and tilting of the specimen, x 15,300
142
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and a thickness of only 0.3 ~tm. The connecting cilium of both penetrates into a pit located in the outer segment. This invagination is eccentric, and a process resembling a microvillus is seen near the invagination. The border of the outer segment shows a thickening on one side. Such cup-shaped outer segments are not a characteristic feature of lateral eye photoreceptors; however, they occur, in addition to regularly formed outer segments, in the pineal organ of Amphibia (Ueck et al., unpublished results). A comparison of transmission and scanning electron micrographs shows that the m e m b r a n e whorls may result from sections through cup-shaped outer segments. Figures 1 c and d show two different images of one outer segment; the electron micrograph in Figure 1 d was taken after rotation and tilting of the specimen. This outer segment differs in size and shape from the outer segments in Figures 1 a and b. It is an elongated structure, 11 Ixm in length, thinner proximally than distally. This extension is not erect; its tip folds back towards the base of the outer segment. A raphe is seen in the middle of the proximal segment and on the lateral aspect of the distal part. The bifurcated curved portion of the outer segment encompasses a finger-like structure bearing a raphe (Fig. 1 c, d). The authors suggest that the demonstration of intact outer segments in the pineal organ of Uroloncha domestica proves morphologically the sensory capacity of the avian pineal organ. Using the acetylcholinesterase method positively reacting nerve cells and nerve fibers, afferent to the brain, could be shown in passeriform birds (Ueck and Kobayashi, 1972); these acetylcholinesterase-positive nervous structures also occur in Uroloncha domestica (Ueck and Wake, unpublished results). However, the number of fully differentiated outer segments per square unit is very low in comparison to the pineal organs of lower vertebrates. The extremely variable and complex ultrastructure of the outer segments of avian pinealocytes makes it difficult to reconstruct them on the basis of transmission electron micrographs. A comparative study of the outer segments in the pineal organs of fish, amphibians and birds is in progress in order to supply more detailed information on the three-dimensional structure of these photoreceptive elements. Acknowledgement. The authors wish to express their gratitude to Miss R. Liesner for technical assistance and to Miss I. Lyncker for secretarial aid.
References
Collin, J.-P,: Contribution a l'6tude de l'organe pin6al. De l'6piphyse sensorielle a la glande pin6ale: modalit6s de transformation et implications fonctionelles, Ann. Stat. Biol. Besse-en Chandesse, Suppl. 1, 1-359 (1969) Dodt, E., Ueck, M., Oksche, A.: Relation of structure and function. The pineal organ of lower vertebrates. J.E. Purkyn6 Centenary Symposium, Prag, 1969 (V. Kruta, ed.), pp. 253-278. Brno: Universita Jana EvangelistyPurkyn~ 1971 Eakin, R.M., Brandenburger, J.L.: Osmic staining of amphibian and gastropod photoreceptors. J. Ultrastruct. Res. 30, 619-641 (1970) Hartwig, H.-G., Pfautsch, M.: RasterelektronenmikroskopischeBeobachtungen an pinealen Sinneszellen der Forelle, Salmo gairdneri (Teleostei). Z. Zellforsch. 138, 585-589 (1973) Herbut6, S., Bayl6, J.D.: Multiple-unit activity in the pineal gland of the Japanese quail: spontaneous firing and responses to photic stimulations. Neuroendocrinol. 16, 52-64 (1974)
Outer Segments in the Pineal Organ of the Lovebird
143
Menaker, M., Oksche, A.: The avian pineal organ. In: D.S. Farrier and J.R. King (eds.), Avian biology, Vol. 4, pp. 79-118. New York-London: Academic Press 1974 Morita, Y. : Absence of electrical activity of the pigeon's pineal organ in response to light. Experientia (Basel) 22, 402 (1966) Morita, Y.: Direct photosensory activity of the pineal. Brain-Endocrine Interaction II. The Ventricular System. 2nd Int. Symp., Shizuoka 1974, pp. 376-387. Basel: Karger 1975 Oksche, A., Kirschstein, H.: Unterschiedlicher elektronenmikroskopischer Feinbau der Sinneszellen im Parietalauge und im Pinealorgan (Epiphysis cerebri) der Lacertilier. Ein Beitrag zum Epiphysenproblem. Z. Zellforsch. 87, 159-192 (1968) Oksche, A., Kirschstein, H.: Elektronenmikroskopische Untersuchungen am Pinealorgan von Passer domesticus. Z. Zellforsch. 102, 214-241 (1969) Ralph, C.L., Dawson, D.C.: Failure of the pineal body of two species of birds (Coturnix coturnix japonica and Passer domesticus) to show electrical responses to illumination. Experientia (Basel) 24, 147-148 (1968) Ueck, M.: Zur Ultrastruktur der Epiphysis der V6gel. Zool. Anz., Suppl. 33, Verh. Zool. Ges. 509-518 (1969) Ueck, M. : Sensorische und sekretorische Strukturelemente des Pinealorgans und ihre funktionelle Bedeutung. Habilitationsschrift, Fachbereich Medizin. Giessen 1972 Ueck, M.: Fluoreszenz- und elektronenmikroskopische Untersuchungen an der Epiphysis cerebri verschiedener Vogelarten. Z. Zellforsch. 137, 37-62 (1973) Ueck, M.: Vergleichende Betrachtungen zur neuroendokrinen Aktivit/it des Pinealorgans. Fortschritte der Zoologic 22, Heft 2/3. Stuttgart: G. Fischer 1974 Ueck, M., Kobayashi, H. : Vergleichende Untersuchungen fiber Acetylcholinesterase-haltige Neurone im Pinealorgan der V6gel. Z. Zellforsch. 129, 140-160 (1972)
Accepted April 19, 1977
Cell and Tissue Research 9 by Springer-Verlag 1977
Short Communication
Photoreceptor-like Outer Segments in the Pineal Organ of the Lovebird, Uroloncha domestica (Ayes: Passeriformes) A Scanning Electron Microscopic Study * ** Manfred Ueck, Reiko Ohnishi, and Kenjiro Wake Department of Anatomy, Osaka City University Medical School, Abenoku, Osaka, Japan
Summary. In the pineal organ of the lovebird, Uroloncha domestica, bulbous, cup-shaped and elongated outer segments of photoreceptor-like pinealocytes are demonstrated by scanning electron microscopy. These scarce outer segments, 4-11 Ixm in length, extend into the pineal lumen. The present structural observations speak in favor of photosensitive pinealocytes in the pineal organ of Uroloncha domestica. The relation of the photoreceptor-like pinealocytes to acetylcholinesterase-positive nerve cells and a nervous connection between the pineal and the brain indicate that the pineal organ of this passeriform species may be the site of neuroendocrine and photoreceptive functions. Key words: Pineal organ - Uroloncha domestica (Aves, Passeriformes) Photoreceptor-like cells - Scanning electron microscopy. Introduction The pinealocytes of lower vertebrates display a sensory (for literature, see Dodt et al., 1971) and a secretory function (for literature, see Ueck, 1974). One of the morphological characteristics of sensory pinealocytes is the presence of cone-like outer segments with regularly arranged membrane disks. These cone-like outer segments are absent from the pinealocytes of mammals. Sensory-like secreting pinealocytes with reduced outer segments were shown in the pineal organs of lizards (cf. Oksche and Kirschstein, 1968; Collin, 1969) and birds (cf. Collin, 1969; Ueck, 1972; Menaker and Oksche, 1974). Two different types of outer segments were observed in avian pineals by use of the transmission electron microscope: (1) a membrane whorl originating from a modified cilium (Oksche and Send offprint requests to: Prof. M. Ueck, Zentrum fiJr Anatomic und Cytobiologie, Aulweg 123, D-6300 Giessen, Federal Republic of Germany, or Prof. K. Wake, Department of Anatomy, Faculty of Medicine, Tokyo Medical and Dental University, Yushima, Bunkyoku, Tokyo, 113 Japan
* Supported by a fellowship from the Japan Society for the Promotion of Science to M. Ueck ** Supported by a grant from the Ministry of Education of Japan to K. Wake and by a grant of the Deutsche Forschungsgemeinschaft to M. Ueck
140
M. Ueck et al.
Kirschstein, 1969), and (2) a bulbous cilium with homogeneous content (Ueck, 1969). The membrane whorls are variable in shape and display an irregular arrangement of their lamellae. Therefore, it is difficult to reconstruct the shape of these complexes from transmission electron micrographs. It has not yet been shown with certainty whether these irregular membrane whorls possess a photoreceptive function. Ueck (1973), using a modified osmification according to Eakin and Brandenburger (1970), demonstrated particles 50A in diameter on the whorl membranes which is in agreement with the observations of Eakin and Brandenburger (1970), who had shown similar particles on the outer segment membrane disks in the retina of the lateral eye. It has therefore been suggested that the osmiophilic particles are somehow correlated to a photopigment. In passeriform birds Ueck and Kobayashi (1972) observed acetylcholinesterase-positive nerve cells in the pineal parenchyma and a tract running through the pineal stalk toward the brain. From these findings they concluded that the pineal organ of passeriform birds displays, in addition to a well developed secretory function, also a sensory function. Ralph and Dawson (1967) in Passer domesticus and Morita (1966) in the pigeon failed to record direct light-dependent impulses from the pineal stalk. Herbut6 and Bayl6 (1974) in the pineal gland of the Japanese quail, however, observed not only spontaneous firing but also responses to photic stimulation. Therefore, there is some indication that the pineal organ of some avian species may have a sensory function in addition to its secretory activity. In the present study using the scanning electron microscope different types of outer segment structures were demonstrated in the pineal organ of Uroloneha domestica.
Material and Methods Four lovebirds, Uroloncha domestica (Aves: Passeriformes), were decapitated; their pineal organs were isolated and fixed in 5 ~ glutaraldehyde in 0.1 M phosphate buffer, pH 7.4, for 90min at 4~ After washing in 0.1 M phosphate buffer containing 0.34 M sucrose for 4 h, the pineals were transferred to a 2~o solution of tannic acid for one day at 4~ followed by washing in 0.22 M phosphate buffer for 2h. The material was postfixed in 1 ~ OsO 4 at 4~ for 4h, then dehydrated. After fracturing in liquid nitrogen and critical point drying, the pineals were coated with gold and observed in a Jeol JSM - 50 A scanning electron microscope.
Results and Discussion
In comparison with the scanning electron microscopic observations on the pineal organ of the teleost fish, Salmo gairdneri (Hartwig and Pfautsch, 1973) and the lamprey, Lampetra japonica (Morita, 1975), only very few outer segments were found to occur in the pineal of the bird, Uroloncha domestica. Scattered outer segments of varying form and size extend beyond the surface of the pineal wall into the pineal lumen. Figure 1 a shows a bulbous outer segment, 4.2 Ixm in length and 2.01xm in diameter. In Figure 1b a structural complex formed by an inner and a corresponding outer segment of a pinealocyte is seen within the pineal lumen. The inner segment is 3.6 ~tm long and 3.3 ~tm thick. The cup-shaped outer segment displays a longitudinal axis of 3.6 ~tm, a transversal axis of approximately 2.0 Ixm
Outer Segments in the Pineal Organ of the Lovebird
141
Fig. 1 a - d . Different types of outer segments in the pineal organ of Uroloncha domestica, a Bulbous outer segment, x 15,300. b Cup-shaped outer segment; i invagination of cilium into the outer segment; t thickening o f the border of the outer segment; m microvillus-like evagination; is inner segment; x 11,700. e Elongated outer segment showing a bifurcation (bl; b2), a raphe (r) and a finger-like process (p); the distal part of the outer segment is bent down toward the base. x 12,600. d The same outer segment as in e after rotation and tilting of the specimen, x 15,300
142
M. Ueck et al.
and a thickness of only 0.3 ~tm. The connecting cilium of both penetrates into a pit located in the outer segment. This invagination is eccentric, and a process resembling a microvillus is seen near the invagination. The border of the outer segment shows a thickening on one side. Such cup-shaped outer segments are not a characteristic feature of lateral eye photoreceptors; however, they occur, in addition to regularly formed outer segments, in the pineal organ of Amphibia (Ueck et al., unpublished results). A comparison of transmission and scanning electron micrographs shows that the m e m b r a n e whorls may result from sections through cup-shaped outer segments. Figures 1 c and d show two different images of one outer segment; the electron micrograph in Figure 1 d was taken after rotation and tilting of the specimen. This outer segment differs in size and shape from the outer segments in Figures 1 a and b. It is an elongated structure, 11 Ixm in length, thinner proximally than distally. This extension is not erect; its tip folds back towards the base of the outer segment. A raphe is seen in the middle of the proximal segment and on the lateral aspect of the distal part. The bifurcated curved portion of the outer segment encompasses a finger-like structure bearing a raphe (Fig. 1 c, d). The authors suggest that the demonstration of intact outer segments in the pineal organ of Uroloncha domestica proves morphologically the sensory capacity of the avian pineal organ. Using the acetylcholinesterase method positively reacting nerve cells and nerve fibers, afferent to the brain, could be shown in passeriform birds (Ueck and Kobayashi, 1972); these acetylcholinesterase-positive nervous structures also occur in Uroloncha domestica (Ueck and Wake, unpublished results). However, the number of fully differentiated outer segments per square unit is very low in comparison to the pineal organs of lower vertebrates. The extremely variable and complex ultrastructure of the outer segments of avian pinealocytes makes it difficult to reconstruct them on the basis of transmission electron micrographs. A comparative study of the outer segments in the pineal organs of fish, amphibians and birds is in progress in order to supply more detailed information on the three-dimensional structure of these photoreceptive elements. Acknowledgement. The authors wish to express their gratitude to Miss R. Liesner for technical assistance and to Miss I. Lyncker for secretarial aid.
References
Collin, J.-P,: Contribution a l'6tude de l'organe pin6al. De l'6piphyse sensorielle a la glande pin6ale: modalit6s de transformation et implications fonctionelles, Ann. Stat. Biol. Besse-en Chandesse, Suppl. 1, 1-359 (1969) Dodt, E., Ueck, M., Oksche, A.: Relation of structure and function. The pineal organ of lower vertebrates. J.E. Purkyn6 Centenary Symposium, Prag, 1969 (V. Kruta, ed.), pp. 253-278. Brno: Universita Jana EvangelistyPurkyn~ 1971 Eakin, R.M., Brandenburger, J.L.: Osmic staining of amphibian and gastropod photoreceptors. J. Ultrastruct. Res. 30, 619-641 (1970) Hartwig, H.-G., Pfautsch, M.: RasterelektronenmikroskopischeBeobachtungen an pinealen Sinneszellen der Forelle, Salmo gairdneri (Teleostei). Z. Zellforsch. 138, 585-589 (1973) Herbut6, S., Bayl6, J.D.: Multiple-unit activity in the pineal gland of the Japanese quail: spontaneous firing and responses to photic stimulations. Neuroendocrinol. 16, 52-64 (1974)
Outer Segments in the Pineal Organ of the Lovebird
143
Menaker, M., Oksche, A.: The avian pineal organ. In: D.S. Farrier and J.R. King (eds.), Avian biology, Vol. 4, pp. 79-118. New York-London: Academic Press 1974 Morita, Y. : Absence of electrical activity of the pigeon's pineal organ in response to light. Experientia (Basel) 22, 402 (1966) Morita, Y.: Direct photosensory activity of the pineal. Brain-Endocrine Interaction II. The Ventricular System. 2nd Int. Symp., Shizuoka 1974, pp. 376-387. Basel: Karger 1975 Oksche, A., Kirschstein, H.: Unterschiedlicher elektronenmikroskopischer Feinbau der Sinneszellen im Parietalauge und im Pinealorgan (Epiphysis cerebri) der Lacertilier. Ein Beitrag zum Epiphysenproblem. Z. Zellforsch. 87, 159-192 (1968) Oksche, A., Kirschstein, H.: Elektronenmikroskopische Untersuchungen am Pinealorgan von Passer domesticus. Z. Zellforsch. 102, 214-241 (1969) Ralph, C.L., Dawson, D.C.: Failure of the pineal body of two species of birds (Coturnix coturnix japonica and Passer domesticus) to show electrical responses to illumination. Experientia (Basel) 24, 147-148 (1968) Ueck, M.: Zur Ultrastruktur der Epiphysis der V6gel. Zool. Anz., Suppl. 33, Verh. Zool. Ges. 509-518 (1969) Ueck, M. : Sensorische und sekretorische Strukturelemente des Pinealorgans und ihre funktionelle Bedeutung. Habilitationsschrift, Fachbereich Medizin. Giessen 1972 Ueck, M.: Fluoreszenz- und elektronenmikroskopische Untersuchungen an der Epiphysis cerebri verschiedener Vogelarten. Z. Zellforsch. 137, 37-62 (1973) Ueck, M.: Vergleichende Betrachtungen zur neuroendokrinen Aktivit/it des Pinealorgans. Fortschritte der Zoologic 22, Heft 2/3. Stuttgart: G. Fischer 1974 Ueck, M., Kobayashi, H. : Vergleichende Untersuchungen fiber Acetylcholinesterase-haltige Neurone im Pinealorgan der V6gel. Z. Zellforsch. 129, 140-160 (1972)
Accepted April 19, 1977
