© 1985S. K argcr A G . Basel (HKM—5180/85/1213-0153 S 2.75/0 Acta anat. 121: 153-162 (1985) Target Cells of Vitamin D in the Vertebrate Retina D. S. Schreiner^'. S. S. Jamie", D. E. M. Lawsonb *Department of Anatomy. University of Ottawa. Ont., Canada; ’’University of Cambridge and Medical Research Council. Cambridge. UK Key Words. Vitamin D •Calcium-binding protein • Vertebrate retina Introduction The classical targets for vitamin D were once thought to comprise only organs that are directly involved in calcium homeostasis, e.g. bone, intestine, kidney, para thyroids, laying hen shell glands, etc. Recently, some new vitamin D target organs have been uncovered by a variety of modern techniques such as: (a) the biochemi cal isolation and characterization of high affinity recep tors for 1.25 (011)2 D3 [Brumbough and Haussler, 1974; Lawson and Wilson, 1974; Christakos and Norman, 1980a, b; Pike et al., 1980] which is the most potent metabolite of vitamin D; (b) the autoradiographic localization of labelled 1.25 (OH)2D3[Stumpf et al., 1979, 1980; Narbaitz et al.. 1981], and finally (c) the detection of a specific protein synthesized as a result of genomic stimulation by vitamin D,. Vitamin D-dependent cal cium-binding protein (D-CaBP) is the only well known protein whose synthesis is de novo under the influence of 1,25 (OI I)2 D3 [Wassennan and Taylor, 1966; Enilage et al., 1973; Spencer et al., 1976; Christakos and Nor man, 1980a. b]. Although the amount of D-CaPB in various tissues can be measured by radioimmunoassay [Christakos et al., 1979; I'homasset et al., 1982], the localization of D-CaBP in specific cell types can be car ried out by immunocytochemical methods [Morrissey et al., 1978; Jande et al.. 1981a, b; Schreiner et al.. 1983]. Results obtained with these three different techniques are in good agreement as to the identity of vitamin D target tissues and cells. Downloaded by: Vanderbilt University Library 184.108.40.206 - 10/26/2017 9:12:12 AM Abstract. Using PAP technique, cellular localization of vitamin D-dependent calcium-binding protein (D-CaBP) was investigated in vertebrate retina with monospecific antisera against chick duodenal D-CaBP. In the chick retina, the receptor cells were positive. In the inner nuclear layer, horizontal cells and some bipolar cells were also positive. Some amacrine cells as well as different levels of the inner plexiform layer were also positive for D-CaBP. A few interspersed ganglion cells were positive but their axons forming the optic tract were negative. Muller's cells were negative. In 1-day-old chicks and 4-week-old rachitic chicks there was paucity and absence, respectively, of DCaBP staining in horizontal cells. In the mouse, rat, and rabbit the receptors had only trace amounts of reaction product in their outer segment and pedicle. Horizontal cells were densely positive throughout their cellular body and processes. Some amacrine cells in the inner nuclear layer were positive. In the mouse and rat three horizontal levels of the outer plexiform layer were very prominent because of their dense staining for D-CaBP. Many ganglion cells were also positive along with their axons forming the optic nerve. In the rabbit, no positive layers were seen in the inner plexiform layer, and ganglion cells with their fibers were negative. In the frog retina there were smaller amounts of D-CaBP in the receptor cells and horizontal cells than that of the chick retina. Also, the fibers of the ganglionic cells were positive for D-CaBP. In all species studied, some amacrine cells were stained for D-CaBP. Because of its possible roles in membrane calcium transport and intracellular Ca++ regulation, it has perhaps similar functions in these positive cells. The synthesis of D-CaBP is dependent upon vitamin D. These positive cells are thus target cells of vitamin D. 154 Schreincr/Jande/Lawsun Fig.l. Vertical section of a chick retina incubated with non-immune rabbit serum (control). All the com ponents of the retina including re ceptor cells (RC). the outer nuclear layer (ONL). the outer plexiform layer, the inner nuclear layer (INL). the inner plexiform layer (IPL). the ganglion cell layer (GC). and the fiber layer (FL) were negative. x225. Material and Methods 4-week-old while Leghorn chicks on normal diel and some on rachitogenic diet [Barnes el at., 19731; I-month-old Sprague-Dawley rats; black mice (C57BL/6.I Jackson Labs); Paris R3 white mice; 2month-old New Zealand white rabbits, and frogs <liana pipiens) were used as source for retina. These were decapitated and each eye was quickly removed and transferred into fixative. The eyes were hemisected at the equator, the anterior half and the vitreous humor was removed. Fixation was carried out in Carnoy's fixative for 3 h at room temperature |Jande et al.. 1981a]. The retinas were then thoroughly washed in 90% ethyl alcohol and processed for paraffin sectioning. 5-um thick sections were used for histochemical staining. The PAP method for paraffin sections according to Sleinberger 11979] was followed. Sections were pre-adsorbed with a 3% solution of normal goat serum (Gibco). The primary antiserum (anti-D-CaBP) was diluted 'Am, the link antiserum (anti-rabbit goat IgG. Miles Laboratories) was diluted Vx and the PAP complex (rabbit peroxidase anti-peroxidase. Miles Laboratories) 'Aw. All dilutions were done with 0.05 M Tris-HCI saline containing 1% normal goat scrum. The slides were incubated in 0.5% diaminobenzidine-HCI containing 0.01% U4), for 20 min at room temperature. Endogenous peroxidase was inhi bited according to the method of Heydermtin and Neville 11977], Con trol sections were stained with non-immune rabbit serum diluted 'A... instead of the antisera. All sera were decomplemented for 30 mm at 56 °C. Monospecific antisera against chick duodenal D-CaBP was prepared according to Spencer et al. 11976] in the laboratory of Dr. !). E. M. l.awson. Observations The localization of D-CaBP was carried out in the chick, rat. mice, rabbit and frog. The best results were obtained for the chick retina and these will be presented first. Normal Chicks Control sections, incubated with normal rabbit serum, did not show any reaction product in the entire retina (fig. 1). The histology of the chick retina is well known [Hodges, 1974] and the positive retinal neurons have been identified according to their location. Inner to the pigment epithelium is the receptor cell layer which has been shown to contain various morphological ly distinct types of photoreceptor cells [Meyer and Cooper, 1966; Morris and Shorey, 1967], All receptor cells have been found to contain the dark reaction prod uct indicative of the presence of D-CaBP. Various struc tural regions of the receptors namely, the inner seg ment. the cytoplasm surrounding the nucleus, some of the nuclei and all synaptic pedicles have been found to be very positive for D-CaBP (fig. 2, 3). There was a sharp demarcation at the outer limiting membrane, be- Downloaded by: Vanderbilt University Library 220.127.116.11 - 10/26/2017 9:12:12 AM D-CaBP which was originally isolated from chick duodenum [ Wasserman and Taylor, 1966] has been very well characterized [Ingersoll and Wasserman, 1971; Bredderman and Wasserman, 1974; Fullmer and Was serman, 1975] and has also been demonstrated in the brain [Taylor, 1974, 1976]. In the central nervous system D-CaBP is confined to neurons and their processes of certain specific nuclei [Jamie et al., 1981a, b; Roth et al., 1981], The retina, which is an extension of the diencepha lon. has been found to also be a target organ of vitamin D and this communication describes the cellular dis tribution of D-CaBP in various retinal cell types and endeavours to discuss the functional significance of these new target cells its it relates to calcium metabolism. Vitamin D. Calcium-Binding Protein and Vertebrate Retina 155 Fig.2. Vertical section of a nor mal chick retina stained with anti-DCaBP. The inner segment of the re ceptor cells (RC) are positive for DCaBP. At the outer limiting mem brane (OLM) there is a sharp bound ary because of the lighter staining of outer nuclear layer (ONL). In the outer plexiform layer (OPL) the positive pedicles of the RC and the positive processes and cellular bodies of horizontal cells (HC) from two distinct layers. In the inner half of the inner nuclear layer (INI.) posi tive amacrine cells (AC) are seen. In the inner plexiform layer (IPL) at least five horizontal bands can be distinguished. A few scattered gan glion cells (GC) are positive. The fiber layer (FL) and the inner limit ing membrane (INC) are negative. x400. cause the outer nuclear region showed smaller amounts of D-CaBP (fig.3). The amount of cytoplasm in this layer is sparse since most of the space is taken up by the nuclei, of which only a few were positive and thus it stained lightly. The synaptic pedicles always stained dark and appear as a distinct layer. Müller cells, which take part in the formation of the external limiting mem brane through their junctional complexes with the re ceptor cells, did not show any staining for D-CaBP (fig-3). The outer plexiform layer where the pedicles of the receptor cells form synaptic connections with the hori zontal and bipolar cells stained quite dark (fig.3). Quite often in the outer plexiform layer two stratifications were distinguishable: an outer one which represents the synaptic pedicles of the photoreceptors and an inner one which is composed of the cell processes of horizontal and bipolar cells. Further inwards is the inner nuclear layer (INL) which contains four types of cells; horizon tal cells, bipolar cells. Müller cells and amacrine cells. Downloaded by: Vanderbilt University Library 18.104.22.168 - 10/26/2017 9:12:12 AM Fig.3. Higher magnification of normal chick retina stained with anti-D-CaBP. Here again the inner segments of the receptor cells RC are clearly positive. The outer nuclear layer (ONL) stains lightly. I'he pedicles of the RC arc positive. In the inner nuclear layer (1NL). horizontal cells (HC) and probably some bipolar cells (BC) are also positive. Amacrine cells (AC) con taining D-CaBP arc present close to the INL. One AC in particular is labeled and its sole process is seen reaching into a D-CaBP positive lay er of the inner plexiform layer, x 562.5 156 Schrciner/Jande/Lawson The main cell body of the horizontal cell, including the nucleus and the cellular processes taking part in the in ner plexiform layer were positive for D-CaBP (fig. 2, 3). It appears that the major amount of reaction product was on the outer side of each nucleus (fig. 3). The nuclei of the bipolar cells are situated in the outer half of the INL. Only a few nuclei in these regions were positive (fig.3). Muller's cells which begin at the outer limiting membrane and end at the inner limiting membrane were completely negative. Further inwards in the INL. closest to the inner plexiform layer (IPL), are the amacrine cell bodies. Although it is not possible to identify the various types of these cells as described by Cajal , it is very apparent that some of the D-CaBP-positive cells are Downloaded by: Vanderbilt University Library 22.214.171.124 - 10/26/2017 9:12:12 AM Fig. 4. Section of a retina from a 4-week-old rachitic chick, stained with anti-D-CaBP. In the outer plcxiform layer (OPL) only the pedicles of the receptor cells are clearly posi tive and the horizontal cells are negative. In the inner nuclear layer (INL). scattered positive cells arc present: some can be clearly iden tified as amacrinc cells, others are probably bipolar cells. Note some positive ganglion cells. x225. Fig.5. Tangential section of a re tina from a 4-week-old rachitic chick stained with anti-D-CaBP. The inner segment of receptor cells (RC) as well as the cytoplasm in the outer nuclear layer (ONI.) are positive. Horizontal cells in the outer plexiform layer (OPL) are not clearly seen and probably some bipolar cells (some of the group are labeled as BC) are positive. x225. Fig. 6. Section of a retina from a 1-day-old chick stained with anti-DCaBP. The receptor cells, probably some bipolar cells (BC). and some amacrinc cells (AC) and ganglion cells are positive. The horizontal cells (HC) are only faintly positive. X365. F’ig.7. Tangential section of a re tina from a I-day-old chick stained with anti-D-CaBP. In the outer nuclear layer (ONL) the cytoplasm and the nuclei of the receptor cells are positive. In the inner nuclear lay er (INL). horizontal cells (HC) are faintly positive while probably some bipolar cells (BC) and some amacrine cells at the bottom of the INL arc positive. X365. Vitamin D. Calcium-Binding Protein and Vertebrate Retina Rachitic Chicks The overall staining for D-CaBP in retina of chicks on rachitogenic diet for 4 weeks w'as lower than that in retina of chicks on normal diet. The receptor cells showed less reaction product but each cell's synapticpedicle was positive and thus the outer region of the outer plexiform layer, as mentioned earlier for the nor mal retina, were darkly stained (fig.4). The horizontal cells, their main cell body and the nucleus and their cellular processes in the outer plexiform layer, were completely negative (fig.4). This is very clear in the tangential section of the retina (fig.5). Thus the inner stratification of the OPL was not visible with D-CaBP staining. Some of the bipolar cells and some amacrine cells were positive (fig.4. 5). The various layers in the IPL were less distinct (fig.4). Some ganglion cells were positive. No other major differences were found from the normal retina. An examination of the retinal development was car ried out on I-day-old chicks and on 2-week-old chicks on normal diet and 2-week-old chicks on rachitogenic diet. At day 1, different parts of the receptor cells, espe cially their regions situated in the outer nuclear region, w'ere positive for D-CaBP (fig.6). The horizontal cell bodies along with their nucleus and their extensions to the OPL were faintly positive (fig.6. 7). Some amacrine cells and probably some bipolar cells were also distinctly positive (fig.6, 7). From the INL inwards, no difference was observed from the normal 4-week-old chick retina. In the retina of the 2-week-old chicks on a normal diet, only some receptor cell bodies in the ONL region were seen to be positive (fig. 8). The two stratifications of the OPL. the outer one formed by receptor cells synaptic pedicles and the innermost layer formed by horizontal cell processes, were visible since the horizontal cells were also positive (fig. 8). No major distinctions w'ere observed in the inner layers of the retina when com pared to 4-week-old chick retina. In the retina from chicks fed a rachitogenic diet for 2 w'eeks the cellular bodies and the cytoplasm in the synaptic pedicle of the receptor cells were positive (fig.9). The soma and the processes of the horizontal cells that take part in the formation of the outer plexiform layer were completely negative. Thus of the two components in the OPL, the outer one formed by synaptic pedicles was distinct while the inner one composed of the horizontal cell processes was not visible (fig.9). Further inwards, amacrine cells and probably some bipolar cells were positive (fig. 9). The horizontal lines in the IPL were present (fig. 9). No major differences we re noted in the inner regions of the normal and rachitic retina. Mouse and Rat In the retina of the white and the black mouse and also in that of the rat. similar neuronal types were posi tive. Thus the following description applies to these three rodents. Only barely detectable amounts of DCaBP were present in the receptor cells -(fig. 10. 11). The horizontal cells soma and their processes stained very darkly, making the OPL very prominent (fig. 10. 11). In the INL. the cell body and nucleus of many amacrine cells were positive (fig. 10, II). In some of these cells, the single main process was seen to reach into the IPL (fig. 10. 11). Three horizontal lines in the IPL were very prominent because of their dense staining for D-CaBP. All the ganglion cells along with their axons, which form the optic tract, were positive (fig. 10. 11 ). Rabbit In the rabbit retina (fig. 12. 13) as in the mouse and rat, only a very faint staining was observed in the inner segment and none was visible in the receptor cell pedi cles. Here again the OPL was the most prominent layer mainly because of the dark positive staining in the hori zontal cells. The INL is very thin and bipolar cells along with Müller cells were negative. However, some amac- Downloaded by: Vanderbilt University Library 126.96.36.199 - 10/26/2017 9:12:12 AM indeed amacrine cells (fig.3). Their location in the INL and their mode of branching in the IPL support such a conclusion. In a number of positive amacrine cells their cellular process can be seen extending into the IPL where they branch and take part in synaptic connections (fig. 3). In the avian retina, the IPL is made up of many hori zontal layers as a consequence of the stratification of the synapses between the processes of bipolar, amacrine and ganglion cells. I lowever. five layers, two being very prominent, were visible because of the positive staining for D-CaBP (fig. 2. 3). Some of the ganglion cells were found to be positive. The reaction product was seen in some nuclei as well as in the cytoplasm (fig.2). The distribution of positive cells varied a great deal and thus in sonic parts of a section only a few while in other regions many positive cells were observed. The cellular processes of the gan glion cells that take part in the IPL and others that form the fiber layer, were negative (fig.2). The inner limiting membrane formed by the processes of Muller's cells was negative (fig. 2). 157 158 Schrcincr/Jandc/Lawson fine cells were positive (fig. 12). No horizontal stratifica tion stood out in the IPL as described in the chick and other rodents (fig. 12). Frog The outer segment of the frog receptor cells as well as their pedicles were faintly positive (fig. 14). Some dis tinct horizontal cells stained darkly. In the INL the cyto plasm as well as nuclei of many bipolar cells were posi tive (fig. 14). Some amacrine cells at the border of the INL and IPL were quite positive (fig. 14). In the IPL many sublayers stood out because of their positive stain ing (fig. 14). Many ganglion cells along with their axons forming the optic tract were positive (fig. 14). Downloaded by: Vanderbilt University Library 188.8.131.52 - 10/26/2017 9:12:12 AM Fig. 8. Section of a 2-week-oid normal chick retina stained with anti-D-CaBP. In the outer nuclear layer (ONL) some of the cell bodies of receptor cells are positive. In the inner nuclear layer (INL) horizontal cells, probably some bipolar cells and some amacrine cells arc positive. In the inner plexiform layer (IPL) three positive bands are visible. X365. Fig. 9. Section of a 2-week-old rachitic chick retina stained with anti-D-CaBP. Receptor cell outer segments and cell bodies in the outer nuclear layer (ONL) are positive. RC pedicles also stained for D-CaBP but horizontal cells (HC) arc nega tive. Probably some bipolar cells (BC). some amacrine cells and some ganglion cells are positive. x365. F'ig. 10. Sections of mouse retina stained with anti-D-CaBP. The re ceptor cells (RC) have but a trace amount of D-CaBP. In the inner nuclear layer (INL) horizontal cell bodies and the processes are densely stained. Some amacrine cells (AC) arc also positive. In the inner plex iform layer three horizontal lines are distinctly stained. Most ganglion cells (GC) and their fibers (FL) arc positive. x225. F'ig. 11. Section of a rat retina stained with anti-D-CaBP. Here again receptor cells are very lightly stained. Horizontal cells (HC) and some amacrine cells (AC) arc posi tive. Most ganglion cells (GC) and their fibers (FL) are positive. x225. Vitamin D, Calcium-Binding Protein and Vertebrate Retina Discussion The presence of D-CaBP in the central nervous sys tem has been well documented [Taylor, 1974: Jmule et ah, 1982; Roth et ah, 1981; Baimbridge et ah, 1980, 1982; Feldman and Christakos, 1983]. Since the retina is an extension of the diencephalon, it is not surprising that some retinal neurons contain D-CaBP. Here is thus a newly discovered target organ for vitamin D in the species investigated. Earlier studies with epithelial tissues such as intes tine, hen shell gland and kidney have connected the function of D-CaBP with transcellular calcium transport as seen in calcium absorption and egg shell formation [Jande et ah, 1981a, b. 1982]. This view is clearly not supported by the presence of D-CaBP in so many cell types such as epithelial reticular cells of the thymus, the macrophage-like cells in spleen, the neuronal cells in the brain, follicular cells of the ovary [Jande et ah, 1982] which apparently are uninvolved in such processes. It seems that D-CaBP is involved in more than one phy siological function in which case it may have a single molecular action which has been incorporated into a variety of processes. The latter almost certainly involves the membrane transport of calcium but whether DCaBP is involved at this point or in the subsequent need to regulate intracellular Ca2+ levels still remains unclear. The presence of D-CaBP in various neurons in the reti na suggests special needs of these cells for calcium which arc dependent on this protein either for membrane transport and/or for intracellular Ca’+ concentration. In all the species observed there was a wide variation in the concentration of D-CaBP in the receptor cells. Staining was deepest in the chick receptor cells and weakest in those of the albino rat. There is evidence that calcium acts as a transmitter in light induction of recep tors [Hagina, 1972; Yoshikami et al.. 1980; Hendriks et al., 1974], The wide difference in D-CaBP observed in chick and rat retina may represent different Ca:+ needs which can only be solved by D-CaBP via its functions outlined above. Our developmental studies on normal and rachitic Downloaded by: Vanderbilt University Library 184.108.40.206 - 10/26/2017 9:12:12 AM Fig. 12. Section of a rabbit retina stained with anti-D-CaBP. Receptor cells (RC) are very faintly positive. Horizontal cells are the most densely stained cells. Some amacrine cells are also positive. x225. Fig. 13. Partially tangential sec tion of a rabbit retina showing the extensive branching of D-CaBP posi tive horizontal cell processes. x225. Fig. 14. Section of a frog retina stained with anti-D-CaBP. The re ceptor cells (RC) outer segments, as well as their cellular bodies in the outer nuclear layer (ONL) and their pedicles are faintly positive. Hori zontal cells (HC). bipolar cells and some amacrine cells (AC) in the in ner nuclear layer (INL) are positive. Four horizontal lines in the inner plexiform layer are positive. Many ganglion cells (GC) and their fibers (FL) are positive. x225. Schreiner/J ande/Lawson 160 ous structures still remains to be solved. Thus in the vertebrate retina various neurons have been demon strated to be the target cells of vitamin D through the localization of D-CaBP in these ceils. The proper func tion of this protein in these cells still remains unclear. Acknowledgements Thanks are due to MRC of Canada (Grant No. MT 3089) and MRC of UK for support of the research. Ms. Schreiner is holder of an MRC Studentship. References Araki. M.; M aeda.T.; Kimura, H.: Dopaminergic cell differentiation in the developing chick retina Rrain Res. 10: 97-103 (1983). Arnold. B.M.; Kuttner, M.; Willis. D .M .; Hitchman. A .J.W .: Har rison, .1. E .: Murray, T. M.: Radioimmunoassay studies of intesti nal calcium-binding protein in the pig. 11. The distribution of intes tinal CaBP in pig tissues. Can. J. Physiol. Pharmacol. 53: 1135-1140 (1975). Baimbridgc. K.G.: Miller. J. J.; Parkcs. C.O .: Calcium-binding pro tein distribution in the rat brain. Brain Res. 239: 519-525 (1982). Baimbridgc, K.G.; Selke, P.A.: Ferguson, M.; Parkcs, C.O.: Hu man calcium-binding protein; in Seigel, Carafoli, Kretsingcr. MacLennan. Wasserman. Calcium-binding proteins; structure and function, pp. 401-404 (Elsevier/North Holland. Amsterdam 1980). Barnes. M .J.: Constable. B.J.; Morten, L .F.; Kodicek. E.: The in fluence of dietary calcium deficiency and parathyroidectomy on bone collagen structure. Biochim. biophys. Acta 328: 373-382 (1973). Buckerfield. M.; Olivier, J.; Chubb, I.W.; Somatostatin-like iinmunoreactivity in amacrine cells of the chicken retina. Neurosci ence 6: 689-695 (1981). Brecha. N.; Karten. H .J.; Laverack. C.: Enkephalin-containing amacrine cells in the avian retina: immunohistochcmical localiza tion. Proc. natn. Acad. Sci. USA 76: 3010-3014 (1979). Bredderman. P. B. ; Wasserman. R. H.: Chemical composition, affini ty for calcium and some related properties of the vitamin D-dependent calcium-binding protein. Biochemistry 13: 1687-1694 (1974). Brumbough. P .F .; Haussler, M R.: la,25-Dihydroxycholecalciferol receptors in intestine. 1. Association of la.25-Dihydroxycholecalciferol with intestinal mucosa chromatin. J. biol. Chem. 249: 1251-1257 (1974). Cajal, S.R.Y .: The structure of the retina (Thomas, Springfield 1972). Christakos, S.; Friedlandcr. E.J.: Frandsen, B.R.: Norman. A.W.: Studies on the mode of action of calciferol. XIII. Development of a radioimmunoassay for vitamin D-dependcnt chick intestinal cal cium-binding protein and tissue distribution. Endocrinology 104: 1495-1503 (1979). Christakos. S.: Norman. A.W.: Vitamin D-dependent calcium-bind ing protein synthesis by chick kidney and duodenal polysomes. Archs Biochem. Biophys. 206: 809-815 (1980a). Downloaded by: Vanderbilt University Library 220.127.116.11 - 10/26/2017 9:12:12 AM retina of chicks clearly demonstrate the dependency for the synthesis of D-CaBP in the horizontal cells on vita min D. The horizontal cells take part in the formation of OPL and the complexity of this layer differs in certain species. These differences are related to the analytical capacity of the retina [Michael, 1969], It appears that horizontal cells in their function have special Ca:+ needs that are dependent on D-CaBP. In all the vertebrates observed, some amacrine cells in the retina were always positive. The vertebrate retina has been shown to consist of several types of amacrine cells containing various different peptides such as: acetylcholine [Nichols and Roelle, 1968), glycine, GABA [Marshall and Voaden, 1974a, b] dopamine [Araki et al., 1983], indolamine [Hauschild and Lalies, 1973; Florén, 1979). enkephalin [Brecha ct ah. 1979; Tornquist et ah, 1981], somatostatin [Kirsch and Leon hardi, 1979; Buckerfield et ah, 1981 ; Eskay et ah, 1980; Tornquist et ah, 1981; Ellis et ah, 1983], TRH [Eskay et ah, 1980; Tornquist et ah, 1981], substance P [Fukuda et ah, 1981 ; Karlen and Brecha, 1980; Eskay et ah, 1980] glucagon and neurotensin [Tornquist et ah, 1981]. Evidence concerning the role of these neuropep tides also found in other areas of the brain has suggested that these may function as neurotransmitters [Tornquist et ah. 1981], In the retina, the release of TRH, somatos tatin and substance P under depolarizing conditions is calcium dependent [Eskay et ah, 1980], and this could be related to the functions of D-CaBP. Experiments are under way in order to find out whether these D-CaBP containing amacrine cells represent those that also con tain these neuropeptides. In all the speeies observed except for the rabbit, some horizontal layers in the IPL were densely positive. These represent the synaptic contacts between D-CaBP positive amacrine, bipolar and ganglion cells. Thus, like the OPL, the IPL formed by the synaptic complexes of bipolar, amacrine and ganglion cells, have special cal cium needs which as stated earlier, may require DCaBP. In the rat, mouse and frog, most of the ganglion cells along with their dendritic fibers were positive for DCaBP. However, in the chick only a few ganglion cells were positive and their fibers were completely negative. This correlates well with the observations of Feldman and Christakos  who noted that the rat optic tract fibers were positive for D-CaBP and of Roth ct al.  who demonstrated that chick optic tract fibers were negative. The functional significance for this dis parity in D-CaBP content between these two homolog Christakos. S.; Norman, A. W.: Vitamin D-dependenl calcium bind ing protein and its relation to 1,25-Dihydroxyvitamin D receptor localization and concentration; in Scigcl. Carafoli, Kretsinger. MacLennan. Wasserman, Calcium-binding proteins: structure and function, pp. 371-378 (Elsevier/North Holland. Amsterdam 1980b). Delorme. A.N.: Danan. J.-L.; Mathieu. H.: Biochemical evidence for the presence of two vitamin D-dcpendent calcium-binding pro teins in mouse kidney. J. biol. Chem. 258: 1878-1884 (1983). Ellis, J .P .; Sullivan. J.M .: Reiner. M.W.: Somatostatin-like immunoreactivity in the retinae of adult and embryonic chickens. Proc. Soc. exp. Biol. Med. 172: 463-471 (1983). Emlage. .1. S.; Lawson. D .E .M .; Kodicek. E.: Vitamin D induced synthesis of mRNA for calcium-binding protein. Nature. Lond. 246: 100-101 (1973). Eskay. R. L .: Long. R .T .: Imvonc. P. M.: Evidence that TRH. somatostatin and substance P are present in neurosecretory ele ments of the vertebrate retina. Brain Res. 196: 554-559 (1980). Feldman. S.C.: Christakos. S.: Vitamin D-dependent calcium-bind ing protein in rat brain: biochemical and immunocytochemical characterization. Endocrinology 112: 290-302 (1983). Floren. I .: Indoleamine accumulating neurons in the retina of chicken and pigeon. A comparison with dopaminergic neurons. Acta ophthai. 57; 198-210 (1979). 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