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Immunohistochemical localization of chick retinal 24 kdalton protein (visinin) in various vertebrate retinae
Brain Research, 331 (1985) 209-215 Elsevier
209
BRE 10644
Immunohistochemical Localization of Chick Retinal 24 kdalton Protein (Visinin) in Various Vertebrate Retinae SACHIKO HATAKENAKA 1, HIROSHI KIYAMA2, MASAYA TOHYAMA 2, NAOMASA MIKI 1
JDepartment of Pharmacology, Cancer Research Institute, Kanazawa University, 13-1 Takaramachi, Kanazawa 920, and 2Department of Neuroanatomy, Institute of Higher Nervous Activity, Osaka University Medical School, 4-3-57, Nakanoshima, Kitaku, Osaka 530 (Japan) (Accepted July 10th, 1984)
Key words: retina - - visinin - - immunohistochemistry - - development - - cone cell
Antiserum against a protein (24,000 daltons, visinin) of chick retina has been provided for immunohistochemicat study on the localization of visinin in chick retinae during development, as well as in various vertebrate retinae. The photoreceptor cells were stained with anti-visinin serum from 7th day embryonic retinae and its intensity was gradually increased with embryonic age. In addition, visinin-like immunoreactivity was found in some kinds of amacrine and displaced amacrine cells from 1lth-day embryonic retinae. When human, cat, frog and carp retinae which contain both rods and cones were examined, staining of cone cells was clearly observed in the photoreceptor cell layer, but not in the rods. Furthermore visinin-like immunoreactivity was barely detectable in the photoreceptor cells of bovine, rat and mouse retinae containing mostly rod cells. These results suggest that visinin is mainly located in the cone cells in various vertebrate retinae and is a good marker for the cone cells.
INTRODUCTION A key to understanding the function of the nervous system is to clarify the biochemical p r o p e r t i e s and histochemical localization of proteins specific to nervous tissues. In our previous report~3, we have analyzed the soluble proteins of chick retina during development and r e p o r t e d that a p e p t i d e of about 24,000 daltons (24 k d a l t o n protein, designated as 'visinin') a p p e a r s in the retina of the 14th day e m b r y o and gradually increases in concentration with embryonic age until hatching. It is not d e t e c t e d in the cerebrum, rectum, pigment epithelium or vitreous body on s o d i u m dodecyl sulfate-polyacrylamide gel analysis. W e p r o p o s e d that visinin is a distinctive protein that increases in concentration during chick retinal d e v e l o p m e n t and would be closely associated with retinal function. In this p a p e r we p r o d u c e d an antibody against visinin and e x a m i n e d the distribution of immunoreactive visinin in chick retina during d e v e l o p m e n t and com-
pared the labeling patterns of various v e r t e b r a t e retinae using an indirect immunofluorescent staining method. MATERIALS AND METHODS
Purification o f visinin The partially purified visinin was p r e p a r e d from soluble proteins of adult chick retina by gel filtration and ion exchange column c h r o m a t o g r a p h y as previously described 13. Visinin was then purified by extracting a corresponding b a n d from sodium dodecyl sulfate-polyacrylamide gels ( S D S - P A G E ) . The purified visinin migrated as a single band on SDSP A G E (Fig. 1A). Protein was assayed by the m e t h o d of Lowry et alA8 with bovine serum albumin as a standard, or by the m e t h o d of B r a d f o r d when Tris was present 4. Preparation o f anti-visinin serum Anti-visinin serum was p r o d u c e d in a New Z e a -
Correspondence: N. Miki, Department of Pharmacology, Cancer Research Institute, Kanazawa University, 13-1 Takaramachi, Kanazawa 920, Japan. 0006-8993/85/$03.30 © 1985 Elsevier Science Publishers B.V. (Biomedical Division)
210 land white rabbit as follows. Purified visinin was mixed with an equal volume of Freund's complete adjuvand and injected into the foot pads and backs in a rabbit (about 3 kg, 300gg/injection) every week for 4 weeks. One week after the last injection, an anti-serum was obtained. The specificity of anti-visinin serum was analyzed by an Ouchterlony double-immunodiffusion test 25 and immunoelectrophoresisl2. Ouchterlony double-immunodiffusion test was carried out in 1% Agar plate containing phosphate-buffered saline (pH 7.4). Immunoelectrophoresis was executed in 1% Agar plate with barbital buffer (pH 8.6, ion strength 0.05). The samples were placed in the wells and electrophoresis was carried out for 1 h at 15 mA (2 mA/cm) at room temperature, and then rabbit anti-visinin serum was placed into the trough. Precipitation lines which developed after 24 h incubation at room temperature were stained with Amide Black 10B. Rabbit IgG was purified by ammonium sulfate precipitation (0-40%) and D E A E cellulose column chromatography eluted with 17.5 mM phosphate buffer (pH 6.3).
Immunohistochemistry Retinae from chick embryos, 1 or 2 week old chicks and frog (Rana catesbiana) were fixed as follows. The animals (after 16th day embryo) were perfused intracardially with ice-cold saline (about 50 ml) followed by ice-cold Zamboni's fixative buffer30 (0.21% picric acid, 2% paraformaldehyde and 130 mM phosphate buffer (pH 7.4), about 500 ml) under sodium pentobarbitone anesthesia (40 mg/kg, i.p.). The eyes were dissected out, post-fixed in the same fixative buffer for 1-2 days and washed in 30% sucrose containing 0.1 M phosphate buffer (pH 7.4) for 2-3 days at 4 °C. The retinae from chick embryo earlier than 16th day or carp (Cyprinus carpio, 700-800 g) were treated by the same method except for the perfusion procedure. Isolated bovine eyes were obtained from a local slaughter house and human retina was from an eye for corneal transplantation. These were fixed with Zamboni's fixative buffer as described above. Frozen sections were cut in a cryostat with section thickness of 10-20/~m. These sections were mounted on chrome-alum-gelatin coated glass slides, air-dried for 2-3 h at room temperature and treated with anti-visinin serum according to CoonsS. All sections were rinsed with ice-cold phosphate buf-
fered saline (PBS) for 10 min prior to the beginning of the immunohistochemical procedure. Some of these sections were incubated in a humid atmosphere at 25 °C with anti-visinin serum (at dilutions of 1:1,000 to 80,000) overnight, and then were rinsed with ice-cold PBS for 10 min, and 1.0% Triton X-100 containing PBS for 10 min. The washing procedure was repeated twice. After washing the sections were incubated with fluorescein isothiocyanate (FITC)conjugated goat anti-rabbit IgG (at a dilution of 1: 1,000) overnight at 25 °C. The sections were rinsed by the previously described washing procedure and mounted in PBS-glycerin mixture. The remaining sections were incubated with preimmune serum, visinin-absorbed antiserum or FITC-conjugated goat anti-rabbit IgG only as a control staining. One section of each retina was stained with hematoxylin and eosin for morphological orientation.
SDS-polyacrylamide PAGE)
gel
electrophoresis (SDS-
SDS-PAGE was carried out by the method of Laemmli 16 with a slight modification as previously described 13.
Chemicals Acrylamide, Agar and other reagents for gel electrophoresis (electrophoresis grade) were from Wako Pure Chemical Industries Ltd. Sodium dodecyl sulfate, Coomassie brilliant blue R-250, Amide black 10B, Triton X-100, picric acid and paraformaldehyde were purchased from Nakarai Chemicals. FITC-conjugated goat anti-rabbit IgG and D E A E cellulose (DE 52) were obtained from Miles Co. Ltd. and Whatman, respectively. RESULTS
Specificity of anti-visinin serum The specificity of anti-visinin serum was examined by an Ouchterlony double-immunodiffusion test and immunoelectrophoresis against electrophoretically purified or partially purified visinin. The anti-serum showed a single precipitation line against purified or partially purified visinin (Fig. 1B, C). This anti-serum did not give any precipitation lines with membrane fractions of chick retina, soluble proteins of chick cerebrum, tectum or bovine retina (data not shown).
211
A
~ ~i~Z¸
B
®
Fig. 1. Preparation of anti-visinin serum. A: electrophoretic pattern of 3/~g (1) and 6/lg (2) of electrophoretically purified visinin on SDS-polyacrylamidegel (11%). The arrow indicates the visinin. Protein was stained with Coomassie Brilliant Blue R-250. B: Ouchterlony double-immunodiffusionof anti-visinin serum against an electrophoretically purified visinin. The center well (1) contained 3/~gof purified visinin. Wells 2 and 4 contained 15/~1 of preimmune serum and anti-visinin serum, respectively. Wells 3 and 5 were filled with 10/~1 (3 mg protein/ml) of IgG from preimmune serum and IgG from anti-visinin serum, respectively. C: immunoelectrophoresis of purified visinin and partially purified visinin. 1: 5/~g of electrophoretically purified visinin. 2:20/~g of partially purified visinin. The trough was filled with 50/A of anti-visinin serum. The anode is to the right. Protein was stained with Amide Black 10B.
lmmunohistochemistry of visinin in developing chick retinae Immunohistochemical examination of 1- or 2week-old chick retina was shown in Fig. 2a. Strong visinin-like immunoreactivity was found in the photoreceptor cell layer. The staining intensity of inner segments was more than that of the outer nuclear layer, but no significant labeling was observed in outer segments of photoreceptor cells (Fig. 2a). In addition, scattering neuronal cells, which are morphologically identified to amacrine (Fig. 2a, arrow) and displaced amacrine cells, were also stained with anti-visinin serum. Some amacrine cells were found to extend the processes into the inner plexiform layer. On the other hand, Miiller cells were not stained. Sections incubated with visinin-absorbed antiserum or preimmune serum were devoid of any immunocytochemical reaction (Fig. 2b). As visinin increased with retinal development 13, we compared the staining patterns of visinin in the retinae from embryo to chicken. No significant staining was detected (Fig. 2c) in 4th-day embryonic retinae. In 7th-day embryonic retinae, putative photo-
receptor cells were apparently stained (Fig. 2d), and the staining population of the peripheral part of the retina was less dense than that of central part (data not shown). Cells migrating from the inner to outer regions of the retina were also stained. In 9th-day embryonic retinae, many connecting fibers between photoreceptor cell layer and ganglion cell layer were also labeled (Fig. 2e), and the staining intensity and population of photoreceptor cells increased. Putative amacrine and other neuronal cells in the inner nuclear layer began to be stained in llth-day embryonic retinae and this gradually increased by 13th-day embryo (Fig. 2f). Although inner segments of photoreceptor cells appear at this stage19, 23, they were not distinguished from the outer nuclear layer in our experiment. In 16th-day embryonic retinae, the staining pattern of whole retinae was essentially the same as that of chick, but inner segments of photoreceptor cells still were not fully developed in this section (Fig. 2g). The processes extending from amacrine cells were stained weakly but apparently observed. In 19th-day embryonic retinae, all inner segments of photoreceptor cells were elongated and labeled as in the adult retina (Fig. 2h). However, outer segments of photoreceptor cells were not stained. The processes of amacrine cells were clearly observed at this stage.
Immunohistochernistry of visinin-like substances in various vertebrate retinae As the dominant cell population of the photoreceptor cell layer in chick retina is thought to be cone cells, we compared the staining patterns of visinin in human, cat, frog and carp retinae which contain both rod and cone cells in various ratios (Fig. 3). A dilution of 1:1,000 serum was used for all animals except chick. The patterns of visinin-like immunoreactivity of human and cat retinae were similar (Fig. 3a, b). Staining of cone cells was clearly observed in the photoreceptor cell layer, but that of rods was not. In the inner nuclear layer, staining was detected in horizontal cells with axonal processes (Fig. 3a, b, arrow). The staining intensity of amacrine cells was rather weak in human retina, although in chick retina some amacrine cells and their processes were clearly observed. In frog and carp retinae, most of the cone cells were stained, but horizontal cells were hardly observed in the inner nuclear layer and not distin-
212 guished from synaptic terminals of cone cells. Furthermore, none of the amacrine cells were stained in frog and carp retinae (Fig. 3c, d). On the other hand, it has been known that a dominant cell group in the photoreceptor layer is that of rod cells in bovine, rat and mouse retinae. I m m u n o histochemical staining with anti-visinin s e r u m in these animals showed that staining was very poor or not observed in the photoreceptor cell layer, but relatively good in horizontal, amacrine and displaced amacrine cells (Fig. 3e). Immunoreactive visinin was not found in MOiler cells in any of these sections. No significant staining was found in visinin-absorbed antiserum or preimmune serum-treated sections. DISCUSSION We have examined the immunohistochemical localization of visinin in developing chick neural retinae and found that the immunoreactive visinin appeared in 7th-day embryonic retinae and thereafter gradually increased with embryonic age, and was mostly located in the photoreceptor cell layer and in part in the inner nuclear layer. No significant staining was observed in Miiller cells in any embryonic ages and adult retinae. Changes in the visinin-stainingpattern during retinal development well support our previous data in which visinin was analyzed by a SDSP A G E 13, although the detection of visinin with immunohistochemical study was much more sensitive than that on a S D S - P A G E . We estimated that visinin of chick retina would be mostly located in photoreceptor cells judging from this immunohistochemical study. But other neuronal cells which are morphologically identified to amacrine and displaced amacrine cells were also reacted with anti-visinin serum. Since the content of visinin
Fig. 2. Indirect immunofluorescent staining of chick retinae during development, a: chick retina (1 or 2 weeks old) stained with anti-visinin serum at a dilution of 1:80,000. The arrow indicates an amacrine cell. b: chick retina stained with visinin-absorbed antiserum at a dilution of 1:1,000. No immunoreactive products were observed, c: 4th-day embryonic retina stained
with anti-visinin serum at a dilution of 1:1,000. d: 7th-day embryonic retina stained with anti-visinin serum at a dilution of 1:80,000. e: 9th-day embryonic retina stained with anti-visinin serum at a dilution of 1:80,000. f: 13th-day embryonic retina stained with anti-visinin serum at a dilution of 1:80,000. g: 16thday embryonic retina stained with anti-visinin serum at a dilution of 1:80,000. h: 19th-day embryonic retina stained with antivisinin serum at a dilution of 1:80,000. OS, outer segments; IS, inner segments; ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer; NL; nuclear layer.
213 ceptor cell layer in chick retina is thought to be cone
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~ ~!~L~ ~
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Fig. 3. Indirect immunofluorescent staining of various vertebrate retinae with anti-visinin serum. These sections were obtained essentially as in Fig. 2. a: human retina stained with antivisinin serum at a dilution of 1:1,000. b: cat retina stained with anti-visinin serum at dilution of 1:1,000. The arrow indicates axonal processes of horizontal cells, c: frog retina stained with anti-visinin serum at a dilution of 1:1,000. d: carp retina stained with anti-visinin serum at a dilution of 1:1,000. The arrow indicates the connecting fibers, e: mouse retina stained with antivisinin serum at a dilution of 1:1,000. Autofluorescence (arrow) was observed in mouse photoreceptor cell layer. All abbreviations are the same as in Fig. 2. was roughly estimated at about 0.5% of soluble protein of retina 13 and the visinin derived only from some amacrine cells and displaced amacrine cells would never reach 0.5% of retinal total soluble protein, we think that visinin mainly originates from p h o t o r e c e p t o r cells. As the dominant cell population of the p h o t o r e -
cells, we considered that visinin was mostly located in cone cells, but the staining intensity of the photoreceptor cell layer was too strong and uniform to confirm it. To clarify this p r o b l e m , we e x a m i n e d other v e r t e b r a t e retinae which contain both rod and cone cells at various ratios (human, cat, frog and carp) or dominantly rod cells (bovine, mouse and rat). Staining of cone cells was clearly found in the p h o t o r e c e p tor cell layer of human, cat, frog and carp retinae, but that of rods was not. On the other hand, no significant stainings were observed in the p h o t o r e c e p t o r cell layer of bovine, mouse and rat retinae. These results may suggest that visinin is located mostly in the cone cells in the v e r t e b r a t e retina b e y o n d species difference, although electron microscopic study is necessary for the precise identification of visinin-containing cells. It is unknown whether or not visinin-like substances in amacrine, horizontal and displaced amacrine cells represent visinin itself, because the antibody against visinin may cross-react with o t h e r related proteins or with many n e u r o p e p t i d e s which exist in the retina, especially in various amacrine cells. Therefore we examined the possibility that neuropeptides cross-react with anti-visinin serum. None of the peptides -tested (substance P, neurotensin, Metenkephalin, Leu-enkephalin, somatostatin and vasoactive intestinal p o l y p e p t i d e , 1-10 /~M) were absorbed by anti-visinin serum when the antisera treated with each peptide were tested for immunohistochemical reactivity. Since recent studies using monoclonal antibodies against retinaZ. 9 show that m a n y of these antibodies react with other neuronal tissues, we are now examining the detection of immunoreactive visinin-like substances in chick brain. Chick liver, kidney and heart were not stained with anti-visinin serum. There are many reports describing the existence of neuropeptides6,10,11,t4,28, neurotransmitter24 and related enzymes 1,5 or some macromolecules 3,7,15,17,20-22,26,27,29 in the retina. Immunohistochemical localization of 2',3'-cyclic nucleotide 3'phosphodiesterase (CNPase) 15, hexokinase 26, L-glutamate aminotransferasel,5 and S-antigen 20,29 are in part similar to that of visinin, but visinin differs from them with respect to its precise distribution, molecular weight and biochemical p r o p e r t i e s such as isoelec-
214 tric point. C a r b o n i c a n h y d r a s e and g l u t a m i n e synthe-
ACKNOWLEDGEMENTS
tase w e r e m a i n l y l o c a t e d on Mtiller cells17, 21. R e t inoid-binding p r o t e i n is l o c a t e d m a i n l y on Miiller cells, but not on n e u r o n a l cells 7. In conclusion, visinin w o u l d be a g o o d m a r k e r for cone cells, and be i n v o l v e d in an i m p o r t a n t f u n c t i o n
W e t h a n k Prof. M. W. H u b e r and Mrs. A . H i n o for reading and typing t h e m a n u s c r i p t . This w o r k was s u p p o r t e d by G r a n t 58570086 for scientific r e s e a r c h f r o m the M i n i s t r y o f E d u c a t i o n , J a p a n .
in the retina, since it is a b u n d a n t in c o n e - c o n t a i n i n g retinae and exists in v e r t e b r a t e r e t i n a e b e y o n d species barriers.
REFERENCES 1 Altschuler, R. A., Mosinger, J. L., Harmison, G. G., Parakkal, M. H. and Wenthold, R. J., Aspartate aminotransferase-like immunoreactivity as a maker for aspartate/glutamate in guinea pig photoreceptors, Nature (Lond.), 298 (1982) 657-659. 2 Barnstable, C. J., Monoclonal antibodies which recognize different cell types in the rat retina, Nature (Lond.), 286 (1980) 231-235. 3 Bloom, W. S. and Puszkin, S., Brain clathrin: immunofluorescent localization in rat retina, J. Histochem. Cytochem., 31 (1983) 46-52. 4 Bradford, M. M., A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding, Analyt. Bioehem., 72 (1976) 248-254. 5 Brandon, C. and Lam, D. M.-K., L-Glutamic acid: a neurotransmitter candidate for cone photoreceptors in human and rat retinas, Proc. nat. Acad. Sci. U.S.A., 80 (1983) 5117-5121. 6 Brecha, N., Karten, H. J. and Laverack, C., Enkephalincontaining amacrine cells in the avian retina: immunohistochemical localization, Proc. nat. Acad. Sci. U.S.A., 76 (1979) 3010-3014. 7 Bunt-Milam, A. H. and Saari, J. C., Immunocytochemical localization of two retinoid-binding proteins in vertebrate retina, J. Cell Biol., 97 (1983) 703-712. 8 Coons, A. H., Fluorescent antibody method. In J. F. Danielli (Eds.), General Cytochernical Methods, Academic, New York, 1958, pp. 399-422. 9 Eisenbarth, G. S., Walsh, F. S. and Nirenberg, M., Monoclonal antibody to a plasma membrane antigen of neurons, Proc. nat. Acad. Sci. U.S.A., 76 (1979) 4913-4917. 10 Ellis, J. P., Sullivan, J. M. and Rana, M. W., Somatostatinlike immunoreactivity in the retinae of adult and embryonic chickens, Proc. Soc. exp. Biol. Med., 172 (1983) 463-471. 11 Fukuda, M., Kuwayama, Y., Shiosaka, S., Inagaki, S., Ishimoto, I., Shimizu, Y., Takagi, H., Sakanaka, M., Takatsuki, K., Senba, E. and Tohyama, M., Localization of vasoactive intestinal polypeptide and neurotensin immunoreactivities in the avian retina, Current Eye Res., 1 (1981) 115-118. 12 Grabar, P. and Williams, C. A., M6thode permettant l'6tude conjugu6e des propri6t6s 61ectrophor6tiques et immunochimiques d'un m61ange de prot6ines. Application au s6rum sanguin, Biochem. biophys. Acta, 10 (1953) 193-194. 13 Hatakenaka, S., Kuo, C.-H. and Miki, N., Analysis of a
distinctive protein in chick retina during development, Develop. Brain Res., 10 (1983) 155-163. 14 Ishimoto, I., Shiosaka, S., Shimizu, Y., Kuwayama, Y., Fukuda, M., Inagaki, S., Takagi, H., Sakanaka, M., Sasaoka, A., Senba, E., Sakiyama, T. and Tohyama, M., Leucine-enkephalin-like immunoreactivity in the chicken retina with a special reference to its fine structures, Invest. Ophthalmol. Vis. Sci., 24 (1983) 879-885. 15 Kohsaka, S., Nishimura, Y., Takamatsu, K., Shimai, K. and Tsukada, Y., Immunohistochemical localization of 2' ,3'-cyclic nucleotide 3'-phosphodiesterase and myelin basic protein in the chick retina, J. Neurochem., 41 (1983) 434-439. 16 Laemmli, U. K., Cleavage of structural proteins during the assembly of the head of bacteriophage T4, Nature (Lond.), 227 (1970) 680-685. 17 Linser, P. and Moscona, A. A., Carbonic anhydrase C in the neural retina: transition from generalized to glia-specific cell localization during embryonic development, Proc. nat. Acad. Sci. U.S.A.. 78 (1981) 7190-7194. 18 Lowry, O. H., Rosebrough, N. J., Farr, A. L. and Randall, R. J., Protein measurement with the Folin phenol reagent, J. biol. Chem., 193 (1951) 265-275. 19 Meller, K. and Tetzlaff, W., Scanning electron microscopic studies on the development of the chick retina, Cell Tiss. Res., 170 (1976) 145-159. 20 Meyers-Elliott, R. H., Jacobs, D. R. and Gammon, R. A., Localization of specific autoantibodies in the retinal photoreceptor cell layer in experimental retinal autoimmunity, J. Neuroimmunol., 4 (1983) 25-34. 21 Moscona, A. A., Mayerson, P., Linser, P. and Moscona, M., Induction of glutamine synthetase in the neural retina of the chick embryo: localization of the enzyme in Miiller fibers and effects of BrdU and cell separation. In E. Giacobini, A. Vernadakis and A. Shahar (Eds.), Tissue Culture in Neurobiology, Raven, New York, 1980, pp. 111-127. 22 Nguyen-Legros, J., Vigny, A. and Gay, M., Post-natal development of TH-like immunoreactivity in the rat retina. Exp. EyeRes., 37 (1983) 23-32. 23 Olson, M. D., Scanning electron microscopy of developing photoreceptors in the chick retina, Anat. Rec., 193 (1979) 423-438. 24 Osborne, N. N., Uptake, localization and release of serotonin in the chick retina, J. Physiol. (Lond.), 331 (1982) 469-479. 25 Ouchterlony, O., Diffusion-in-gel methods for immunological analysis, II. In P. Kallosnd and B. H. Waksman (Eds.), Progress in Allergy. Vol. VI, Karger, Basel, 1962, pp. 30-154.
215 26 Simurda, M. and Wilson, J. E., Localization of hexokinase in neural tissue: immunofluorescence studies on the developing cerebellum and retina of the rat, J. Neurochem., 35 (1980) 58-66. 27 Terenghi, G., Cocchia, D.I Micherri, F., Diani, A. R., Peterson, T., Cole, D. F., Bloom, S. R. and P01ak, J. M., Localization of S-100 protein in MOiler cells of the retina, 1. Light microscopical immunocytochemistry, Invest. Ophthalmol. Vis. Sci., 24 (1983) 976-980. 28 Tornqvist, K., Lor6n, I., HAkanson, R. and Sundler, F.,
Peptide-containing neurons in the chicken retina, Exp. Eye Res, 33 (1981) 55-64. 29 Wacker, W. B., Donoso, L. A., Kalsow, C. M., Yankeelov, Jr., J. A. and Organisciak, D. T., Experimental allergic uveitis. Isolation, characterization and localization of a soluble uveitopathogenic antigen from bovine retina, J. lmmunol., 119 (1977) 1949-1958. 30 Zamboni, L. and De Martino, C., Buffered picric-acid formaldehyde: a new rapid fixative for electromicroscopy, J. Cell Biol., 35 (1967) 148A.
209
BRE 10644
Immunohistochemical Localization of Chick Retinal 24 kdalton Protein (Visinin) in Various Vertebrate Retinae SACHIKO HATAKENAKA 1, HIROSHI KIYAMA2, MASAYA TOHYAMA 2, NAOMASA MIKI 1
JDepartment of Pharmacology, Cancer Research Institute, Kanazawa University, 13-1 Takaramachi, Kanazawa 920, and 2Department of Neuroanatomy, Institute of Higher Nervous Activity, Osaka University Medical School, 4-3-57, Nakanoshima, Kitaku, Osaka 530 (Japan) (Accepted July 10th, 1984)
Key words: retina - - visinin - - immunohistochemistry - - development - - cone cell
Antiserum against a protein (24,000 daltons, visinin) of chick retina has been provided for immunohistochemicat study on the localization of visinin in chick retinae during development, as well as in various vertebrate retinae. The photoreceptor cells were stained with anti-visinin serum from 7th day embryonic retinae and its intensity was gradually increased with embryonic age. In addition, visinin-like immunoreactivity was found in some kinds of amacrine and displaced amacrine cells from 1lth-day embryonic retinae. When human, cat, frog and carp retinae which contain both rods and cones were examined, staining of cone cells was clearly observed in the photoreceptor cell layer, but not in the rods. Furthermore visinin-like immunoreactivity was barely detectable in the photoreceptor cells of bovine, rat and mouse retinae containing mostly rod cells. These results suggest that visinin is mainly located in the cone cells in various vertebrate retinae and is a good marker for the cone cells.
INTRODUCTION A key to understanding the function of the nervous system is to clarify the biochemical p r o p e r t i e s and histochemical localization of proteins specific to nervous tissues. In our previous report~3, we have analyzed the soluble proteins of chick retina during development and r e p o r t e d that a p e p t i d e of about 24,000 daltons (24 k d a l t o n protein, designated as 'visinin') a p p e a r s in the retina of the 14th day e m b r y o and gradually increases in concentration with embryonic age until hatching. It is not d e t e c t e d in the cerebrum, rectum, pigment epithelium or vitreous body on s o d i u m dodecyl sulfate-polyacrylamide gel analysis. W e p r o p o s e d that visinin is a distinctive protein that increases in concentration during chick retinal d e v e l o p m e n t and would be closely associated with retinal function. In this p a p e r we p r o d u c e d an antibody against visinin and e x a m i n e d the distribution of immunoreactive visinin in chick retina during d e v e l o p m e n t and com-
pared the labeling patterns of various v e r t e b r a t e retinae using an indirect immunofluorescent staining method. MATERIALS AND METHODS
Purification o f visinin The partially purified visinin was p r e p a r e d from soluble proteins of adult chick retina by gel filtration and ion exchange column c h r o m a t o g r a p h y as previously described 13. Visinin was then purified by extracting a corresponding b a n d from sodium dodecyl sulfate-polyacrylamide gels ( S D S - P A G E ) . The purified visinin migrated as a single band on SDSP A G E (Fig. 1A). Protein was assayed by the m e t h o d of Lowry et alA8 with bovine serum albumin as a standard, or by the m e t h o d of B r a d f o r d when Tris was present 4. Preparation o f anti-visinin serum Anti-visinin serum was p r o d u c e d in a New Z e a -
Correspondence: N. Miki, Department of Pharmacology, Cancer Research Institute, Kanazawa University, 13-1 Takaramachi, Kanazawa 920, Japan. 0006-8993/85/$03.30 © 1985 Elsevier Science Publishers B.V. (Biomedical Division)
210 land white rabbit as follows. Purified visinin was mixed with an equal volume of Freund's complete adjuvand and injected into the foot pads and backs in a rabbit (about 3 kg, 300gg/injection) every week for 4 weeks. One week after the last injection, an anti-serum was obtained. The specificity of anti-visinin serum was analyzed by an Ouchterlony double-immunodiffusion test 25 and immunoelectrophoresisl2. Ouchterlony double-immunodiffusion test was carried out in 1% Agar plate containing phosphate-buffered saline (pH 7.4). Immunoelectrophoresis was executed in 1% Agar plate with barbital buffer (pH 8.6, ion strength 0.05). The samples were placed in the wells and electrophoresis was carried out for 1 h at 15 mA (2 mA/cm) at room temperature, and then rabbit anti-visinin serum was placed into the trough. Precipitation lines which developed after 24 h incubation at room temperature were stained with Amide Black 10B. Rabbit IgG was purified by ammonium sulfate precipitation (0-40%) and D E A E cellulose column chromatography eluted with 17.5 mM phosphate buffer (pH 6.3).
Immunohistochemistry Retinae from chick embryos, 1 or 2 week old chicks and frog (Rana catesbiana) were fixed as follows. The animals (after 16th day embryo) were perfused intracardially with ice-cold saline (about 50 ml) followed by ice-cold Zamboni's fixative buffer30 (0.21% picric acid, 2% paraformaldehyde and 130 mM phosphate buffer (pH 7.4), about 500 ml) under sodium pentobarbitone anesthesia (40 mg/kg, i.p.). The eyes were dissected out, post-fixed in the same fixative buffer for 1-2 days and washed in 30% sucrose containing 0.1 M phosphate buffer (pH 7.4) for 2-3 days at 4 °C. The retinae from chick embryo earlier than 16th day or carp (Cyprinus carpio, 700-800 g) were treated by the same method except for the perfusion procedure. Isolated bovine eyes were obtained from a local slaughter house and human retina was from an eye for corneal transplantation. These were fixed with Zamboni's fixative buffer as described above. Frozen sections were cut in a cryostat with section thickness of 10-20/~m. These sections were mounted on chrome-alum-gelatin coated glass slides, air-dried for 2-3 h at room temperature and treated with anti-visinin serum according to CoonsS. All sections were rinsed with ice-cold phosphate buf-
fered saline (PBS) for 10 min prior to the beginning of the immunohistochemical procedure. Some of these sections were incubated in a humid atmosphere at 25 °C with anti-visinin serum (at dilutions of 1:1,000 to 80,000) overnight, and then were rinsed with ice-cold PBS for 10 min, and 1.0% Triton X-100 containing PBS for 10 min. The washing procedure was repeated twice. After washing the sections were incubated with fluorescein isothiocyanate (FITC)conjugated goat anti-rabbit IgG (at a dilution of 1: 1,000) overnight at 25 °C. The sections were rinsed by the previously described washing procedure and mounted in PBS-glycerin mixture. The remaining sections were incubated with preimmune serum, visinin-absorbed antiserum or FITC-conjugated goat anti-rabbit IgG only as a control staining. One section of each retina was stained with hematoxylin and eosin for morphological orientation.
SDS-polyacrylamide PAGE)
gel
electrophoresis (SDS-
SDS-PAGE was carried out by the method of Laemmli 16 with a slight modification as previously described 13.
Chemicals Acrylamide, Agar and other reagents for gel electrophoresis (electrophoresis grade) were from Wako Pure Chemical Industries Ltd. Sodium dodecyl sulfate, Coomassie brilliant blue R-250, Amide black 10B, Triton X-100, picric acid and paraformaldehyde were purchased from Nakarai Chemicals. FITC-conjugated goat anti-rabbit IgG and D E A E cellulose (DE 52) were obtained from Miles Co. Ltd. and Whatman, respectively. RESULTS
Specificity of anti-visinin serum The specificity of anti-visinin serum was examined by an Ouchterlony double-immunodiffusion test and immunoelectrophoresis against electrophoretically purified or partially purified visinin. The anti-serum showed a single precipitation line against purified or partially purified visinin (Fig. 1B, C). This anti-serum did not give any precipitation lines with membrane fractions of chick retina, soluble proteins of chick cerebrum, tectum or bovine retina (data not shown).
211
A
~ ~i~Z¸
B
®
Fig. 1. Preparation of anti-visinin serum. A: electrophoretic pattern of 3/~g (1) and 6/lg (2) of electrophoretically purified visinin on SDS-polyacrylamidegel (11%). The arrow indicates the visinin. Protein was stained with Coomassie Brilliant Blue R-250. B: Ouchterlony double-immunodiffusionof anti-visinin serum against an electrophoretically purified visinin. The center well (1) contained 3/~gof purified visinin. Wells 2 and 4 contained 15/~1 of preimmune serum and anti-visinin serum, respectively. Wells 3 and 5 were filled with 10/~1 (3 mg protein/ml) of IgG from preimmune serum and IgG from anti-visinin serum, respectively. C: immunoelectrophoresis of purified visinin and partially purified visinin. 1: 5/~g of electrophoretically purified visinin. 2:20/~g of partially purified visinin. The trough was filled with 50/A of anti-visinin serum. The anode is to the right. Protein was stained with Amide Black 10B.
lmmunohistochemistry of visinin in developing chick retinae Immunohistochemical examination of 1- or 2week-old chick retina was shown in Fig. 2a. Strong visinin-like immunoreactivity was found in the photoreceptor cell layer. The staining intensity of inner segments was more than that of the outer nuclear layer, but no significant labeling was observed in outer segments of photoreceptor cells (Fig. 2a). In addition, scattering neuronal cells, which are morphologically identified to amacrine (Fig. 2a, arrow) and displaced amacrine cells, were also stained with anti-visinin serum. Some amacrine cells were found to extend the processes into the inner plexiform layer. On the other hand, Miiller cells were not stained. Sections incubated with visinin-absorbed antiserum or preimmune serum were devoid of any immunocytochemical reaction (Fig. 2b). As visinin increased with retinal development 13, we compared the staining patterns of visinin in the retinae from embryo to chicken. No significant staining was detected (Fig. 2c) in 4th-day embryonic retinae. In 7th-day embryonic retinae, putative photo-
receptor cells were apparently stained (Fig. 2d), and the staining population of the peripheral part of the retina was less dense than that of central part (data not shown). Cells migrating from the inner to outer regions of the retina were also stained. In 9th-day embryonic retinae, many connecting fibers between photoreceptor cell layer and ganglion cell layer were also labeled (Fig. 2e), and the staining intensity and population of photoreceptor cells increased. Putative amacrine and other neuronal cells in the inner nuclear layer began to be stained in llth-day embryonic retinae and this gradually increased by 13th-day embryo (Fig. 2f). Although inner segments of photoreceptor cells appear at this stage19, 23, they were not distinguished from the outer nuclear layer in our experiment. In 16th-day embryonic retinae, the staining pattern of whole retinae was essentially the same as that of chick, but inner segments of photoreceptor cells still were not fully developed in this section (Fig. 2g). The processes extending from amacrine cells were stained weakly but apparently observed. In 19th-day embryonic retinae, all inner segments of photoreceptor cells were elongated and labeled as in the adult retina (Fig. 2h). However, outer segments of photoreceptor cells were not stained. The processes of amacrine cells were clearly observed at this stage.
Immunohistochernistry of visinin-like substances in various vertebrate retinae As the dominant cell population of the photoreceptor cell layer in chick retina is thought to be cone cells, we compared the staining patterns of visinin in human, cat, frog and carp retinae which contain both rod and cone cells in various ratios (Fig. 3). A dilution of 1:1,000 serum was used for all animals except chick. The patterns of visinin-like immunoreactivity of human and cat retinae were similar (Fig. 3a, b). Staining of cone cells was clearly observed in the photoreceptor cell layer, but that of rods was not. In the inner nuclear layer, staining was detected in horizontal cells with axonal processes (Fig. 3a, b, arrow). The staining intensity of amacrine cells was rather weak in human retina, although in chick retina some amacrine cells and their processes were clearly observed. In frog and carp retinae, most of the cone cells were stained, but horizontal cells were hardly observed in the inner nuclear layer and not distin-
212 guished from synaptic terminals of cone cells. Furthermore, none of the amacrine cells were stained in frog and carp retinae (Fig. 3c, d). On the other hand, it has been known that a dominant cell group in the photoreceptor layer is that of rod cells in bovine, rat and mouse retinae. I m m u n o histochemical staining with anti-visinin s e r u m in these animals showed that staining was very poor or not observed in the photoreceptor cell layer, but relatively good in horizontal, amacrine and displaced amacrine cells (Fig. 3e). Immunoreactive visinin was not found in MOiler cells in any of these sections. No significant staining was found in visinin-absorbed antiserum or preimmune serum-treated sections. DISCUSSION We have examined the immunohistochemical localization of visinin in developing chick neural retinae and found that the immunoreactive visinin appeared in 7th-day embryonic retinae and thereafter gradually increased with embryonic age, and was mostly located in the photoreceptor cell layer and in part in the inner nuclear layer. No significant staining was observed in Miiller cells in any embryonic ages and adult retinae. Changes in the visinin-stainingpattern during retinal development well support our previous data in which visinin was analyzed by a SDSP A G E 13, although the detection of visinin with immunohistochemical study was much more sensitive than that on a S D S - P A G E . We estimated that visinin of chick retina would be mostly located in photoreceptor cells judging from this immunohistochemical study. But other neuronal cells which are morphologically identified to amacrine and displaced amacrine cells were also reacted with anti-visinin serum. Since the content of visinin
Fig. 2. Indirect immunofluorescent staining of chick retinae during development, a: chick retina (1 or 2 weeks old) stained with anti-visinin serum at a dilution of 1:80,000. The arrow indicates an amacrine cell. b: chick retina stained with visinin-absorbed antiserum at a dilution of 1:1,000. No immunoreactive products were observed, c: 4th-day embryonic retina stained
with anti-visinin serum at a dilution of 1:1,000. d: 7th-day embryonic retina stained with anti-visinin serum at a dilution of 1:80,000. e: 9th-day embryonic retina stained with anti-visinin serum at a dilution of 1:80,000. f: 13th-day embryonic retina stained with anti-visinin serum at a dilution of 1:80,000. g: 16thday embryonic retina stained with anti-visinin serum at a dilution of 1:80,000. h: 19th-day embryonic retina stained with antivisinin serum at a dilution of 1:80,000. OS, outer segments; IS, inner segments; ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer; NL; nuclear layer.
213 ceptor cell layer in chick retina is thought to be cone
~
~ ~!~L~ ~
¸¸
Fig. 3. Indirect immunofluorescent staining of various vertebrate retinae with anti-visinin serum. These sections were obtained essentially as in Fig. 2. a: human retina stained with antivisinin serum at a dilution of 1:1,000. b: cat retina stained with anti-visinin serum at dilution of 1:1,000. The arrow indicates axonal processes of horizontal cells, c: frog retina stained with anti-visinin serum at a dilution of 1:1,000. d: carp retina stained with anti-visinin serum at a dilution of 1:1,000. The arrow indicates the connecting fibers, e: mouse retina stained with antivisinin serum at a dilution of 1:1,000. Autofluorescence (arrow) was observed in mouse photoreceptor cell layer. All abbreviations are the same as in Fig. 2. was roughly estimated at about 0.5% of soluble protein of retina 13 and the visinin derived only from some amacrine cells and displaced amacrine cells would never reach 0.5% of retinal total soluble protein, we think that visinin mainly originates from p h o t o r e c e p t o r cells. As the dominant cell population of the p h o t o r e -
cells, we considered that visinin was mostly located in cone cells, but the staining intensity of the photoreceptor cell layer was too strong and uniform to confirm it. To clarify this p r o b l e m , we e x a m i n e d other v e r t e b r a t e retinae which contain both rod and cone cells at various ratios (human, cat, frog and carp) or dominantly rod cells (bovine, mouse and rat). Staining of cone cells was clearly found in the p h o t o r e c e p tor cell layer of human, cat, frog and carp retinae, but that of rods was not. On the other hand, no significant stainings were observed in the p h o t o r e c e p t o r cell layer of bovine, mouse and rat retinae. These results may suggest that visinin is located mostly in the cone cells in the v e r t e b r a t e retina b e y o n d species difference, although electron microscopic study is necessary for the precise identification of visinin-containing cells. It is unknown whether or not visinin-like substances in amacrine, horizontal and displaced amacrine cells represent visinin itself, because the antibody against visinin may cross-react with o t h e r related proteins or with many n e u r o p e p t i d e s which exist in the retina, especially in various amacrine cells. Therefore we examined the possibility that neuropeptides cross-react with anti-visinin serum. None of the peptides -tested (substance P, neurotensin, Metenkephalin, Leu-enkephalin, somatostatin and vasoactive intestinal p o l y p e p t i d e , 1-10 /~M) were absorbed by anti-visinin serum when the antisera treated with each peptide were tested for immunohistochemical reactivity. Since recent studies using monoclonal antibodies against retinaZ. 9 show that m a n y of these antibodies react with other neuronal tissues, we are now examining the detection of immunoreactive visinin-like substances in chick brain. Chick liver, kidney and heart were not stained with anti-visinin serum. There are many reports describing the existence of neuropeptides6,10,11,t4,28, neurotransmitter24 and related enzymes 1,5 or some macromolecules 3,7,15,17,20-22,26,27,29 in the retina. Immunohistochemical localization of 2',3'-cyclic nucleotide 3'phosphodiesterase (CNPase) 15, hexokinase 26, L-glutamate aminotransferasel,5 and S-antigen 20,29 are in part similar to that of visinin, but visinin differs from them with respect to its precise distribution, molecular weight and biochemical p r o p e r t i e s such as isoelec-
214 tric point. C a r b o n i c a n h y d r a s e and g l u t a m i n e synthe-
ACKNOWLEDGEMENTS
tase w e r e m a i n l y l o c a t e d on Mtiller cells17, 21. R e t inoid-binding p r o t e i n is l o c a t e d m a i n l y on Miiller cells, but not on n e u r o n a l cells 7. In conclusion, visinin w o u l d be a g o o d m a r k e r for cone cells, and be i n v o l v e d in an i m p o r t a n t f u n c t i o n
W e t h a n k Prof. M. W. H u b e r and Mrs. A . H i n o for reading and typing t h e m a n u s c r i p t . This w o r k was s u p p o r t e d by G r a n t 58570086 for scientific r e s e a r c h f r o m the M i n i s t r y o f E d u c a t i o n , J a p a n .
in the retina, since it is a b u n d a n t in c o n e - c o n t a i n i n g retinae and exists in v e r t e b r a t e r e t i n a e b e y o n d species barriers.
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