THE JOURNAL OF COMPARATIVE NEUROLOGY 302:417-424 (1990)
Localization of Two Calcium Binding Proteins,Calbindin (281)) and ParValbumin (12 kD),in the Vertebrate Retina K. HAMANO, H. KIYAMA, P.C. EMSON, R. MANABE, M. NAKAUCHI, AND M. TOHYAMA Department of Ophthalmology (K.H., R.M.), Biomedical Center (H.K.), Department of Anatomy (M.N.), Osaka University Medical School, Kitaku, Osaka 530, Japan; MRC Group (P.C.E.), Institute of Animal Physiology and Genetics Research, Babraham, Cambridge CB2 4AT, United Kingdom
ABSTRACT We used immunocytochemistry to locate two calcium binding proteins, calbindin (CaB) and parvalbumin (PV), in the retina of goldfish, frog, chick, rat, guinea pig, dog, and man. The location of CaB depended on the type of dominant photoreceptor cells in birds and mammals. In cone-dominant retinas such as those of the chick, CaB-like immunoreactivity was found in the cones, cone bipolars, and ganglion cells. Amacrine cells 5-12 p,m across were also labeled. In rod-dominant retinas, such as those of the rat, guinea pig, and dog, horizontal cells, small amacrine cells (about 6 pm across), and cells in the ganglion cell layer were labeled. In the human retina, which has both cones and rods in abundance, cones, cone bipolars, ganglion cells, horizontal cells, and small and large amacrine cells were labeled. In the frog and goldfish, the level of CaB-like immunoreactivity was low. In the frog, a few cones, amacrine cells, and cells in the ganglion cell layer were labeled. No immunoreactive structures were seen in the goldfish retina. PV-like immunoreactivity was found in chicks, rats, and dogs. No such immunoreactive structures were seen in the other species. In the chick, only amacrine cells were labeled. In the rat, amacrine cells and several displaced amacrine cells were labeled. In the dog, in addition to amacrine cells and displaced amacrine cells, horizontal cells were strongly labeled. Thus, PV-like immunoreactivity was found in those elements relating to the modulation of the main pathway of the visual transmission system. Key words: calbindin, parvalbumin, retina, immunohistochemistry
Intracellular Caz+is important as a "second messenger" postsynaptically. It has various metabolic effects when bound to the regulatory subunit of cyclic-AMP-dependent protein kinases in the central and peripheral nervous systems. Calcium-binding proteins seem to maintain the intracellular Ca2+concentration at an appropriate level. In the nervous system, two calcium binding proteins, calbindin (CaB), which is vitamin-D-dependent (Wasserman and Taylor, '661, and parvalbumin (PV), which is water-soluble are widely but unevenly distrib(Celio and Heizmann, %l), uted (Jande and Maler, '81; Roth et al., '81; Legrand et al., '83; Heizmann, '84). CaB and PV have been found in the retina (Endo et al., '85; Rabie et al., '85; Schreiner et al., '85; Endo et al., '86; Verstappen et al., '86; Rohrenbeck et al., '87). In chick retina, photoreceptor cells, horizontal cells, and some bipolar cells contain CaB-like immunoreac-
o 1990 WILEY-LISS, INC.
tive (CaBI) structures (Schreiner et al., '85). In rat, mouse, and rabbit retinas, horizontal cells and some amacrine cells contained CaB-like immunoreactivity. In mouse and rat, some ganglion cells are also labeled by antiserum against CaB (Schreiner et al., '85). Cats and monkeys contains CaBI horizontal cells (Rohrenbeck et al., '87). In the human retina, photoreceptor, horizontal, some amacrine, some ganglion, and some bipolar cells are labeled (Verstappen et al., '86). PV-like immunoreactivity has also been examined in retinas of several vertebrates (Endo et al., '85, '86). In the rat retina, PV is found in the amacrine cells. In monkeys and humans, in addition to amacrine cells, PV immunoreactivity has also been found in horizontal cells (Endo et al., Accepted August 29,1990.
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'86). Thus, there are species differences in the location of retinal CaB and PV. Here, we examined the location of CaB and PV in the retina of seven species, from fish to human, in an endeavour to understand the reasons for their distribution.
MATEXIALSANDMETHODS Preparation of tissues The eyeballs of three goldfish and three dogs were removed while the animals were under sodium pentobarbital anesthesia (10 mg/kg). Three frogs, three chicks, four rats, and three guinea pigs were perfused transcardially under sodium pentobarbital anesthesia (10 mgkg) with ice-cold saline (50-100 ml) followed by Zamboni's fixative (300 ml/lOO g) at 4°C (Zamboni and Martino, '76). The eyes were removed, cut open, and immersed in the same fixative for 2-3 days. Two human eyes enucleated because of intra-orbital metastasis of malignant tumors were placed in the same fixative for about a week. All the specimens were then immersed overnight in 0.1 M phosphate buffer containing 30% sucrose, frozen, and cut into sections 10-15 Fm thick on a cryostat.
Definition of sublayers in the inner pldorm layer of retinas The process which defined the sublayers of the inner plexiform layer in chick retina was based on that of Cajal (Cajal, '72) and Mark (Mark, '86). Since the classification of this layer in other animals is confusing or difficult at present (Mark, '86), we divided the inner plexiform layer into five equal sublayers for all retinas and numbered them in distal-proximal sequence (sublayer 1, sublayer 2, . . . sublayer 5) in this study.
RESULTS 1) Distribution of CaBI structures in vertebrate retina
Goldfish retina. No immunoreactive structures were found. Frog retina (Fig. 1). A few immunoreactive photoreceptor cells were seen (Fig. 1A,B, arrowheads). Although positive cells were distributed throughout the retina, the positive cells in the central area (about 30% cells were labeled) outnumbered those in the peripheral area (less than 5%).The outer segment of these cells lacked immunoreactivity. Strong immunoreactivity was found in the inner segment. The bodies of these cells were weakly labeled. To Immunohistochemidprocedures judge from the location of the labeled bodies, these cells To stain CaBI structures, sections were rinsed in 0.02 M seemed to be cones (Cajal, '72; Llinas and Precht, '76). A phosphate-buffered saline (PBS), 0.3% Triton X-100 contain- few immunoreactive cells and fibers were seen in the ing 0.02 M PBS, and 0.02 M PBS for 10 minutes each, innermost part of the inner nuclear (Fig. lA,B, arrows) and incubated in a humid chamber with antichick intestinal inner plexiform layers. These cells seemed to be amacrine CaB antiserum raised in a rabbit (Ichimiya et al., '88) at 4°C cells. There were also some immunoreactive cells in the overnight, rinsed again as above, and incubated with goat ganglion cell layer (Fig. lA, double arrow). No immunoreacantirabbit immunoglobulin G (IgG) serum conjugated with tive fibers were seen in the nerve fiber layer (Fig. 1). fluorescein isothiocyanate (FITC) (Miles) at 4°C overnight. Chick retina (Fig. 2). A number of photoreceptor To stain PVI structures, sections were rinsed as for CaB cells were labeled by the antiserum (Fig. 2A,B). Immunoreimmunoreactivity, incubated with antirat muscle PV antise- active bodies of the photoreceptor cells were long and rum raised in sheep (donated by Dr. P.C. Emson) at 4°C columnar, and were arranged more or less in a row. Stout, overnight, rinsed as above, and then incubated with rabbit smooth immunoreactive processes projected from the inner antisheep IgG serum conjugated with FITC (Cappel, U.S.A.) bases of these bodies, particularly in the central retina. at 4°C overnight. Samples were all rinsed once more before They reached the middle part of the outer plexiform layer being mounted in a 1:l mixture of glycerin and PBS. The and ended in a thick pedicle. The shape of the immunoreacantisera were diluted before use with 0.02 M PBS as tive photoreceptors suggested that they are cones (Meyer follows, CaB, PV, and goat antirabbit IgG serum, 1:1,000; and Cooper, '66; Morris and Shorey, '67; Cajal, '72; Fig. rabbit antisheep IgG serum, 1500. lA,B). In the inner nuclear layer, three groups of immunoreactive cells were seen (Fig. 2A). The first group formed an immunoreactive band of two to three rows of cells at the Specificityof the primary antisera outer part of this layer. Singular immunoreactive processes Rabbit anti-CaB antiserum was produced against CaB, arose from the outer base of the immunoreactive cell bodies M.W. 28,000, purified by high-pressure liquid chromatogra- (Fig. 2A,B) and passed through the outer part of this layer phy after isolation from chick intestine (Spencer et al., '78). terminating on the cone pedicles (Fig. 2B, arrows). Thus, Western blotting has shown that the antiserum recognizes these cells were cone bipolars. The second group consisted CaB of molecular weights of 28,000 and 30,000 in mamma- of a few immunoreactive cells in the middle of this layer lian brains (Ichimiya et al., '88) and also calretinin, M.W. (Fig. 2A, arrowheads). They were probably bipolar cells 29,000 (Rogers, '87). Sheep anti-PV antiserum was raised because their cell bodies were similar to those of the first against purified PV from rat muscle and demonstrated group in size and shape, and because there were both satisfactory specificity by Western blotting (Emson, per- descending and ascending processes from their cell bodies. sonal communication).The specificities of the antisera were The last group of cells was scattered in the inner half of the also checked by absorption tests. Structures were not inner nuclear layer (Fig. 2A). These cells varied in size: stained with either CaB antiserum in those sections stained most of them were small to medium and a few were large. with antiserum pre-absorbed by purified CaB from chick The large immunoreactive cells and some of the small to M or PV antiserum medium-sized immunoreactive cells at the inner surface of intestine a t a final concentration of in sections stained with the antiserum pre-absorbed by the inner nuclear layer seemed to be stratified amacrine cells because single immunoreactive processes appeared M. purified rat muscle PV at a final concentration of
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Fig. 1. Fluorescence photomicrographs showing the distribution of CaBI structures in the frog retina. Some cones (arrowheads) and a few amacrine cells (arrows) were labeled. In addition, immunoreactive cells were seen in the ganglion cell layer (double arrow),but none were found
in the nerve fiber layer. ONL: Outer nuclear layer. INL: Inner nuclear layer. IPL: Inner plexiform layer. GL: Ganglion cell layer. A,B: Central part of the frog retina. Bar, 50 pm.
from the inner bases of the cell bodies to form immunoreactive fiber bands of different densities at sublayers 1 , 2 , and 4 in the inner plexiform layer (Cajal, '72) (Fig. 2C, arrows). The highest density was in sublayer 2. Sublayer 4 was moderately dense, but sublayers 3 and 5 contained disseminated immunoreactive fibers (Fig. 2A,C). In the ganglion cell layer, a number of immunoreactive cells were seen. Medium-sized immunoreactive cells were most abundant. Immunoreactive varicose fibers were often seen in the nerve fiber layer (Fig. 2A,D, arrows), so it was likely that at least some of them were ganglion cells that project into the central nervous system.
R a t retina (Fig. 3A). No immunoreactive cells were detected in the photoreceptor cell layer. In the outermost part of the inner nuclear layer, several large immunoreactive cells (about 12 pm in diameter) were found. Processes from these cells formed an immunoreactive fiber plexus of high density in the inner part of the outer plexiform layer. To judge from these findings, they were horizontal cells. On the inner surface of the inner nuclear layer, several small round cells were observed. Single processes from these cells formed fiber plexuses with low density in sublayers 1,3,and 4 of the inner plexiform layer. In the ganglion cell layer, some cells were labeled. They were small (about 5 pm) to
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Fig. 2. Fluorescence photomicrographs showing the distribution of CaBI structures in the chick retina. A number of cones (A and B), cells in the outer half of the INL, a few cells in the middle of the INL (A, arrowheads), several cells of various sizes in the inner half of the INL (A-C), and some cells in the GL (A, C, and D) contained CaB-like immunoreactivity. Immunoreactive fibers formed fiber bands in sublayers 1, 2, and 4 in the IPL (A, C). In B, singular immunoreactive processes arising from the immunoreactive cells in the outer half of the
INL extended to the outer plexiform layer (OPL) and terminated on single immunoreactive cone pedicles (arrows), indicating that these cells were cone bipolars. In C , single processes (arrows) arisingfrom the immunoreactive cells in the inner half of the INL terminated in the IPL, indicating that these cells were amacrine cells. In the nerve fiber layer (NFL), a group of immunoreactive varicose fibers were seen (A and D, arrows), suggesting that the immunoreactive cells in the GL were ganglion cells. A-D: Central part of the chick retina. Bar, 50 pm.
large (about 10 pm) but medium-sized cells were most abundant. There were no immunoreactive fibers in the nerve fiber layer; therefore most of these cells seemed to be displaced amacrine cells. Guinea p i g retina (Fig. 3B). The distribution pattern of CaBI structures in guinea pig retina was similar to that of rats. Immunoreactive neurons were horizontal, amacrine, and those in the ganglion cell layer. Labeled amacrine cells were more numerous in the guinea pig retina than in that of the rat, so the immunoreactive fiber bands found in the inner plexiform layer were denser than in the rat. In the outer nuclear layer of the guinea pig retina, slender fiber-like or pillar-like structures that extended
from outer to inner limiting membranes were often seen. These may belong to Muller cells (Fig. 4B, arrows). Dog retina (Fig. 3C,D). The distribution pattern of CaBI structures in dog retina was similar to that of rats. However, there were more labeled amacrine cells in the dog retina than in the rat, and the immunoreactive fiber plexus in the inner plexiform layer of dogs was denser than that in the rat. Human retina (Fig. 4). As in the chick retina, many cone-like photoreceptor cells were strongly labeled. The highest number of positive cells was identified in the central portion. Immunoreactivity was found in their bodies and inner fibers. In the inner nuclear layer, three groups of
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Fig. 3. Fluorescence photomicrographs showing the distribution of CaBI structures in rod-dominant retinas of (A) rat, (B) guinea pig, and (C and D) dog. In these retinas, horizontal cells were strongly labeled. A dense immunoreactive fiber plexus from these cells was seen in the inner part of the OPL. Several small amacrine cells in the INL and some cells in the GL were labeled. No immunoreactive fibers were seen in the GL. The distribution and density of the immunoreactive fiber plexuses
in the IPL were different in different species. In rat retina, the plexuses were in sublayers 1, 4,and 5, and faintly labeled. In guinea pig retina, they were in sublayers 2, 3, and 4,and densely labeled. In dog retina, they formed two lines, but were distributed diffusely. In the guinea pig retina, some Muller cells were stained by the antiserum (B, arrows). A-D, central retina. Bar, 50 km.
immunoreactive neurons were seen. One was of horizontal cells. There were fewer immunoreactive horizontal cells in the human retina than in the retinas of rat, guinea pig, or dog. Processes of the immunoreactive horizontal cells formed a fiber plexus of low density at the inner part of the outer plexiform layer. The second group was of a few cells in the middle part of the inner nuclear layer (Fig. 4A,B, arrows). Two processes projected from opposite sides of the soma, indicating that these neurons were bipolar cells. The third group was of amacrine cells, which were in the innermost part of the inner nuclear layer. Ascending processes from these neurons terminated diffusely in the inner plexiform layer. The ganglion cell layer contained labeled cells of various sizes. Fiber processes from large labeled cells in the innermost part of the ganglion cell layer were
seen in the nerve fiber layer (Fig. 4C, arrowhead). These labeled cells seemed to be ganglion cells.
2) Distributionof PVI s t r u b in the vertebrate retina Goldfish and frog retinas. No immunoreactive structures were found. Chick retina (Fig. 5A). Several amacrine cells were labeled by PV antiserum. Their somata were about 7 km in diameter and were located in the inner half of the inner nuclear layer (Fig. 5A, arrows). Single processes of labeled amacrine cells ascended the inner plexiform layer to form immunoreactive fiber bands of low density at sublayers 1,3, and 4. Other retinal elements lacked PV immunoreactivity.
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Guinea p i g retina. No immunoreactive structures were found. Rat retina (Fig. 5B). A few amacrine cells were labeled. These cells could be classified into two types by the
K. HAMANO ET AL. arborization pattern of their processes (Fig. 5B, arrows). Somata of both types of amacrine cells were about 7 p,m across and located in the innermost part of the inner nuclear layer. One type emitted a single process straight to sublayer 5 of the inner plexiform layer without ramification. The other had processes that arborized immediately in sublayer 1 and did not ascend further. No other elements were labeled. A few immunoreactive cells were seen in the ganglion cell layer, but no immunoreactive fibers were observed. Dog retina (Fig. 5C). A number of horizontal cells were labeled in which the processes formed a densely packed immunoreactive fiber plexus at the inner part of the outer plexiform layer. A few amacrine cells were also labeled (Fig. 5C, arrows). Their processes terminated diffusely in the inner plexiform layer. No immunoreactive fibers were observed in the nerve fiber layer. Human retina. No immunoreactive structures were observed.
DISCUSSION The presence of two CaBI systemsinthe vertebrate retina CaB-like immunoreactivity can be divided into two systems in the birds and mammals studied here: cone-CaBI and rod-CaBI systems. Photoreceptor cells in the chick retina are mostly cones (Duke-Elder, '58). In these retinas, elements directly related to phototransmission were labeled: cones, cone bipolars with descending processes terminating on the cone pedicles, and some ganglion cells. Amacrine cells of various sizes were also labeled. In retinas, of which the photoreceptor cells are mainly rods, such as in the rat, guinea pig, and dog, the following elements were labeled: horizontal cells, small to medium-sized amacrine cells, and cells in the ganglion cell layer. Unlike the chick retina, large amacrine cells were not labeled. In the human retina, which contains both cones and rods in abundance, both cone-CaBI and rod-CaBI systems were found. Elements related to both systems were labeled. Accordingly, cones, cone bipolars, small to large amacrine cells, horizontal cells, and cells in the ganglion cell layer were labeled. Furthermore, there exist nonlabeled amacrine, bipolar, horizontal, and ganglion cells in the retinas, suggesting heterogeneity of these cells based upon the localization of the CaB-like immunoreactivity. In cone-dominant retinas, there were labeled cells and fibers in the ganglion cell and nerve fiber layers, respectively, showing that at least some of the labeled cells in the ganglion cell layer were the ganglion cells. On the other hand, in rod-dominant retinas such as rat, guinea pig, and dog, although labeled cells were found in the ganglion cell layer, none or only a few labeled fibers were seen in the nerve fiber layer. Schreiner et al. ('85) have reported the
Fig. 4. Fluorescence photomicrographs showing the distribution of CaBI structures in the human retina, which contains both cones and rods in abundance. Elements found both in the cone dominant retina and the rod dominant retina were labeled in the human retina: cones, bipolar cells (arrows), cells in the GL, horizontal cells, and amacrine cells of various sizes. In the nerve fiber layer, immunoreactive fibers were seen (B and C), suggesting that some of the immunoreactive cells in the GL are ganglion cells (C, arrowhead). A Perimacular region. B Central retina. C: Peripheral retina. Bar, 50 pm.
Fig. 5. Fluorescent photomicrographs of PVI structures in the chick (A), rat (B), and dog (C) retina. Immunoreactive amacrine cells were always present in these species (arrows). In the dog, horizontal cells
were also strongly labeled. Furthermore, some immunoreactive cells were seen in the GL of rat and dog, but no immunoreactive fibers were seen in the NFL in these animals. Bar, 50 p,m.
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presence of CaBI cells in the ganglion cell layer of mouse and rat retinas (rod-dominant retinas). They suggested that these cells are ganglion cells and not displaced amacrine cells, since a small number of immunoreactive fibers were observed in the nerve fiber layer. However, it should be noted that the number of the labeled cells in the ganglion cell layer was much more numerous than that of immunoreactive nerve fibers in their study. Rohrenbeck et al. ('89) also found labeled cells in the ganglion cell layer of monkey, and suggested that they were displaced amacrine cells because of a lack of immunoreactive fibers in the nerve fiber layer. In any case, in order to clarify the category of the labeled cells in the ganglion cell layer, an experiment that uses immunocytochemistry combined with retrograde tracing will be required. There are a few discrepancies about the location of retinal CaB in this study and previous studies. For example, in the monkey retina which belongs to the same category as the human retina (mixed type of cones and rods), the nerve fiber layer contained none or only a few immunoreactive fibers (Rohrenbeck et al., '891, while numerous positive fibers were seen in the nerve fiber layer of the human retina as shown in this study. Aprevious study demonstrated the presence of CaBI cones and a few bipolar cells in the cat (Rohrenbeck et al., '89) but we failed to demonstrate this in the dog. CaBI horizontal cells have been found in chick (Schreiner et al., '85) and pigeon (Pasteels et al., '87) retinas, but we could not support these findings. In contrast, we have demonstrated the presence of many CaBI bipolar cells, ganglion cells, and positive fibers in the nerve fiber layer in the chick retina, while previous studies (Pasteels et al., '87; Schreiner et al., '85) indicated the presence of a few positive bipolar cells, cells in the ganglion cell layer, and no positive fibers in the nerve fiber layer in chick and pigeon retinas. In frog retina, which contains both rods and cones (2:1, according to Llinas and Precht, '76), cones, some amacrine cells, and some cells in the ganglion cell layer were labeled. The immunoreactivity in these cones was less than that seen in those of chick or human. No immunoreactive bipolar cells were detected. There were a few discrepancies between our and the previous study. Although Schreiner et al. ('85) showed CaBI structures in some horizontal cells, many bipolar cells, and many ganglion cells along with their axons forming the optic tract, we did not find any positive bipolar, horizontal cells, nor any positive fibers in the nerve fiber layer. Perhaps the differences arose because of the different antisera used or species differences. In the guinea pig retina we found some positive, slender, fiber-like or pillar-like structures that seemed to be Muller cells. The reason why Muller cells only in the guinea pig retina were stained is open to discussion.
PVI struureS in the vertebrate retina Findings common to all species examined with PV in the vertebrate retina were that photoreceptor cells and bipolar cells lacked PV-like immunoreactivity. There were species differences in the location of PV. There were no immunoreactive structures in the goldfish, frog, guinea pig, or human retina. Chick, rat, and dog retina, on the other hand, contained PV-like immunoreactivity, but PVI structures were in slightly different locations in these species. Horizontal cells were labeled in dog retina only and cells in the ganglion cell layer were labeled in rat and dog retinas only.
Our results are somewhat different from those of Rohrenbeck et al. ('87). We did not find labeled horizontal cells in rat and human retinas, while they found labeled horizontal cells in rat, cat, monkey, and human retinas. We did not find labeled ganglion cells in any of the species examined, but they have found labeled ganglion cells in cat and monkey. These discrepancies may have arisen because of the different antisera used, because of the heterogeneity of PV-like immunoreactive substances, or because of species differences.
IAITFaAmclTED Cajal, S.R.Y. (1972) The Structure of the Retina. Springfield: Thomas. Celio, M.R., and C.W. Heizmann (1981) Calcium-binding protein parvalbumin as a neuronal marker. Nature 293:300-302. Duke-Elder, S (1958) System of Ophthalmology. Vol. 1, London: Henry Kimpton, pp. 397427. Endo, T., S. Kobayashi, and T. Onaya (1985) Parvalbumin in rat cerebellum and retina during postnatal development. Neurosci. Lett. 60279-282. Endo, T., M. Kobayashi, S. Kobayashi, and T. Onaya (1986) Immunocytochemical and biochemical localization of parvalbumin in the retina. Cell Tissue Res. 243913-217. Heizmann, C.W. (1984) Parvalbumin, an intracellular calcium-binding protein; distribution, properties and possible roles in mammalian cells. Experientia 40r9 1 0 9 21. Ichimiya, Y., P.C. Emson, C.Q. Mountjoy, D.E.M. Lawson, and C.W. Heizmann (1988) Brain Res. 475:156-159. Jande, S.S., and L. Maler (1981) Immunohistochemical mapping of vitamin D-dependent calcium-binding protein in brain. Nature 294:765-767. Legrand, C., M. Thomasset, C.O. Parkes, M.C. Clavel, and A. Rabie (1983) Calcium-binding protein in the developing rat cerebellum. Cell Tissue Res. 233:389402. Llinas, R., and W. Precht (1976) Frog Neurology. A Handbook. Berlin, Heidelberg, New York: Springer-Verlag, pp. 251-277. Mark, R.E. (1986) Neurochemical stratification in the inner plexiform layer of the vertebrate retina. Vision Res. 26:223-238. Meyer, D.B., and T.G. Cooper (1966) The visual cells of the chicken as revealed by phase contrast microscopy. Am. J. Anat. 118:723-734. Morris, V.B., and C.D. Shorey (1967) An electron microscope study of types of receptor in the chick retina. J. Comp. Neurol. 129;313-340. Pasteels, B., M. Parmentier, E.M. Lowson, A. Verstappen, and R. Pochet (1987) Calcium binding protein immunoreactivity in pigeon retina. Invest. Ophthalmol. Vis. Sci. 28:658-664. Rabie, A,, M. Thomasset, C.O. Parkes, and M.C. Clavel (1985) lmmunocytochemical detection of 28000-MW calcium-binding protein in horizontal cells of the rat retina. Cell Tissue Res. 2401493-496. Rogers, J.H. (1987) Calretinin: A gene for a novel calcium-binding protein expressed principally in neurons. J. Cell Biol. 105:1343-1353. Rohrenbeck, J.,H. Wassle, and C.W. Heizmann (1987) Immunocytochemical labelling of horizontal cells in mammalian retina using antibodies against calcium-binding proteins. Neurosci. Lett. 771255-260. Rohrenbeck, J., H. Wassle, and B.B. Boycott (1989) Horizontal cells in the monkey retina: Immunocytochemical staining with antibodies against calcium binding proteins. Eur. J. Neurosci. 1:407A20. Roth, J., D. Baetens, A.W. Norman, and L.M. Garciasegura (1981) Specific neurons in chick central nervous system stain with an antibody against chick intestinal vitamin D-dependent calcium-binding protein. Brain Res. 222:452457. Schreiner, D.S., S.S. Jande, and D.E.M. Lawson (1985) Target cells of vitamin D in the vertebrate retina. Acta Anat. 121r153-162. Spencer, R., M. Charman, P.W. Wilson, and D.E.M. Lawson (1978) The relationship between vitamin D-stimulated calcium transport and intestinal calcium-binding protein in the chicken. Biochem. J. 170:93-101. Verstappen, A., M. Parmentier, M. Chirnoaga, D.E.M. Lawson, J.L. Pasteels, and R. Pochet (1986) Vitamin D-dependent calcium binding protein immunoreactivity in human retina. Ophthalmic Res. 18,209-214. Wasserman, R.H., and A.N. Taylor (1966) Vitamin D-induced calciumbinding protein in chick intestinal mucosa. Science 15.2791-793. Zamboni, L., and C.D. Martino (1976) Buffered picric acid formaldehyde: a new rapid fixative for electron microscopy. J. Cell Biol. 35:148A.
Localization of Two Calcium Binding Proteins,Calbindin (281)) and ParValbumin (12 kD),in the Vertebrate Retina K. HAMANO, H. KIYAMA, P.C. EMSON, R. MANABE, M. NAKAUCHI, AND M. TOHYAMA Department of Ophthalmology (K.H., R.M.), Biomedical Center (H.K.), Department of Anatomy (M.N.), Osaka University Medical School, Kitaku, Osaka 530, Japan; MRC Group (P.C.E.), Institute of Animal Physiology and Genetics Research, Babraham, Cambridge CB2 4AT, United Kingdom
ABSTRACT We used immunocytochemistry to locate two calcium binding proteins, calbindin (CaB) and parvalbumin (PV), in the retina of goldfish, frog, chick, rat, guinea pig, dog, and man. The location of CaB depended on the type of dominant photoreceptor cells in birds and mammals. In cone-dominant retinas such as those of the chick, CaB-like immunoreactivity was found in the cones, cone bipolars, and ganglion cells. Amacrine cells 5-12 p,m across were also labeled. In rod-dominant retinas, such as those of the rat, guinea pig, and dog, horizontal cells, small amacrine cells (about 6 pm across), and cells in the ganglion cell layer were labeled. In the human retina, which has both cones and rods in abundance, cones, cone bipolars, ganglion cells, horizontal cells, and small and large amacrine cells were labeled. In the frog and goldfish, the level of CaB-like immunoreactivity was low. In the frog, a few cones, amacrine cells, and cells in the ganglion cell layer were labeled. No immunoreactive structures were seen in the goldfish retina. PV-like immunoreactivity was found in chicks, rats, and dogs. No such immunoreactive structures were seen in the other species. In the chick, only amacrine cells were labeled. In the rat, amacrine cells and several displaced amacrine cells were labeled. In the dog, in addition to amacrine cells and displaced amacrine cells, horizontal cells were strongly labeled. Thus, PV-like immunoreactivity was found in those elements relating to the modulation of the main pathway of the visual transmission system. Key words: calbindin, parvalbumin, retina, immunohistochemistry
Intracellular Caz+is important as a "second messenger" postsynaptically. It has various metabolic effects when bound to the regulatory subunit of cyclic-AMP-dependent protein kinases in the central and peripheral nervous systems. Calcium-binding proteins seem to maintain the intracellular Ca2+concentration at an appropriate level. In the nervous system, two calcium binding proteins, calbindin (CaB), which is vitamin-D-dependent (Wasserman and Taylor, '661, and parvalbumin (PV), which is water-soluble are widely but unevenly distrib(Celio and Heizmann, %l), uted (Jande and Maler, '81; Roth et al., '81; Legrand et al., '83; Heizmann, '84). CaB and PV have been found in the retina (Endo et al., '85; Rabie et al., '85; Schreiner et al., '85; Endo et al., '86; Verstappen et al., '86; Rohrenbeck et al., '87). In chick retina, photoreceptor cells, horizontal cells, and some bipolar cells contain CaB-like immunoreac-
o 1990 WILEY-LISS, INC.
tive (CaBI) structures (Schreiner et al., '85). In rat, mouse, and rabbit retinas, horizontal cells and some amacrine cells contained CaB-like immunoreactivity. In mouse and rat, some ganglion cells are also labeled by antiserum against CaB (Schreiner et al., '85). Cats and monkeys contains CaBI horizontal cells (Rohrenbeck et al., '87). In the human retina, photoreceptor, horizontal, some amacrine, some ganglion, and some bipolar cells are labeled (Verstappen et al., '86). PV-like immunoreactivity has also been examined in retinas of several vertebrates (Endo et al., '85, '86). In the rat retina, PV is found in the amacrine cells. In monkeys and humans, in addition to amacrine cells, PV immunoreactivity has also been found in horizontal cells (Endo et al., Accepted August 29,1990.
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'86). Thus, there are species differences in the location of retinal CaB and PV. Here, we examined the location of CaB and PV in the retina of seven species, from fish to human, in an endeavour to understand the reasons for their distribution.
MATEXIALSANDMETHODS Preparation of tissues The eyeballs of three goldfish and three dogs were removed while the animals were under sodium pentobarbital anesthesia (10 mg/kg). Three frogs, three chicks, four rats, and three guinea pigs were perfused transcardially under sodium pentobarbital anesthesia (10 mgkg) with ice-cold saline (50-100 ml) followed by Zamboni's fixative (300 ml/lOO g) at 4°C (Zamboni and Martino, '76). The eyes were removed, cut open, and immersed in the same fixative for 2-3 days. Two human eyes enucleated because of intra-orbital metastasis of malignant tumors were placed in the same fixative for about a week. All the specimens were then immersed overnight in 0.1 M phosphate buffer containing 30% sucrose, frozen, and cut into sections 10-15 Fm thick on a cryostat.
Definition of sublayers in the inner pldorm layer of retinas The process which defined the sublayers of the inner plexiform layer in chick retina was based on that of Cajal (Cajal, '72) and Mark (Mark, '86). Since the classification of this layer in other animals is confusing or difficult at present (Mark, '86), we divided the inner plexiform layer into five equal sublayers for all retinas and numbered them in distal-proximal sequence (sublayer 1, sublayer 2, . . . sublayer 5) in this study.
RESULTS 1) Distribution of CaBI structures in vertebrate retina
Goldfish retina. No immunoreactive structures were found. Frog retina (Fig. 1). A few immunoreactive photoreceptor cells were seen (Fig. 1A,B, arrowheads). Although positive cells were distributed throughout the retina, the positive cells in the central area (about 30% cells were labeled) outnumbered those in the peripheral area (less than 5%).The outer segment of these cells lacked immunoreactivity. Strong immunoreactivity was found in the inner segment. The bodies of these cells were weakly labeled. To Immunohistochemidprocedures judge from the location of the labeled bodies, these cells To stain CaBI structures, sections were rinsed in 0.02 M seemed to be cones (Cajal, '72; Llinas and Precht, '76). A phosphate-buffered saline (PBS), 0.3% Triton X-100 contain- few immunoreactive cells and fibers were seen in the ing 0.02 M PBS, and 0.02 M PBS for 10 minutes each, innermost part of the inner nuclear (Fig. lA,B, arrows) and incubated in a humid chamber with antichick intestinal inner plexiform layers. These cells seemed to be amacrine CaB antiserum raised in a rabbit (Ichimiya et al., '88) at 4°C cells. There were also some immunoreactive cells in the overnight, rinsed again as above, and incubated with goat ganglion cell layer (Fig. lA, double arrow). No immunoreacantirabbit immunoglobulin G (IgG) serum conjugated with tive fibers were seen in the nerve fiber layer (Fig. 1). fluorescein isothiocyanate (FITC) (Miles) at 4°C overnight. Chick retina (Fig. 2). A number of photoreceptor To stain PVI structures, sections were rinsed as for CaB cells were labeled by the antiserum (Fig. 2A,B). Immunoreimmunoreactivity, incubated with antirat muscle PV antise- active bodies of the photoreceptor cells were long and rum raised in sheep (donated by Dr. P.C. Emson) at 4°C columnar, and were arranged more or less in a row. Stout, overnight, rinsed as above, and then incubated with rabbit smooth immunoreactive processes projected from the inner antisheep IgG serum conjugated with FITC (Cappel, U.S.A.) bases of these bodies, particularly in the central retina. at 4°C overnight. Samples were all rinsed once more before They reached the middle part of the outer plexiform layer being mounted in a 1:l mixture of glycerin and PBS. The and ended in a thick pedicle. The shape of the immunoreacantisera were diluted before use with 0.02 M PBS as tive photoreceptors suggested that they are cones (Meyer follows, CaB, PV, and goat antirabbit IgG serum, 1:1,000; and Cooper, '66; Morris and Shorey, '67; Cajal, '72; Fig. rabbit antisheep IgG serum, 1500. lA,B). In the inner nuclear layer, three groups of immunoreactive cells were seen (Fig. 2A). The first group formed an immunoreactive band of two to three rows of cells at the Specificityof the primary antisera outer part of this layer. Singular immunoreactive processes Rabbit anti-CaB antiserum was produced against CaB, arose from the outer base of the immunoreactive cell bodies M.W. 28,000, purified by high-pressure liquid chromatogra- (Fig. 2A,B) and passed through the outer part of this layer phy after isolation from chick intestine (Spencer et al., '78). terminating on the cone pedicles (Fig. 2B, arrows). Thus, Western blotting has shown that the antiserum recognizes these cells were cone bipolars. The second group consisted CaB of molecular weights of 28,000 and 30,000 in mamma- of a few immunoreactive cells in the middle of this layer lian brains (Ichimiya et al., '88) and also calretinin, M.W. (Fig. 2A, arrowheads). They were probably bipolar cells 29,000 (Rogers, '87). Sheep anti-PV antiserum was raised because their cell bodies were similar to those of the first against purified PV from rat muscle and demonstrated group in size and shape, and because there were both satisfactory specificity by Western blotting (Emson, per- descending and ascending processes from their cell bodies. sonal communication).The specificities of the antisera were The last group of cells was scattered in the inner half of the also checked by absorption tests. Structures were not inner nuclear layer (Fig. 2A). These cells varied in size: stained with either CaB antiserum in those sections stained most of them were small to medium and a few were large. with antiserum pre-absorbed by purified CaB from chick The large immunoreactive cells and some of the small to M or PV antiserum medium-sized immunoreactive cells at the inner surface of intestine a t a final concentration of in sections stained with the antiserum pre-absorbed by the inner nuclear layer seemed to be stratified amacrine cells because single immunoreactive processes appeared M. purified rat muscle PV at a final concentration of
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Fig. 1. Fluorescence photomicrographs showing the distribution of CaBI structures in the frog retina. Some cones (arrowheads) and a few amacrine cells (arrows) were labeled. In addition, immunoreactive cells were seen in the ganglion cell layer (double arrow),but none were found
in the nerve fiber layer. ONL: Outer nuclear layer. INL: Inner nuclear layer. IPL: Inner plexiform layer. GL: Ganglion cell layer. A,B: Central part of the frog retina. Bar, 50 pm.
from the inner bases of the cell bodies to form immunoreactive fiber bands of different densities at sublayers 1 , 2 , and 4 in the inner plexiform layer (Cajal, '72) (Fig. 2C, arrows). The highest density was in sublayer 2. Sublayer 4 was moderately dense, but sublayers 3 and 5 contained disseminated immunoreactive fibers (Fig. 2A,C). In the ganglion cell layer, a number of immunoreactive cells were seen. Medium-sized immunoreactive cells were most abundant. Immunoreactive varicose fibers were often seen in the nerve fiber layer (Fig. 2A,D, arrows), so it was likely that at least some of them were ganglion cells that project into the central nervous system.
R a t retina (Fig. 3A). No immunoreactive cells were detected in the photoreceptor cell layer. In the outermost part of the inner nuclear layer, several large immunoreactive cells (about 12 pm in diameter) were found. Processes from these cells formed an immunoreactive fiber plexus of high density in the inner part of the outer plexiform layer. To judge from these findings, they were horizontal cells. On the inner surface of the inner nuclear layer, several small round cells were observed. Single processes from these cells formed fiber plexuses with low density in sublayers 1,3,and 4 of the inner plexiform layer. In the ganglion cell layer, some cells were labeled. They were small (about 5 pm) to
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Fig. 2. Fluorescence photomicrographs showing the distribution of CaBI structures in the chick retina. A number of cones (A and B), cells in the outer half of the INL, a few cells in the middle of the INL (A, arrowheads), several cells of various sizes in the inner half of the INL (A-C), and some cells in the GL (A, C, and D) contained CaB-like immunoreactivity. Immunoreactive fibers formed fiber bands in sublayers 1, 2, and 4 in the IPL (A, C). In B, singular immunoreactive processes arising from the immunoreactive cells in the outer half of the
INL extended to the outer plexiform layer (OPL) and terminated on single immunoreactive cone pedicles (arrows), indicating that these cells were cone bipolars. In C , single processes (arrows) arisingfrom the immunoreactive cells in the inner half of the INL terminated in the IPL, indicating that these cells were amacrine cells. In the nerve fiber layer (NFL), a group of immunoreactive varicose fibers were seen (A and D, arrows), suggesting that the immunoreactive cells in the GL were ganglion cells. A-D: Central part of the chick retina. Bar, 50 pm.
large (about 10 pm) but medium-sized cells were most abundant. There were no immunoreactive fibers in the nerve fiber layer; therefore most of these cells seemed to be displaced amacrine cells. Guinea p i g retina (Fig. 3B). The distribution pattern of CaBI structures in guinea pig retina was similar to that of rats. Immunoreactive neurons were horizontal, amacrine, and those in the ganglion cell layer. Labeled amacrine cells were more numerous in the guinea pig retina than in that of the rat, so the immunoreactive fiber bands found in the inner plexiform layer were denser than in the rat. In the outer nuclear layer of the guinea pig retina, slender fiber-like or pillar-like structures that extended
from outer to inner limiting membranes were often seen. These may belong to Muller cells (Fig. 4B, arrows). Dog retina (Fig. 3C,D). The distribution pattern of CaBI structures in dog retina was similar to that of rats. However, there were more labeled amacrine cells in the dog retina than in the rat, and the immunoreactive fiber plexus in the inner plexiform layer of dogs was denser than that in the rat. Human retina (Fig. 4). As in the chick retina, many cone-like photoreceptor cells were strongly labeled. The highest number of positive cells was identified in the central portion. Immunoreactivity was found in their bodies and inner fibers. In the inner nuclear layer, three groups of
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Fig. 3. Fluorescence photomicrographs showing the distribution of CaBI structures in rod-dominant retinas of (A) rat, (B) guinea pig, and (C and D) dog. In these retinas, horizontal cells were strongly labeled. A dense immunoreactive fiber plexus from these cells was seen in the inner part of the OPL. Several small amacrine cells in the INL and some cells in the GL were labeled. No immunoreactive fibers were seen in the GL. The distribution and density of the immunoreactive fiber plexuses
in the IPL were different in different species. In rat retina, the plexuses were in sublayers 1, 4,and 5, and faintly labeled. In guinea pig retina, they were in sublayers 2, 3, and 4,and densely labeled. In dog retina, they formed two lines, but were distributed diffusely. In the guinea pig retina, some Muller cells were stained by the antiserum (B, arrows). A-D, central retina. Bar, 50 km.
immunoreactive neurons were seen. One was of horizontal cells. There were fewer immunoreactive horizontal cells in the human retina than in the retinas of rat, guinea pig, or dog. Processes of the immunoreactive horizontal cells formed a fiber plexus of low density at the inner part of the outer plexiform layer. The second group was of a few cells in the middle part of the inner nuclear layer (Fig. 4A,B, arrows). Two processes projected from opposite sides of the soma, indicating that these neurons were bipolar cells. The third group was of amacrine cells, which were in the innermost part of the inner nuclear layer. Ascending processes from these neurons terminated diffusely in the inner plexiform layer. The ganglion cell layer contained labeled cells of various sizes. Fiber processes from large labeled cells in the innermost part of the ganglion cell layer were
seen in the nerve fiber layer (Fig. 4C, arrowhead). These labeled cells seemed to be ganglion cells.
2) Distributionof PVI s t r u b in the vertebrate retina Goldfish and frog retinas. No immunoreactive structures were found. Chick retina (Fig. 5A). Several amacrine cells were labeled by PV antiserum. Their somata were about 7 km in diameter and were located in the inner half of the inner nuclear layer (Fig. 5A, arrows). Single processes of labeled amacrine cells ascended the inner plexiform layer to form immunoreactive fiber bands of low density at sublayers 1,3, and 4. Other retinal elements lacked PV immunoreactivity.
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Guinea p i g retina. No immunoreactive structures were found. Rat retina (Fig. 5B). A few amacrine cells were labeled. These cells could be classified into two types by the
K. HAMANO ET AL. arborization pattern of their processes (Fig. 5B, arrows). Somata of both types of amacrine cells were about 7 p,m across and located in the innermost part of the inner nuclear layer. One type emitted a single process straight to sublayer 5 of the inner plexiform layer without ramification. The other had processes that arborized immediately in sublayer 1 and did not ascend further. No other elements were labeled. A few immunoreactive cells were seen in the ganglion cell layer, but no immunoreactive fibers were observed. Dog retina (Fig. 5C). A number of horizontal cells were labeled in which the processes formed a densely packed immunoreactive fiber plexus at the inner part of the outer plexiform layer. A few amacrine cells were also labeled (Fig. 5C, arrows). Their processes terminated diffusely in the inner plexiform layer. No immunoreactive fibers were observed in the nerve fiber layer. Human retina. No immunoreactive structures were observed.
DISCUSSION The presence of two CaBI systemsinthe vertebrate retina CaB-like immunoreactivity can be divided into two systems in the birds and mammals studied here: cone-CaBI and rod-CaBI systems. Photoreceptor cells in the chick retina are mostly cones (Duke-Elder, '58). In these retinas, elements directly related to phototransmission were labeled: cones, cone bipolars with descending processes terminating on the cone pedicles, and some ganglion cells. Amacrine cells of various sizes were also labeled. In retinas, of which the photoreceptor cells are mainly rods, such as in the rat, guinea pig, and dog, the following elements were labeled: horizontal cells, small to medium-sized amacrine cells, and cells in the ganglion cell layer. Unlike the chick retina, large amacrine cells were not labeled. In the human retina, which contains both cones and rods in abundance, both cone-CaBI and rod-CaBI systems were found. Elements related to both systems were labeled. Accordingly, cones, cone bipolars, small to large amacrine cells, horizontal cells, and cells in the ganglion cell layer were labeled. Furthermore, there exist nonlabeled amacrine, bipolar, horizontal, and ganglion cells in the retinas, suggesting heterogeneity of these cells based upon the localization of the CaB-like immunoreactivity. In cone-dominant retinas, there were labeled cells and fibers in the ganglion cell and nerve fiber layers, respectively, showing that at least some of the labeled cells in the ganglion cell layer were the ganglion cells. On the other hand, in rod-dominant retinas such as rat, guinea pig, and dog, although labeled cells were found in the ganglion cell layer, none or only a few labeled fibers were seen in the nerve fiber layer. Schreiner et al. ('85) have reported the
Fig. 4. Fluorescence photomicrographs showing the distribution of CaBI structures in the human retina, which contains both cones and rods in abundance. Elements found both in the cone dominant retina and the rod dominant retina were labeled in the human retina: cones, bipolar cells (arrows), cells in the GL, horizontal cells, and amacrine cells of various sizes. In the nerve fiber layer, immunoreactive fibers were seen (B and C), suggesting that some of the immunoreactive cells in the GL are ganglion cells (C, arrowhead). A Perimacular region. B Central retina. C: Peripheral retina. Bar, 50 pm.
Fig. 5. Fluorescent photomicrographs of PVI structures in the chick (A), rat (B), and dog (C) retina. Immunoreactive amacrine cells were always present in these species (arrows). In the dog, horizontal cells
were also strongly labeled. Furthermore, some immunoreactive cells were seen in the GL of rat and dog, but no immunoreactive fibers were seen in the NFL in these animals. Bar, 50 p,m.
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presence of CaBI cells in the ganglion cell layer of mouse and rat retinas (rod-dominant retinas). They suggested that these cells are ganglion cells and not displaced amacrine cells, since a small number of immunoreactive fibers were observed in the nerve fiber layer. However, it should be noted that the number of the labeled cells in the ganglion cell layer was much more numerous than that of immunoreactive nerve fibers in their study. Rohrenbeck et al. ('89) also found labeled cells in the ganglion cell layer of monkey, and suggested that they were displaced amacrine cells because of a lack of immunoreactive fibers in the nerve fiber layer. In any case, in order to clarify the category of the labeled cells in the ganglion cell layer, an experiment that uses immunocytochemistry combined with retrograde tracing will be required. There are a few discrepancies about the location of retinal CaB in this study and previous studies. For example, in the monkey retina which belongs to the same category as the human retina (mixed type of cones and rods), the nerve fiber layer contained none or only a few immunoreactive fibers (Rohrenbeck et al., '891, while numerous positive fibers were seen in the nerve fiber layer of the human retina as shown in this study. Aprevious study demonstrated the presence of CaBI cones and a few bipolar cells in the cat (Rohrenbeck et al., '89) but we failed to demonstrate this in the dog. CaBI horizontal cells have been found in chick (Schreiner et al., '85) and pigeon (Pasteels et al., '87) retinas, but we could not support these findings. In contrast, we have demonstrated the presence of many CaBI bipolar cells, ganglion cells, and positive fibers in the nerve fiber layer in the chick retina, while previous studies (Pasteels et al., '87; Schreiner et al., '85) indicated the presence of a few positive bipolar cells, cells in the ganglion cell layer, and no positive fibers in the nerve fiber layer in chick and pigeon retinas. In frog retina, which contains both rods and cones (2:1, according to Llinas and Precht, '76), cones, some amacrine cells, and some cells in the ganglion cell layer were labeled. The immunoreactivity in these cones was less than that seen in those of chick or human. No immunoreactive bipolar cells were detected. There were a few discrepancies between our and the previous study. Although Schreiner et al. ('85) showed CaBI structures in some horizontal cells, many bipolar cells, and many ganglion cells along with their axons forming the optic tract, we did not find any positive bipolar, horizontal cells, nor any positive fibers in the nerve fiber layer. Perhaps the differences arose because of the different antisera used or species differences. In the guinea pig retina we found some positive, slender, fiber-like or pillar-like structures that seemed to be Muller cells. The reason why Muller cells only in the guinea pig retina were stained is open to discussion.
PVI struureS in the vertebrate retina Findings common to all species examined with PV in the vertebrate retina were that photoreceptor cells and bipolar cells lacked PV-like immunoreactivity. There were species differences in the location of PV. There were no immunoreactive structures in the goldfish, frog, guinea pig, or human retina. Chick, rat, and dog retina, on the other hand, contained PV-like immunoreactivity, but PVI structures were in slightly different locations in these species. Horizontal cells were labeled in dog retina only and cells in the ganglion cell layer were labeled in rat and dog retinas only.
Our results are somewhat different from those of Rohrenbeck et al. ('87). We did not find labeled horizontal cells in rat and human retinas, while they found labeled horizontal cells in rat, cat, monkey, and human retinas. We did not find labeled ganglion cells in any of the species examined, but they have found labeled ganglion cells in cat and monkey. These discrepancies may have arisen because of the different antisera used, because of the heterogeneity of PV-like immunoreactive substances, or because of species differences.
IAITFaAmclTED Cajal, S.R.Y. (1972) The Structure of the Retina. Springfield: Thomas. Celio, M.R., and C.W. Heizmann (1981) Calcium-binding protein parvalbumin as a neuronal marker. Nature 293:300-302. Duke-Elder, S (1958) System of Ophthalmology. Vol. 1, London: Henry Kimpton, pp. 397427. Endo, T., S. Kobayashi, and T. Onaya (1985) Parvalbumin in rat cerebellum and retina during postnatal development. Neurosci. Lett. 60279-282. Endo, T., M. Kobayashi, S. Kobayashi, and T. Onaya (1986) Immunocytochemical and biochemical localization of parvalbumin in the retina. Cell Tissue Res. 243913-217. Heizmann, C.W. (1984) Parvalbumin, an intracellular calcium-binding protein; distribution, properties and possible roles in mammalian cells. Experientia 40r9 1 0 9 21. Ichimiya, Y., P.C. Emson, C.Q. Mountjoy, D.E.M. Lawson, and C.W. Heizmann (1988) Brain Res. 475:156-159. Jande, S.S., and L. Maler (1981) Immunohistochemical mapping of vitamin D-dependent calcium-binding protein in brain. Nature 294:765-767. Legrand, C., M. Thomasset, C.O. Parkes, M.C. Clavel, and A. Rabie (1983) Calcium-binding protein in the developing rat cerebellum. Cell Tissue Res. 233:389402. Llinas, R., and W. Precht (1976) Frog Neurology. A Handbook. Berlin, Heidelberg, New York: Springer-Verlag, pp. 251-277. Mark, R.E. (1986) Neurochemical stratification in the inner plexiform layer of the vertebrate retina. Vision Res. 26:223-238. Meyer, D.B., and T.G. Cooper (1966) The visual cells of the chicken as revealed by phase contrast microscopy. Am. J. Anat. 118:723-734. Morris, V.B., and C.D. Shorey (1967) An electron microscope study of types of receptor in the chick retina. J. Comp. Neurol. 129;313-340. Pasteels, B., M. Parmentier, E.M. Lowson, A. Verstappen, and R. Pochet (1987) Calcium binding protein immunoreactivity in pigeon retina. Invest. Ophthalmol. Vis. Sci. 28:658-664. Rabie, A,, M. Thomasset, C.O. Parkes, and M.C. Clavel (1985) lmmunocytochemical detection of 28000-MW calcium-binding protein in horizontal cells of the rat retina. Cell Tissue Res. 2401493-496. Rogers, J.H. (1987) Calretinin: A gene for a novel calcium-binding protein expressed principally in neurons. J. Cell Biol. 105:1343-1353. Rohrenbeck, J.,H. Wassle, and C.W. Heizmann (1987) Immunocytochemical labelling of horizontal cells in mammalian retina using antibodies against calcium-binding proteins. Neurosci. Lett. 771255-260. Rohrenbeck, J., H. Wassle, and B.B. Boycott (1989) Horizontal cells in the monkey retina: Immunocytochemical staining with antibodies against calcium binding proteins. Eur. J. Neurosci. 1:407A20. Roth, J., D. Baetens, A.W. Norman, and L.M. Garciasegura (1981) Specific neurons in chick central nervous system stain with an antibody against chick intestinal vitamin D-dependent calcium-binding protein. Brain Res. 222:452457. Schreiner, D.S., S.S. Jande, and D.E.M. Lawson (1985) Target cells of vitamin D in the vertebrate retina. Acta Anat. 121r153-162. Spencer, R., M. Charman, P.W. Wilson, and D.E.M. Lawson (1978) The relationship between vitamin D-stimulated calcium transport and intestinal calcium-binding protein in the chicken. Biochem. J. 170:93-101. Verstappen, A., M. Parmentier, M. Chirnoaga, D.E.M. Lawson, J.L. Pasteels, and R. Pochet (1986) Vitamin D-dependent calcium binding protein immunoreactivity in human retina. Ophthalmic Res. 18,209-214. Wasserman, R.H., and A.N. Taylor (1966) Vitamin D-induced calciumbinding protein in chick intestinal mucosa. Science 15.2791-793. Zamboni, L., and C.D. Martino (1976) Buffered picric acid formaldehyde: a new rapid fixative for electron microscopy. J. Cell Biol. 35:148A.
