Horizontal Cells of the Pigeon Retina 1 ANDREW P. MARIAN1 AND ALPHONSE E. LEURE-DuPREE Lkpartmnt of Anatomy, The Pennsylvania Skate University College of Medicine, Hershey, Pennsylvania 17033
ABSTRACT Two types of horizontal cells are seen in Golgi-impregnated retinas of the pigeon. Type I horizontal cells are compact, “brush-shaped,” and have a n axon ending a s a n irregular spinous arborization. The majority of the dendrites terminate in the distal part of the outer plexiform layer (OPL) as clusters which contact cones, but some terminate as single expansions in the proximal part of the OPL. The axon terminal spines are found only in the distal part of the OPL and contact both rods and cones. Pigeon Type I horizontal cells are Cajal’s “brush-shaped” cells, and their axon terminals resemble Cajal’s “stellate” cells. Type I1 horizontal cells have irregular, wavy, multi-branched dendrites, appear horizontally flattened, and lack axons. The dendrites terminate in the proximal part of the OPL a s isolated spines and contact only cones. The Type I1 horizontal cells of the pigeon have not been previously described in the avian retina. Horizontal cells are neurons with somata in the inner nuclear layer of the retina. They contribute processes to the outer plexiform layer where they are involved in lateral interactions a t the level of the first synapse in the visual system (Dowling, ’70; Stell, ’72). Tissues processed by Golgi techniques have provided the most information on the location of horizontal cells and the spatial distribution of their processes in the vertebrate retina (Cajal, ’33). Recent studies dealing solely with the morphology of horizontal cells, or describing them as part of larger studies of retinal organization, include work on a cartilaginous fish (Stell and Witkovsky, ’731, teleosts (Parthe, ’72; Naka and Carraway, ’74; Stell, ’751, amphibians (Dowling and Werblin, ’69; Lasansky, ’731, reptiles (Lasansky, ’71; Saito et al., ’73), and mammals (Polyak, ’57; Dowling et al., ’66; Boycott and Dowling, ’69; Gallego, ’71; Boycott and Kolb, ’73; Kolb, ’74; Ogden, ’74; Gallego and Sobrino, ’75; West and Dowling, ’75). However, except for Cajal’s (1889) initial study and Gallego’s (‘75b1, recent report, there is little information dealing with the types of horizontal cells in the avian retina and the relationship of their processes to the outer plexiform layer. In gallinaceous and passerine birds, Cajal (‘33) described two types of horizontal cells: the “brush-shaped” cells with relatively thick protoplasmic branches and a thin axon which J. COMP. NEUR., 175: 13-26.
terminated a s a flattened thickening with spines; and the “stellate” cells with branches which were thinner but longer than the “brush-shaped” cells, and a short axon which could not be followed to its termination. Recently, Gallego et al. (‘75b) reported only a single type of horizontal cell in a number of diurnal and nocturnal avian species. They concluded that the “stellate” cells of Cajal are actually the axonal expansions of the “brushshaped” cells. The pigeon is a diurnal species with a duplex retina (Cohen, ’63) organized into distinct red and yellow fields. Although red oil droplets are found in both fields the color of the red field (dorso-temporal quadrant) is due to the larger size of the red oil droplets a t the distal end of the photoreceptor inner segments and to the presence of small red microdroplets in the inner segments of these cones (Schultze, 1866; van Genderen Stort, 1887; Pedler and Boyle, ’69). The pigeon retina is reported to have a relatively complex cellular and synaptic organization (Binggeli and Paule, ’69; Dubin, ’70). I t was used in this study since it has been the most common subject of physiological investigations concerning the visual systems of birds. This report describes two distinct horizontal cell types ob-
’ Supported in part by NIH Research Grant EYO 1438-02 and the Josiah Macy. Jr. Foundation. Macy Faculty Fellow.
13
14
ANDREW P. MARIAN1 AND ALPHONSE E. LEURE-DuPREE
sewed in Golgi-impregnated retinal tissues of the pigeon and takes into account variations of the cells’ dimensions in the central area, the more peripheral yellow fields and the area dorsalis or red field (Galifret, ’68). MATERIALS AND METHODS
Adult domestic pigeons (Columbia livia) were anesthetized with pentobarbital sodium, and the globes excised and equatorially bissected. Staining of the posterior eyecups containing the retina, in situ, was by the rapid-Golgi triple impregnation (Valverde, ’69) or a variation of the Colonnier (‘64) modification of the Golgi-Kopsch technique using 4% potassium dichromate and 5% glutaraldehyde for fixation. Other pigeons, lightly anesthetized with pentobarbital sodium, were sacrificed by intracardiac perfusion of 3% glutaraldehyde in 0.1 M Na cacodylate. Within one hour after perfusion, the globes were excised and impregnated according to the Colonnier technique. The tissues were rapidly dehydrated, cleared and embedded in celloidin or Epon 812. Celloidin embedded retinas were cut a t a thickness of 80-120 p m on a sliding microtome. Epon embedded retinas were cut by hand with a razor after gentle warming with a metal spatula. The preparation of flat mounts was according to the technique described by Boycott and Kolb (‘73). Three well impregnated horizontal cells of
each type and the same number of axon terminals were remounted on Epon blanks, serially resectioned at 1p m on a Sorvall MT-2B ultramicrotome using glass knives (Stell and Witkovsky, ’731, and counterstained with Methylene Blue-Azure I1 (Richardson et al., ’60). The preparations were studied by light microscopy, photographed, and drawn with the aid of a camera lucida. RESULTS
Two types of horizontal cells are found in Golgi preparations of the pigeon retina. Type I horizontal cells are small, compact, “brushshaped,” and clearly have a n axon; a process of considerable length which terminates as a n irregular spinous arborization. Type I1 horizontal cells have multi-branched dendrites, appear horizontally flattened, and have a greater dendritic span than Type I cells, but have no observable axons. Representative types of these horizontal cells are illustrated in camera lucida drawings (fig. 1). Qpe I horizontal cells The soma of the Type I cell is located in the distal-most layer of nuclei of the inner nuclea r layer (figs. 2,4). The cell body is round and has a n average diameter of 7.5 p m in the yellow fields, 5.5 p m in the red area and 6 p m in the central area. Usually about six dendrites, 1.5-2 p m thick, arise from the soma at TYPE I1
TYPE I
50~1lFig. 1 Camera lucida drawings of horizontal cells in the pigeon retina aa viewed in vertical sections (above) and flat preparations (below).
15
PIGEON HORIZONTAL CELLS
its junction with the outer plexiform layer (fig. 4).Two to six processes terminate singly a t the proximal level of the outer plexiform layer (figs. 5,6).The majority of the dendrites have a cluster of “knob-like” swellings (fig. 51, each of which is less than 1 p m in size and terminates in the most distal portion of the outer plexiform layer. Viewed in flat preparations, the dendritic span of the Type I horizontal cells is circular to slightly elliptical (fig. 71, and, like the soma, the diameter of the dendritic span varies. Average field diameters are 26 p m in the yellow fields, 14 p m in the red area and 20 p m in the central area. The dendrites do not fill the field homogeneously, but are segregated into eight to ten discrete groups or clusters, each of which averages 3 pm (fig. 7). The clusters may contain as many as nine terminal dendritic expansions, but some dendrites terminate in single expansions a t the same level as the clusters (fig. 7). The dendritic clusters terminating in the distal layer of photoreceptor terminals contact cones, while those terminating a s single expansions in the proximal layers also contact cones (fig. 13). The single axonal process of a Type I horizontal cell (figs. 2,3)is about 0.7 p m in diameter and consistently originates from the base of a primary dendrite. The distance from the point of origin to the termination of the axon is from 15-500pm. The average length, however, is over 100 pm, except in the red area where it is 25-50pm. The course of the axon, which has no collaterals, is within the outer plexiform layer just distal to the inner nuclear layer, is often tortuous and may cross itself several times before terminating as an expansion adjacent to the dendrites and soma of its origin (fig. 12).Varicosities as thick as 2 p m often occur along the length of this process (fig. 3). At its termination, the axon rapid-
ly increases to 3-5p m in diameter and forms an irregular tuberous structure which occupies a n intermediate position in the outer plexiform layer (figs. 2,8). Although irregular and variable in shape, the axon terminal usually approximates in diameter of span, the dendritic span of its cell of origin. Numerous spines, a s thick as 1 p m a t the base and narrowing to less than 0.5 p m a t the tip, project from the terminal expansion (fig. 9). However, unlike the dendrites from the soma which have dendritic terminals throughout the outer plexiform layer, these spines end only in the most distal portion of the outer plexiform layer. While many of the spines are isolated structures, some branch and form clusters (fig. 9). The terminal spines contact rods and cones in the distal layer of photoreceptor terminals (fig. 14). Both single spines and clusters contact cones, but only single spines contact rods.
Type II horizontal cells The Type I1 horizontal cells are considerably larger than the Type I horizontal cells (see table 1 and fig. 12 for comparison). The perikaryon of the Type I1 horizontal cell, like that of the Type I cell, is located in the distal layer of nuclei in the inner nuclear layer. It appears rounded when viewed in a flat mount (fig. 111, but is more flattened than the soma of the Type I cell when viewed in a vertical section (fig. 10). In spite of this difference in shape, the Type I1 somata are approximately the same size as the Type I somata in the yellow fields and central area, but they appear to be larger (6pm) in the red field than the Type I somata of this area. The most characteristic feature of Type I1 horizontal cells are irregular wavy dendrites. There are four to eight primary dendrites, which are often as thick as 3 p m as they radiate from the soma. They
TABLE 1
Differencesbetween horizontal cell types of the pigeon retina Dendritic span (pm) Cell type
area
Yellow fields
Red area
18-20
22-32
13-17
Central
Dendritic diameter em)
Level of termination of dendrites in OPL
Mode of termination of dendrites in OPL
Distal
Clusters
Proximal
Single expansions Single SDines
Axon
~
Type I
Type 11
33-40
48-75
30-39
Present
1.6-2
1
Proximal
Not Present
16
ANDREW P. MARIAN1 AND ALPHONSE E. LEURE-DUPREE
quickly narrow to about 1 pm, but may be slightly larger than this along their course especially a t points where branching occurs (fig. 11). When viewed in a flat preparation, the field formed by the span of dendrites is round and is usually 54 p m in diameter in the yellow field; however, in the more peripheral parts of this area i t may reach 75 pm. The dendritic span in the red field is 33 p m in diameter and only slightly larger in the central area (38 pm). Sectioning of retinas in a vertical plane (fig. 101, shows both the primary dendrites and their branches are confined to the most proximal margin of the outer plexiform layer. In contrast to the terminal clusters of the Type I horizontal cells, the terminations of the Type I1 dendritic processes are typically single small (0.5 pm) spines (fig. ll), which are found also at various points along the length of the dendrites (fig. 10).The Type I1 horizontal cell has never been observed to have an axonal process. DISCUSSION
Two distinct horizontal cell types are described in Golgi-impregnated pigeon retina: a small "brush-shaped'' cell (Type I) with an axon expanding into a spinous terminal arborization; and a larger horizontally flattened cell (Type 11) with irregular dendrites and no apparent axon. The horizontal cells of the pigeon retina can be distinguished from one another, not only on the basis of an axon and diameter of dendritic span, but also by levels and patterns of termination of dendrites in the outer plexiform layer. The majority of dendrites of the Type I cells terminate in the outermost level of the outer plexiform layer in clusters. In contrast, the dendrites of the Type I1 horizontal cells terminate in the innermost level of the outer plexiform layer. The Type I horizontal cells of pigeon retina are clearly the "brush-shaped" type described in birds by Cajal (1889, '33). The terminal expansions of the Type I cells of the pigeon resemble Cajal's ('33) "stellate" cells, not only in appearance, but also level of termination of spines in the distal part of the outer plexiform layer. This finding substantiates the observations of Gallego et al. ('75b1, in other avian species. However, the Type I1 horizontal cells reported in this paper, have not been previously described in the avian retina. Of all reported types of mammalian horizontal cells, the Type I cells of the pigeon
most closely resemble the foveal horizontal cells of monkey retina (Polyak, '57) and the horizontal cells of diurnal squirrels (West and Dowling, '75). The Type I1 horizontal cells of pigeon retina are most like the A type described in cat and rabbit (Dowling et al., '66; Fisher and Boycott, '74). In the retinas of the passerine and gallinaceous species studied by Cajal ('331, the photoreceptor terminals were found in a number of concentric plexuses in the retinal plane, with rod terminals present in the outermost layer and cone terminals present in all layers. These results have been confirmed in chick (Morris and Shorey, '671, and chicken, eagles and hawks (Gallego et al., '75a). Such a distinct stratification of photoreceptor terminals and laminar organization of horizontal cell processes in the outer plexiform layer of the pigeon retina has aided identification of photoreceptor-horizontal cell connections. The dendrites of the Type I horizontal cells, which end as single terminals in the proximal part of the outer plexiform layer, contact cones, since there are no rod terminals a t the proximal level. Other dendrites of this cell type which terminate in clusters in the distal layer of photoreceptor terminals also contact cones and are similar to the clusters of dendritic terminals of horizontal cells observed in primates (Polyak, '57; Boycott and Kolb, '73; Ogden, '74; Gallego, '751, and the A and B horizontal cells of cat (Dowling et al., '66; Fisher and Boycott, '741, which are known from Golgi-EM studies to contact only cones (Kolb, '70; '74; Boycott and Kolb, '73). The dendrites terminating as single expansions, a t the same (distal) level as the clusters contact cones, and are located a t the periphery of the dendritic field. This arrangement is also present in goldfish cone horizontal cells (Stell and Lightfoot, '75). The axon terminal spines of the Type I horizontal cells are located in the distal layer of the outer plexiform layer, where rod-type photoreceptor terminals are located, and it might be attractive to suppose that they are similar to mammalian axon terminal systems which are known to contact only rods (Kolb, '70, '74; Boycott and Kolb, '73). However, in the pigeon retina these terminals contact both rods and cones. The same horizontal cell process in contact with rods and cones has been observed previously in tiger salamander retina from serial EM reconstruction (Las-
PIGEON HORIZONTAL CELLS
ansky, ’73), and is also reported for the L1cells of the turtle retina (Saito e t al., ‘74). The Type I1 horizontal cells of pigeon retina are not unique among vertebrate horizontal cells in lacking a n axon. The absence of such a process is reported in smooth dogfish retina (Stell and Witkovsky, ,731, the rod horizontal cells of goldfish (Stell, ’751, the A-type horizontal cells of cat and rabbit (Dowling et al., ’66; Fisher and Boycott, ’74), and a horizontal cell type (termed “amacrine” of the outer plexiform layer) in dog, rat and guinea pig retinas (Gallego, ’71). The dendrites of the Type I1 horizontal cells of the pigeon retina are located in the proximal part of the outer plexiform layer, and contact cones, since only cone terminals are located at this level of the outer plexiform layer. The horizontal cells in various species of fish are known to be arranged in layers (Parthe, ‘72; Stell and Witkovsky, ’73; Naka and Carraway, ’75; Stell, ’751, while the photoreceptor terminals are present in a single stratum. In goldfish retina the layering of the horizontal cells has been shown to be related to color-coded connections with the photoreceptors (Stell and Lightfoot, ’75). Conversely, in the pigeon retina, the photoreceptor terminals are distributed in different strata in the outer plexiform layer while the horizontal cells are present in a single level of the inner nuclear layer. The dendritic processes of the horizontal cells are arranged in the sublayers of the outer plexiform layer in a manner specific to each of the horizontal cell types. Therefore, it is reasonable to expect that the distribution of horizontal cell processes in this layer may be related to the presence of specific cone color types in each of the strata of the outer plexiform layer of the pigeon. ACKNOWLEDGMENTS
The authors gratefully acknowledge Doctors E. V. Famiglietti, Jr. and Helga Kolb for their criticisms and suggestions. This material was presented in part in a preliminary report a t the 1976 Meeting of the Association for Research in Vision and Ophthalmology held at Sarasota, Florida. LITERATURE CITED Binggeli, R. L., and W. J. Paule 1969 The pigeon retina: Quantitative aspects of the optic nerve and ganglion cell layer. J. Comp. Neur., 137: 1-17. Boycott, B. B., and J. E. Dowling 1969 Organization of the
17
primate retina: Light Microscopy. Phil. Trans. R. SOC. Lond. B, 255: 109-184. Boycott, B. B., and H.Kolb 1973 The horizontal cells of the rhesus monkey retina. J. Comp. Neur., 148: 115-140. Cajal, 5.R. 1889 Sur la morphologie et les connexionsde la retine des oiseaux. Anat. Am.,4: 111-121. 1933 La d i n e des vertebres. Trav. d. Labor. d. Rech. biol. L’Univ. d. Madrid. 28. As translated in: The Structure of the Retina. S. A. Thorpe and M. Glickstein, transls. Charles C Thomas, Springfield, Ill. Cohen, A. I. 1963 The fine structure of the visual recep tors of the pigeon. Exp. Eye Res., 2: 88-97. Colonnier, M. 1964 The tangential organization of visual cortex. J. Anat. (London), 98: 327-344. Dowling, J. E. 1970 Organization of vertebrate retinas. Invest. Ophth., 9: 655-680. Dowling, J. E., J. E. Brown and D. Major 1966 Synapses of horizontal cells in the rabbit and cat retinas. Science, 153: 1639-1641.
Dowling. J. E.. and F. S. Werblin 1969 Organization of the retina of the mudpuppy Necturua macuklrs. I. Synaptic structure. J. Neurophysiol., 32: 315-338. Dubin, M.W. 1970 The inner plexiform layer of the vertebrate retina: A quantitative and comparative electron microscopic analysis. J. Comp. Neur., 140: 479-505. Fisher, 5. K., and B. B. Boycott 1974 Synaptic connexions made by horizontal cells within the outer plexiform layer of the retina of the cat and the rabbit. Proc. R. SOC.Lond. B, 186: 317-331. Galifret, Y. 1968 Les diverses aires fonctionnelles de la retine du pigeon. 2. Zellforsch., 86: 535-545. Gallego, A. 1971 Horizontal and amacrine cells in the mammal’s retina. Vision Res. (Suppl. 3), 11: 33-50. Gallego, A, M. Baron and M. Gay= 1975a Organization of the outer plexiform layer of the diurnal and nocturnal bird retinae. Vision Res., 15: 1027-1028. 1975b Horizontal cells of the avian retina. Vision Res., 15: 1029-1030. Gallego, A., and J. A. Sobrino 1975 Short-axon horizontal cells of the monkey’s retina. Vision Res., 15: 747-748. Kolb, H. 1970 Organization of the outer plexiform layer of the primate retina: Electron microscopy of Golgiimpregnated cells. Phil. Trans. R. Soc. Lond. B, 258: 261283. 1974 The connections between horizontal cells and photoreceptors in the retina of the cat: Electron microscopy of Golgi preparations. J. Comp. Neur., 155: 1-14. Lasansky, A. 1971 Synaptic organization of cone cells in the turtle retina. Phil Trans. R. Soc. Lond. B, 262: 365381. 1973
Organization of the outer synaptic layer in the retina of the larval tiger salamander. Phil. Trans. R. Soc. Lond. B, 265:471-489. Morris, V. B., and C. D. Shorey 1967 An electron microscope study of types of receptor in chick retina; J. Comp. Neur., 129: 313-340. Naka, K., and N. R. G. Carraway 1975 Morphological and functional identifications of catfish retinal neurons. I. Classical morphology. J. Neurophysiol., 38: 53-71. Ogden, T. E. 1974 The morphology of retinal neurons of the owl monkey Aotes. J. Comp. Neur., 153: 399-428. Parthe, V. 1972 Horizontal, bipolar and oligopolar cells in the teleost retina. Vision Rea., 12: 395-406. Pedler, C., and M. Boyle 1969 Multiple oil droplets in the photoreceptors of the pigeon. Vision Res., 9: 525-528. Polyak, S. 1957 Structure of the vertebrate retina. In: The Vertebrate Visual System. The University of Chicago Press,Chicago, pp. 207-287. Richardson, K. C., L. Jarett and G. H.Finke 1960 Embed-
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ANDREW P. MARIAN1 AND ALPHONSE E. LEURE-DuPREE
ding in epoxy resins for ultrathin sectioning in electron microscopy. Stain Technol., 35: 313-323. Saito, T., W.H. Miller and T. Tomita 1974 C- and L-type horizontal cells in the turtle retina. Vision Res., 14: 119123.
Schultze, M. 1866 Zur anatomie und physiologie der retina. Archiv fur Microsk. Anatomie, 2: 175-286. Stell. W.K. 1972 The morphological organization of the vertebrate retina. In: Handbook of Sensory Physiology. VW2. Physiology of photoreceptor organs. M. G. F. Fourtes, ed. Springer-Verlag, Berlin, pp. 111-213. 1975 Horizontal cell axons and axon terminals in goldfish retina. J. Comp. Neur., 159: 503-520. Stell. W K.. and D. 0. Lightfoot 1975 Color-specificinterconnections of cones and horizontal cells in the retina of the goldfish. J. Comp. Neur., 159: 473-502.
Stell, W. K., and P. Witkovsky 1973 Retinal structure in the smooth dofish, Mustelus canis: Light microscopy of photoreceptor and horizontal cells. J. Camp. Neur., 148: 33-46.
Valverde, F. 1970 The Golgi method. A tool for comparative structural analyses. In: Contemporary Research Methods in Neuroanatomy. W. J. Nauta and S. 0. F. Ebbesson, eds. Springer-Verlag, New York, pp. 12-31. van Genderen Stort, A. G. H. 1887 Ueber form- und ortsveriinderungen der netzhautelemente unter einfluss von licht und dunkel. v. Graefe's Arch. Ophthal., 33: 229292.
West, R. W., and J. E. Dowling 1975 Anatomical evidence for cone and rod-like receptors in the gray squirrel, ground squirrel, and prairie dog retinas. J. Comp. Neur., 159: 439-460.
PLATES
N 0
X
896.
3 Type I horizontal cell viewed from scleral side in a flat preparation in a montage at two different levels of focus. X 1,024.
The vitreal surface is oriented towards the bottom of the figure.
2 Type I horizontal cell viewed in a vertical section in a montage of micrographs a t different levels of focus.
Light micrographs of Golgi-impregnated horizontal cells of the pigeon retina.
EXPLANATION OF FIGURES
PLATE 1
PIGEON HORIZONTAL CELL3 Andrew P. Mariani and Alphonae E. Leure-duPree PLATE 1
PLATE 2 EXPLANATION OF FIGURES
Light micrographs of Golgi-impregnated horizontal cells of the pigeon retina. 4 Type I horizontal cell soma and dendrites in a vertical section. X 1,920. 5 Different focus of same cell as figure 4. Note the termination of the dendrite in the dis-
tal portion of OPL as a cluster of “knob-like” swellings, while the proximal termination is as a single expansion. X 1,920. 6 Type I horizontal cell soma and dendrites which terminate in the proximal area of the OPL, as viewed from the scleral surface of the retina in a flat preparation. X 2,205.
7 Different level of focus of the same Type I horizontal cell 88 figure 6. The dendrites terminate in clusters at the distal level of the OPL. X 2,205.
8 Type I horizontal cell axon terminal as viewed in a vertical section.
X
1.890.
9 Type I horizontal cell axon terminal viewed from scleral side in a flat preparation. x 1.890.
22
PIGEON HORIZONTAL CELLS Andrew P. Mariani and Alphonse E. Leure-duPme
PLATE 2
23
PLATE 3 EXPLANATION OF FIGURES
Light micrographs of Golgi-impregnated horizontal cells of the pigeon retina.
10 Type I1 horizontal cell viewed in a vertical section. The dendrites terminate as single spines which are also found along the length of the dendrites. Note that the termination of the dendrites is only in the proximal area of the OPL. X 1,964. 11 Type I1 horizontal cell viewed from vitreal side in a flat preparation.
X
2,112.
12 Type I and Type I1 horizontal cells viewed from scleral side in a flat preparation. Note that the Type I1 (11) horizontal cell has a much wider dendritic span than the Type I (1) horizontal cell. at, Type I horizontal cell axon terminal. X 892.
24
PIGEON HORIZONTAL CELLS Andrew P. Mariani and Alphonse E. Leure-duRee
PLATE 3
25
PLATE 4
PIGEON HORIZONTAL CELLS Andrew
P. Muriani and Alphonse E. Leure-duPree
EXPLANATION OF FlGURES
Light micrographs of 1 pm sections of Golgi-impregnated horizontal cells of the pigeon retina.
13 Type I horizontal cell soma and dendrites in vertical section. Clusters (double arrows) of dendritic terminals in distal layer of OPL contact cones. Dendrites terminating in proximal part of OPL (single arrow) also contact cones. X 1,520. 14 Type I horizontal cell axon terminal in vertical section. Terminal clusters (double
arrows) contact cones, while single terminal (single arrow) contacts a rod.
26
X
1,520.
ABSTRACT Two types of horizontal cells are seen in Golgi-impregnated retinas of the pigeon. Type I horizontal cells are compact, “brush-shaped,” and have a n axon ending a s a n irregular spinous arborization. The majority of the dendrites terminate in the distal part of the outer plexiform layer (OPL) as clusters which contact cones, but some terminate as single expansions in the proximal part of the OPL. The axon terminal spines are found only in the distal part of the OPL and contact both rods and cones. Pigeon Type I horizontal cells are Cajal’s “brush-shaped” cells, and their axon terminals resemble Cajal’s “stellate” cells. Type I1 horizontal cells have irregular, wavy, multi-branched dendrites, appear horizontally flattened, and lack axons. The dendrites terminate in the proximal part of the OPL a s isolated spines and contact only cones. The Type I1 horizontal cells of the pigeon have not been previously described in the avian retina. Horizontal cells are neurons with somata in the inner nuclear layer of the retina. They contribute processes to the outer plexiform layer where they are involved in lateral interactions a t the level of the first synapse in the visual system (Dowling, ’70; Stell, ’72). Tissues processed by Golgi techniques have provided the most information on the location of horizontal cells and the spatial distribution of their processes in the vertebrate retina (Cajal, ’33). Recent studies dealing solely with the morphology of horizontal cells, or describing them as part of larger studies of retinal organization, include work on a cartilaginous fish (Stell and Witkovsky, ’731, teleosts (Parthe, ’72; Naka and Carraway, ’74; Stell, ’751, amphibians (Dowling and Werblin, ’69; Lasansky, ’731, reptiles (Lasansky, ’71; Saito et al., ’73), and mammals (Polyak, ’57; Dowling et al., ’66; Boycott and Dowling, ’69; Gallego, ’71; Boycott and Kolb, ’73; Kolb, ’74; Ogden, ’74; Gallego and Sobrino, ’75; West and Dowling, ’75). However, except for Cajal’s (1889) initial study and Gallego’s (‘75b1, recent report, there is little information dealing with the types of horizontal cells in the avian retina and the relationship of their processes to the outer plexiform layer. In gallinaceous and passerine birds, Cajal (‘33) described two types of horizontal cells: the “brush-shaped” cells with relatively thick protoplasmic branches and a thin axon which J. COMP. NEUR., 175: 13-26.
terminated a s a flattened thickening with spines; and the “stellate” cells with branches which were thinner but longer than the “brush-shaped” cells, and a short axon which could not be followed to its termination. Recently, Gallego et al. (‘75b) reported only a single type of horizontal cell in a number of diurnal and nocturnal avian species. They concluded that the “stellate” cells of Cajal are actually the axonal expansions of the “brushshaped” cells. The pigeon is a diurnal species with a duplex retina (Cohen, ’63) organized into distinct red and yellow fields. Although red oil droplets are found in both fields the color of the red field (dorso-temporal quadrant) is due to the larger size of the red oil droplets a t the distal end of the photoreceptor inner segments and to the presence of small red microdroplets in the inner segments of these cones (Schultze, 1866; van Genderen Stort, 1887; Pedler and Boyle, ’69). The pigeon retina is reported to have a relatively complex cellular and synaptic organization (Binggeli and Paule, ’69; Dubin, ’70). I t was used in this study since it has been the most common subject of physiological investigations concerning the visual systems of birds. This report describes two distinct horizontal cell types ob-
’ Supported in part by NIH Research Grant EYO 1438-02 and the Josiah Macy. Jr. Foundation. Macy Faculty Fellow.
13
14
ANDREW P. MARIAN1 AND ALPHONSE E. LEURE-DuPREE
sewed in Golgi-impregnated retinal tissues of the pigeon and takes into account variations of the cells’ dimensions in the central area, the more peripheral yellow fields and the area dorsalis or red field (Galifret, ’68). MATERIALS AND METHODS
Adult domestic pigeons (Columbia livia) were anesthetized with pentobarbital sodium, and the globes excised and equatorially bissected. Staining of the posterior eyecups containing the retina, in situ, was by the rapid-Golgi triple impregnation (Valverde, ’69) or a variation of the Colonnier (‘64) modification of the Golgi-Kopsch technique using 4% potassium dichromate and 5% glutaraldehyde for fixation. Other pigeons, lightly anesthetized with pentobarbital sodium, were sacrificed by intracardiac perfusion of 3% glutaraldehyde in 0.1 M Na cacodylate. Within one hour after perfusion, the globes were excised and impregnated according to the Colonnier technique. The tissues were rapidly dehydrated, cleared and embedded in celloidin or Epon 812. Celloidin embedded retinas were cut a t a thickness of 80-120 p m on a sliding microtome. Epon embedded retinas were cut by hand with a razor after gentle warming with a metal spatula. The preparation of flat mounts was according to the technique described by Boycott and Kolb (‘73). Three well impregnated horizontal cells of
each type and the same number of axon terminals were remounted on Epon blanks, serially resectioned at 1p m on a Sorvall MT-2B ultramicrotome using glass knives (Stell and Witkovsky, ’731, and counterstained with Methylene Blue-Azure I1 (Richardson et al., ’60). The preparations were studied by light microscopy, photographed, and drawn with the aid of a camera lucida. RESULTS
Two types of horizontal cells are found in Golgi preparations of the pigeon retina. Type I horizontal cells are small, compact, “brushshaped,” and clearly have a n axon; a process of considerable length which terminates as a n irregular spinous arborization. Type I1 horizontal cells have multi-branched dendrites, appear horizontally flattened, and have a greater dendritic span than Type I cells, but have no observable axons. Representative types of these horizontal cells are illustrated in camera lucida drawings (fig. 1). Qpe I horizontal cells The soma of the Type I cell is located in the distal-most layer of nuclei of the inner nuclea r layer (figs. 2,4). The cell body is round and has a n average diameter of 7.5 p m in the yellow fields, 5.5 p m in the red area and 6 p m in the central area. Usually about six dendrites, 1.5-2 p m thick, arise from the soma at TYPE I1
TYPE I
50~1lFig. 1 Camera lucida drawings of horizontal cells in the pigeon retina aa viewed in vertical sections (above) and flat preparations (below).
15
PIGEON HORIZONTAL CELLS
its junction with the outer plexiform layer (fig. 4).Two to six processes terminate singly a t the proximal level of the outer plexiform layer (figs. 5,6).The majority of the dendrites have a cluster of “knob-like” swellings (fig. 51, each of which is less than 1 p m in size and terminates in the most distal portion of the outer plexiform layer. Viewed in flat preparations, the dendritic span of the Type I horizontal cells is circular to slightly elliptical (fig. 71, and, like the soma, the diameter of the dendritic span varies. Average field diameters are 26 p m in the yellow fields, 14 p m in the red area and 20 p m in the central area. The dendrites do not fill the field homogeneously, but are segregated into eight to ten discrete groups or clusters, each of which averages 3 pm (fig. 7). The clusters may contain as many as nine terminal dendritic expansions, but some dendrites terminate in single expansions a t the same level as the clusters (fig. 7). The dendritic clusters terminating in the distal layer of photoreceptor terminals contact cones, while those terminating a s single expansions in the proximal layers also contact cones (fig. 13). The single axonal process of a Type I horizontal cell (figs. 2,3)is about 0.7 p m in diameter and consistently originates from the base of a primary dendrite. The distance from the point of origin to the termination of the axon is from 15-500pm. The average length, however, is over 100 pm, except in the red area where it is 25-50pm. The course of the axon, which has no collaterals, is within the outer plexiform layer just distal to the inner nuclear layer, is often tortuous and may cross itself several times before terminating as an expansion adjacent to the dendrites and soma of its origin (fig. 12).Varicosities as thick as 2 p m often occur along the length of this process (fig. 3). At its termination, the axon rapid-
ly increases to 3-5p m in diameter and forms an irregular tuberous structure which occupies a n intermediate position in the outer plexiform layer (figs. 2,8). Although irregular and variable in shape, the axon terminal usually approximates in diameter of span, the dendritic span of its cell of origin. Numerous spines, a s thick as 1 p m a t the base and narrowing to less than 0.5 p m a t the tip, project from the terminal expansion (fig. 9). However, unlike the dendrites from the soma which have dendritic terminals throughout the outer plexiform layer, these spines end only in the most distal portion of the outer plexiform layer. While many of the spines are isolated structures, some branch and form clusters (fig. 9). The terminal spines contact rods and cones in the distal layer of photoreceptor terminals (fig. 14). Both single spines and clusters contact cones, but only single spines contact rods.
Type II horizontal cells The Type I1 horizontal cells are considerably larger than the Type I horizontal cells (see table 1 and fig. 12 for comparison). The perikaryon of the Type I1 horizontal cell, like that of the Type I cell, is located in the distal layer of nuclei in the inner nuclear layer. It appears rounded when viewed in a flat mount (fig. 111, but is more flattened than the soma of the Type I cell when viewed in a vertical section (fig. 10). In spite of this difference in shape, the Type I1 somata are approximately the same size as the Type I somata in the yellow fields and central area, but they appear to be larger (6pm) in the red field than the Type I somata of this area. The most characteristic feature of Type I1 horizontal cells are irregular wavy dendrites. There are four to eight primary dendrites, which are often as thick as 3 p m as they radiate from the soma. They
TABLE 1
Differencesbetween horizontal cell types of the pigeon retina Dendritic span (pm) Cell type
area
Yellow fields
Red area
18-20
22-32
13-17
Central
Dendritic diameter em)
Level of termination of dendrites in OPL
Mode of termination of dendrites in OPL
Distal
Clusters
Proximal
Single expansions Single SDines
Axon
~
Type I
Type 11
33-40
48-75
30-39
Present
1.6-2
1
Proximal
Not Present
16
ANDREW P. MARIAN1 AND ALPHONSE E. LEURE-DUPREE
quickly narrow to about 1 pm, but may be slightly larger than this along their course especially a t points where branching occurs (fig. 11). When viewed in a flat preparation, the field formed by the span of dendrites is round and is usually 54 p m in diameter in the yellow field; however, in the more peripheral parts of this area i t may reach 75 pm. The dendritic span in the red field is 33 p m in diameter and only slightly larger in the central area (38 pm). Sectioning of retinas in a vertical plane (fig. 101, shows both the primary dendrites and their branches are confined to the most proximal margin of the outer plexiform layer. In contrast to the terminal clusters of the Type I horizontal cells, the terminations of the Type I1 dendritic processes are typically single small (0.5 pm) spines (fig. ll), which are found also at various points along the length of the dendrites (fig. 10).The Type I1 horizontal cell has never been observed to have an axonal process. DISCUSSION
Two distinct horizontal cell types are described in Golgi-impregnated pigeon retina: a small "brush-shaped'' cell (Type I) with an axon expanding into a spinous terminal arborization; and a larger horizontally flattened cell (Type 11) with irregular dendrites and no apparent axon. The horizontal cells of the pigeon retina can be distinguished from one another, not only on the basis of an axon and diameter of dendritic span, but also by levels and patterns of termination of dendrites in the outer plexiform layer. The majority of dendrites of the Type I cells terminate in the outermost level of the outer plexiform layer in clusters. In contrast, the dendrites of the Type I1 horizontal cells terminate in the innermost level of the outer plexiform layer. The Type I horizontal cells of pigeon retina are clearly the "brush-shaped" type described in birds by Cajal (1889, '33). The terminal expansions of the Type I cells of the pigeon resemble Cajal's ('33) "stellate" cells, not only in appearance, but also level of termination of spines in the distal part of the outer plexiform layer. This finding substantiates the observations of Gallego et al. ('75b1, in other avian species. However, the Type I1 horizontal cells reported in this paper, have not been previously described in the avian retina. Of all reported types of mammalian horizontal cells, the Type I cells of the pigeon
most closely resemble the foveal horizontal cells of monkey retina (Polyak, '57) and the horizontal cells of diurnal squirrels (West and Dowling, '75). The Type I1 horizontal cells of pigeon retina are most like the A type described in cat and rabbit (Dowling et al., '66; Fisher and Boycott, '74). In the retinas of the passerine and gallinaceous species studied by Cajal ('331, the photoreceptor terminals were found in a number of concentric plexuses in the retinal plane, with rod terminals present in the outermost layer and cone terminals present in all layers. These results have been confirmed in chick (Morris and Shorey, '671, and chicken, eagles and hawks (Gallego et al., '75a). Such a distinct stratification of photoreceptor terminals and laminar organization of horizontal cell processes in the outer plexiform layer of the pigeon retina has aided identification of photoreceptor-horizontal cell connections. The dendrites of the Type I horizontal cells, which end as single terminals in the proximal part of the outer plexiform layer, contact cones, since there are no rod terminals a t the proximal level. Other dendrites of this cell type which terminate in clusters in the distal layer of photoreceptor terminals also contact cones and are similar to the clusters of dendritic terminals of horizontal cells observed in primates (Polyak, '57; Boycott and Kolb, '73; Ogden, '74; Gallego, '751, and the A and B horizontal cells of cat (Dowling et al., '66; Fisher and Boycott, '741, which are known from Golgi-EM studies to contact only cones (Kolb, '70; '74; Boycott and Kolb, '73). The dendrites terminating as single expansions, a t the same (distal) level as the clusters contact cones, and are located a t the periphery of the dendritic field. This arrangement is also present in goldfish cone horizontal cells (Stell and Lightfoot, '75). The axon terminal spines of the Type I horizontal cells are located in the distal layer of the outer plexiform layer, where rod-type photoreceptor terminals are located, and it might be attractive to suppose that they are similar to mammalian axon terminal systems which are known to contact only rods (Kolb, '70, '74; Boycott and Kolb, '73). However, in the pigeon retina these terminals contact both rods and cones. The same horizontal cell process in contact with rods and cones has been observed previously in tiger salamander retina from serial EM reconstruction (Las-
PIGEON HORIZONTAL CELLS
ansky, ’73), and is also reported for the L1cells of the turtle retina (Saito e t al., ‘74). The Type I1 horizontal cells of pigeon retina are not unique among vertebrate horizontal cells in lacking a n axon. The absence of such a process is reported in smooth dogfish retina (Stell and Witkovsky, ,731, the rod horizontal cells of goldfish (Stell, ’751, the A-type horizontal cells of cat and rabbit (Dowling et al., ’66; Fisher and Boycott, ’74), and a horizontal cell type (termed “amacrine” of the outer plexiform layer) in dog, rat and guinea pig retinas (Gallego, ’71). The dendrites of the Type I1 horizontal cells of the pigeon retina are located in the proximal part of the outer plexiform layer, and contact cones, since only cone terminals are located at this level of the outer plexiform layer. The horizontal cells in various species of fish are known to be arranged in layers (Parthe, ‘72; Stell and Witkovsky, ’73; Naka and Carraway, ’75; Stell, ’751, while the photoreceptor terminals are present in a single stratum. In goldfish retina the layering of the horizontal cells has been shown to be related to color-coded connections with the photoreceptors (Stell and Lightfoot, ’75). Conversely, in the pigeon retina, the photoreceptor terminals are distributed in different strata in the outer plexiform layer while the horizontal cells are present in a single level of the inner nuclear layer. The dendritic processes of the horizontal cells are arranged in the sublayers of the outer plexiform layer in a manner specific to each of the horizontal cell types. Therefore, it is reasonable to expect that the distribution of horizontal cell processes in this layer may be related to the presence of specific cone color types in each of the strata of the outer plexiform layer of the pigeon. ACKNOWLEDGMENTS
The authors gratefully acknowledge Doctors E. V. Famiglietti, Jr. and Helga Kolb for their criticisms and suggestions. This material was presented in part in a preliminary report a t the 1976 Meeting of the Association for Research in Vision and Ophthalmology held at Sarasota, Florida. LITERATURE CITED Binggeli, R. L., and W. J. Paule 1969 The pigeon retina: Quantitative aspects of the optic nerve and ganglion cell layer. J. Comp. Neur., 137: 1-17. Boycott, B. B., and J. E. Dowling 1969 Organization of the
17
primate retina: Light Microscopy. Phil. Trans. R. SOC. Lond. B, 255: 109-184. Boycott, B. B., and H.Kolb 1973 The horizontal cells of the rhesus monkey retina. J. Comp. Neur., 148: 115-140. Cajal, 5.R. 1889 Sur la morphologie et les connexionsde la retine des oiseaux. Anat. Am.,4: 111-121. 1933 La d i n e des vertebres. Trav. d. Labor. d. Rech. biol. L’Univ. d. Madrid. 28. As translated in: The Structure of the Retina. S. A. Thorpe and M. Glickstein, transls. Charles C Thomas, Springfield, Ill. Cohen, A. I. 1963 The fine structure of the visual recep tors of the pigeon. Exp. Eye Res., 2: 88-97. Colonnier, M. 1964 The tangential organization of visual cortex. J. Anat. (London), 98: 327-344. Dowling, J. E. 1970 Organization of vertebrate retinas. Invest. Ophth., 9: 655-680. Dowling, J. E., J. E. Brown and D. Major 1966 Synapses of horizontal cells in the rabbit and cat retinas. Science, 153: 1639-1641.
Dowling. J. E.. and F. S. Werblin 1969 Organization of the retina of the mudpuppy Necturua macuklrs. I. Synaptic structure. J. Neurophysiol., 32: 315-338. Dubin, M.W. 1970 The inner plexiform layer of the vertebrate retina: A quantitative and comparative electron microscopic analysis. J. Comp. Neur., 140: 479-505. Fisher, 5. K., and B. B. Boycott 1974 Synaptic connexions made by horizontal cells within the outer plexiform layer of the retina of the cat and the rabbit. Proc. R. SOC.Lond. B, 186: 317-331. Galifret, Y. 1968 Les diverses aires fonctionnelles de la retine du pigeon. 2. Zellforsch., 86: 535-545. Gallego, A. 1971 Horizontal and amacrine cells in the mammal’s retina. Vision Res. (Suppl. 3), 11: 33-50. Gallego, A, M. Baron and M. Gay= 1975a Organization of the outer plexiform layer of the diurnal and nocturnal bird retinae. Vision Res., 15: 1027-1028. 1975b Horizontal cells of the avian retina. Vision Res., 15: 1029-1030. Gallego, A., and J. A. Sobrino 1975 Short-axon horizontal cells of the monkey’s retina. Vision Res., 15: 747-748. Kolb, H. 1970 Organization of the outer plexiform layer of the primate retina: Electron microscopy of Golgiimpregnated cells. Phil. Trans. R. Soc. Lond. B, 258: 261283. 1974 The connections between horizontal cells and photoreceptors in the retina of the cat: Electron microscopy of Golgi preparations. J. Comp. Neur., 155: 1-14. Lasansky, A. 1971 Synaptic organization of cone cells in the turtle retina. Phil Trans. R. Soc. Lond. B, 262: 365381. 1973
Organization of the outer synaptic layer in the retina of the larval tiger salamander. Phil. Trans. R. Soc. Lond. B, 265:471-489. Morris, V. B., and C. D. Shorey 1967 An electron microscope study of types of receptor in chick retina; J. Comp. Neur., 129: 313-340. Naka, K., and N. R. G. Carraway 1975 Morphological and functional identifications of catfish retinal neurons. I. Classical morphology. J. Neurophysiol., 38: 53-71. Ogden, T. E. 1974 The morphology of retinal neurons of the owl monkey Aotes. J. Comp. Neur., 153: 399-428. Parthe, V. 1972 Horizontal, bipolar and oligopolar cells in the teleost retina. Vision Rea., 12: 395-406. Pedler, C., and M. Boyle 1969 Multiple oil droplets in the photoreceptors of the pigeon. Vision Res., 9: 525-528. Polyak, S. 1957 Structure of the vertebrate retina. In: The Vertebrate Visual System. The University of Chicago Press,Chicago, pp. 207-287. Richardson, K. C., L. Jarett and G. H.Finke 1960 Embed-
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ANDREW P. MARIAN1 AND ALPHONSE E. LEURE-DuPREE
ding in epoxy resins for ultrathin sectioning in electron microscopy. Stain Technol., 35: 313-323. Saito, T., W.H. Miller and T. Tomita 1974 C- and L-type horizontal cells in the turtle retina. Vision Res., 14: 119123.
Schultze, M. 1866 Zur anatomie und physiologie der retina. Archiv fur Microsk. Anatomie, 2: 175-286. Stell. W.K. 1972 The morphological organization of the vertebrate retina. In: Handbook of Sensory Physiology. VW2. Physiology of photoreceptor organs. M. G. F. Fourtes, ed. Springer-Verlag, Berlin, pp. 111-213. 1975 Horizontal cell axons and axon terminals in goldfish retina. J. Comp. Neur., 159: 503-520. Stell. W K.. and D. 0. Lightfoot 1975 Color-specificinterconnections of cones and horizontal cells in the retina of the goldfish. J. Comp. Neur., 159: 473-502.
Stell, W. K., and P. Witkovsky 1973 Retinal structure in the smooth dofish, Mustelus canis: Light microscopy of photoreceptor and horizontal cells. J. Camp. Neur., 148: 33-46.
Valverde, F. 1970 The Golgi method. A tool for comparative structural analyses. In: Contemporary Research Methods in Neuroanatomy. W. J. Nauta and S. 0. F. Ebbesson, eds. Springer-Verlag, New York, pp. 12-31. van Genderen Stort, A. G. H. 1887 Ueber form- und ortsveriinderungen der netzhautelemente unter einfluss von licht und dunkel. v. Graefe's Arch. Ophthal., 33: 229292.
West, R. W., and J. E. Dowling 1975 Anatomical evidence for cone and rod-like receptors in the gray squirrel, ground squirrel, and prairie dog retinas. J. Comp. Neur., 159: 439-460.
PLATES
N 0
X
896.
3 Type I horizontal cell viewed from scleral side in a flat preparation in a montage at two different levels of focus. X 1,024.
The vitreal surface is oriented towards the bottom of the figure.
2 Type I horizontal cell viewed in a vertical section in a montage of micrographs a t different levels of focus.
Light micrographs of Golgi-impregnated horizontal cells of the pigeon retina.
EXPLANATION OF FIGURES
PLATE 1
PIGEON HORIZONTAL CELL3 Andrew P. Mariani and Alphonae E. Leure-duPree PLATE 1
PLATE 2 EXPLANATION OF FIGURES
Light micrographs of Golgi-impregnated horizontal cells of the pigeon retina. 4 Type I horizontal cell soma and dendrites in a vertical section. X 1,920. 5 Different focus of same cell as figure 4. Note the termination of the dendrite in the dis-
tal portion of OPL as a cluster of “knob-like” swellings, while the proximal termination is as a single expansion. X 1,920. 6 Type I horizontal cell soma and dendrites which terminate in the proximal area of the OPL, as viewed from the scleral surface of the retina in a flat preparation. X 2,205.
7 Different level of focus of the same Type I horizontal cell 88 figure 6. The dendrites terminate in clusters at the distal level of the OPL. X 2,205.
8 Type I horizontal cell axon terminal as viewed in a vertical section.
X
1.890.
9 Type I horizontal cell axon terminal viewed from scleral side in a flat preparation. x 1.890.
22
PIGEON HORIZONTAL CELLS Andrew P. Mariani and Alphonse E. Leure-duPme
PLATE 2
23
PLATE 3 EXPLANATION OF FIGURES
Light micrographs of Golgi-impregnated horizontal cells of the pigeon retina.
10 Type I1 horizontal cell viewed in a vertical section. The dendrites terminate as single spines which are also found along the length of the dendrites. Note that the termination of the dendrites is only in the proximal area of the OPL. X 1,964. 11 Type I1 horizontal cell viewed from vitreal side in a flat preparation.
X
2,112.
12 Type I and Type I1 horizontal cells viewed from scleral side in a flat preparation. Note that the Type I1 (11) horizontal cell has a much wider dendritic span than the Type I (1) horizontal cell. at, Type I horizontal cell axon terminal. X 892.
24
PIGEON HORIZONTAL CELLS Andrew P. Mariani and Alphonse E. Leure-duRee
PLATE 3
25
PLATE 4
PIGEON HORIZONTAL CELLS Andrew
P. Muriani and Alphonse E. Leure-duPree
EXPLANATION OF FlGURES
Light micrographs of 1 pm sections of Golgi-impregnated horizontal cells of the pigeon retina.
13 Type I horizontal cell soma and dendrites in vertical section. Clusters (double arrows) of dendritic terminals in distal layer of OPL contact cones. Dendrites terminating in proximal part of OPL (single arrow) also contact cones. X 1,520. 14 Type I horizontal cell axon terminal in vertical section. Terminal clusters (double
arrows) contact cones, while single terminal (single arrow) contacts a rod.
26
X
1,520.
