LI ow-resistance intercellular pathways

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1 Interreceptoral junctions in the teleost retina P. Witkovsky, M. Shakib, and H. Ripps Junctions between photoreceptors of carp and catfish were examined to determine the potential pathways for interaction in the distal retina. In the carp, each cone emits many basal processes which course horizontally for up to 15 i*m in the outer plexiform layer. These make both en passant and terminal contacts, which resemble gap junctions, with both the pedicles and basal processes of neighboring cones. Another possible route for communication between carp cones is suggested by the shallow penetration of a basal process of one cone into the invagination of another. Carp rods emit short basal processes which contact the spherules and basal processes of neighboring rods. These junctions form punctate regions of close apposition. Light microscopic observations indicate the presence of rod-cone contacts in carp retina, but their five structure has not been elucidated. In the catfish retina, numerous, typical gap junctions are found between adjacent receptor bases, their short basal processes, and between perinuclear regiom of neighboring receptors. Due to the similar electron microscopic appearance of rod and cone bases in this retina, the receptor type participating in a given junction could not be identified with certainty. LI ow-resistance intercellular pathways are thought to constitute an important avenue for spread of excitation in the distal layers of some vertebrate retinae. In many species of fish, for example, the closely apposed membranes of neighboring horizontal cells'- - exhibit structural details characteristic of so-called "gap" junctions. 1 Moreover, there is good evidence that such intercellular junctions provide channels of enhanced ionic permeability through which horizontal cells are electrically coupled.' From the Departments of Physiology and Ophthalmology, Columbia University, and the Departments of Physiology and Ophthalmology, New York University, New York. Supported by Grants EY and EY Submitted for publication April 12, Reprint requests: Dr. P. Witkovsky, Department of Ophthalmology Research, Columbia University, College of Physicians and Surgeons, 630 W. 168 St., New York, N. Y There is far less certainty as regards the nature and functional significance of the lateral contacts between contiguous membranes of photoreceptor terminals detected in almost every class of vertebrate. 5 " Although it has been suggested that intercellular communication may occur at these sites, 7 - * '" " their structural features are unlike chemical synapses and, except in turtle, 12 none have been identified by conventional electron microscopy as typical gap junctions, i.e., membranes in close apposition separated by a central lucent gap of 20 to 30 A. 11 ' " It is noteworthy, however, that Raviola and Gilula 15 have interpreted the freeze-fracture appearance of terminal contacts in monkeys, rabbit, and turtle retinae as representing a variant of the gap junction. Our interest in the physiology of photoreceptors in carp and catfish retinas has dictated a need to determine the possible pathways for information flow in the outer

2 Volume 13 Number 12 Teleost retina 997 plexiform layer of the retina. In this report, some features of photoreceptor morphology in these two teleost species are described, with emphasis placed on the ultrastructure of the junctional complexes that link neighboring photoreceptors. Materials and methods Carp (Cyprinus carpio) and channel catfish {Ictalurus punctatus) were maintained in a dark room in aerated, filtered tap water at 14 to 15 C. (catfish) or 17 to 18 C. (carp). The fish were immobilized by spinal section just posterior to the gill slits and the eyes enucleated and opened by incision at the ora serrata. Darkadapted retinas were either dissected free of the underlying tissue or removed with attached pigment epithelium and choroid. Tissue prepared for electron microscopy was cut into small pieces and placed immediately in ice-cold fixative; good preservation of the catfish retina was obtained by fixing for 60 to 90 minutes at 4 C. in a freshly-prepared mixture of 2 per cent glutaraldehyde, 1 per cent osmium tetroxide, 0.1 M phosphate buffet at ph 7.1, and 3 per cent sucrose.'" Carp retinas were fixed in cold 4 per cent glutaraldehyde, 0.1 mm phosphate buffer, and 3 per cent sucrose for one hour, then postfixed for one hour in cold 1 per cent OsOj solution containing identical concentration of phosphate buffer and sucrose. A few retinas of both species were fixed directly in buffered osmium; the appearance and location of interreceptoral contacts were similar with and without primary aldehyde fixation. Following fixation, some of the specimens were stained en bloc with uranyl acetate. After washing for 30 minutes in 0.1 M maleate buffer (ph 5.0) at 4 C, the retinas were stained in freshly prepared 2 per cent uranyl acetate in the same buffer and at the same temperature for 60 to 90 minutes in darkness. All tissues were dehydrated in graded alcohols, transferred to propylene oxide, and embedded flat in Epon 812 or Adaldite (Cargille 6005). One micron sections were taken from each specimen and stained with toluidine blue or methylgne blue- Azure II for light microscopy. Thin sections were collected on naked 200 mesh grids or on single slot grids coated with formvar film. Retinal pieces treated en bloc with uranyl acetate were subsequently stained with lead citrate; others were double stained with uranyl acetate and lead citrate. The sections were examined in a Zeiss EH9A or a Siemens Elmiskop I electron microscope. Golgi-impregnated material was prepared from dark-adapted specimens. The retinas were immersed first in a mixture of 0.2 per cent Osd in 3 per cent potassium dichromate for two to four days, and then in 0.75 per cent silver nitrate for two to three days at room temperature in the dark (rapid Golgi method); they were embedded in soft plastic and sectioned at 40 to 50 f-m on a sliding microtome. Some of these sections were remounted on plastic blanks, resectioned at 0.5 to 1.0 pm and counterstained with Azure II. Further details of the method are provided in reference 17. Results Light microscopy carp. Two classes of photoreceptor are readily discerned in the Golgi-impregnated carp retina (Plate I, Figs. 1 through 5). Although the outer segments of the impregnated cells were not usually identifiable, the shapes of the proximal portions of the receptors were clearly identical with those described in carp by Schultze, is and similar also to corresponding regions of the rods and cones of perch 1 " and Eugerres.- 0 The rod (Plate I, Figs. 4 and 5) is characterized by a slender stalk about 1 (xm in diameter which swells to enclose the nucleus, and again to terminate in a spherule about 2 /xm in diameter. Rod nuclei occupy a proximal position with respect to cone nuclei, but rod spherules terminate in an irregular line situated distal to the row of cone pedicles (Plate I, Figs. 1 and 4). Unlike the rods of the catfish retina (see below), the rod spherule in the carp generally is aligned with the long axis of the cell. The spherule emits several short processes less than 0.5 fim in diameter, and generally about 0.5 /xiri in length, but occasionally as long as 3.0 ju.ni; all of the processes have a terminal swelling (Plate I, Figs. 4 and 5). The longer processes are often directed proximally, ending in the region of the cone pedicles (Plate I, Fig. 4). Carp cones are characterized by a somewhat stouter vertical stalk, about 2 p.m in diameter which terminates in a pyramidally shaped pedicle that abuts the outer plexiform layer. Pedicles are typically 5 to 6 pm wide at the base and give rise to numerous telodendritic processes. Short, stout processes 0.3 to 1.0 /xm wide and

3 998 Witkovsky, Shakib, and Ripps Investigative Ophthalmology December 1974 Fig. 1. Two neighboring carp cones impregnated by the Rapid-Golgi method. Note the laterally extending cone basal processes with terminal swellings. The profile of a rod is seen out of focus to the left of the cones. x2,400. Marker is 10.0 ^m for Figs. 1 through 5. Fig. 2. A 1 tim thick section through a Golgi-impregnated carp cone. Note short basal processes extending proximally from cone base. Section counterstained with Azure II. x2,400. Fig. 3. A 1 Mm thick section through Golgi-impregnated carp photoreceptors, counterstained with Azure II. A basal process from the impregnated carp cone terminates on a rod spherule (arrow). x2,400. about 1 juin long are directed proximally toward the layer of external horizontal cells (Plate I, Fig. 2) whereas others 0.2 to 0.4 ;u.m in thickness and up to 15 /xm in length, course horizontally in the outer plexiform layer (Plate I, Figs. 1 and 3). The latter overlap extensively with the processes of nearby cones (Plate I, Fig. 1). In light microscopic examination of the 40 to 60 fj.m thick plastic sections, it was impossible to establish the terminal point of a cellular process when many Golgi-impregnated profiles lay in close proximity. Much better resolution was obtained by resectioning appropriate portions of thick plastic sections at a thickness of 0.5 to 1.0 /j.m, then counterstaining with toluidine blue to establish the position of the retinal layers. In fortunate sections, apparent rod-cone contacts were noted. Plate I, Fig. 3 shows a cone basal process ending with a terminal swelling on a rod spherule, whereas in Plate I, Fig. 4, a rod basal process appears to contact a cone pedicle. Light microscopy catfish. The photoreceptor layer of the catfish retina also contains rods and cones. Both classes of receptor are capable of photomechanical movement, rods contracting in the dark and expanding in the light, cones doing the reverse. 21 In the light-adapted retina (Plate I, Fig. 6), there is a clear separation of rod and cone outer segments, due to a dramatic extension of the rod myoids. The distinctions between rods and cones at the level of their outer segments are readily perceived. Cones have a narrow tapering outer segment about 10 pm long, whereas the rod outer segment is broad, cylindrical, and approximately 30 ^m in length. The nuclei of the receptors form two irregular layers and it appears from serial reconstructions that most of the nuclei in the distal layer belong to cones, those in the proximal layer to rods (G. Adomian, personal communication). The synaptic bases of the photoreceptors form a single row just distal to the outer plexiform layer and the layer of external horizontal cells (Plate I, Fig. 6). However,

4 Volume 13 Number 12 Teleost retina 999 Fig. 4. A 1 /im thick section through Golgi-impregnated carp photoreceptors, counterstained with Azure II, A rod basal process appears to end on a cone pedicle (arrow). A well impregnated rod profile with basal processes is seen out of focus at left. x2,400. Fig. 5, A carp rod impregnated by the Rapid-Golgi method. Note the short basal processes. x2,400. Fig. 6, A 1Mm thick, Azure H-stained section of the photoreceptor layer of a light-adapted catfish retina. Rod outer segments (ROS), fully extended through photomechanical movements, lie distal to the layer of cone outer segments (COS). Receptor bases (RB) form a single row. xl,280. Marker 10.0 wn. Fig. 7. A catfish rod impregnated by the rapid Golgi method. *2,000. Marker is 10.0 /*m for Figs. 7 and 8. Fig. 8. A catfish cone impregnated by the rapid Golgi method. x2,000. Note the short basal processes of both rods and cones (arrows).

5 , * * 1000 Witkovsky, Shakib, and Ripps investigative Ophthalmology December 1974 tl s. Fig. 9, A section through two adjacent carp rod spherules. At two points (arrows) intervening processes of Miiller fibers are excluded and the bases are joined by junctional complexes. Aldehyde fixed. x24,000. Marker 1.0 Am. Fig. 10. A punctate junction joining two carp rods. Aldehyde plus osmium-fixed tissue, stained en bloc with uranyl acetate. x300? 000. Marker 500 A. in toluidine blue-stained sections we could not distinguish rod from cone terminals, a situation that persisted for the most part in examination of sections by electron microscopy. ' With the Golgi method, silver chromate impregnation did not extend to the outer segments of the catfish receptors. Consequently, we relied upon criteria based on the differences in nuclear position referred to above and the location of the synaptic base of the cell with respect to its nucleus. According to Adomian (personal communication), the perikaryal region of the rod cell and its synaptic base are connected by a short and usually obliquely oriented fiber, whereas the cone terminal and its nuclear region are joined by a relatively long and vertically oriented stalk. These criteria enabled us to distinguish the terminal portions of the two types (Plate I, Figs. 7 and 8). The synaptic bases of catfish photoreceptors are spherical to pyramidal in shape, but either form could be found in association with rods or cones. Indeed, as shown in Plate I, Figs. 7 and 8, the more spherically shaped terminal belongs to a cone. Fine processes less than 0.5 //.m in diameter and 0.5 to 3.0 jum in length were emitted by all receptor bases, but occasionally also arose from the region of the perikaryon. Electron microscopy, carp. Partial reconstruction of photoreceptor bases through examination of serial sections was employed to trace junction al contacts in the outer plexiform layer. The short processes emitted from rods invariably terminated on a similar process from a neighboring rod spherule or on the spherule itself. Occasionally, two contacts between a pair of cells were observed (Plate II, Fig. 9); in addition, groups of junctions were seen in which more than two rod spherules participated. Serial sections through these junctions established that they consisted of

6 Vo/imi<; 13 Number 12 Teleost retina Fig. 11. A carp cone base showing the entry of a horizontal cell dendrite (H) and a bipolar cell dendrite (B, arrow). Note the punctate densities within horizontal cell terminals (arrows). Aldehyde fixed. x20,000. Marker 1.0 jum. Fig. 12. Two adjacent carp cones. A basal process from one (arrow) makes a shallow penetration into the invagination of its neighbor. Aldehyde fixed. x20,000. Marker 1.0 Mm. punctate regions generally less than 0.5 //.m in either plane normal to that of membrane apposition. At higher magnification, the junctional region at times had the sevenlayered appearance of a gap junction. However, as shown in Plate II, Fig. 10, the dimension of the union were 110 A instead of the usually encountered 140 A width. 34 We were not successful in following the course of the relatively long processes of rod spherules. The problem of tracing cone telodendria was particularly difficult in that processes of several cell types course in the region of the outer plexiform layer, and some of these participate in close membrane appositions. Thus we had first to establish reliable criteria for distinguishing among processes of receptor, bipolar, horizontal, and Miillerian cells. This was achieved through examination of serial sections in combination with the cellular profiles established by Golgi-silver impregnation. Our findings are fully consistent with the earlier study of the goldfish outer plexiform layer by Stell a - to which we can add some further details. The cone pedicles of the carp retina

7 1002 Witkovsky, ShaMb, and Ripps Investigative Ophthalmology December 1974 H Fig. 13. A carp cone into which penetrates a horizontal cell dendrite (H) containing two dense bodies. Note that the stout, electron-lucent horizontal cell dendrites are the outer layer of processes entering the cone base. Long, narrow processes, mainly bipolar cell dendrites, are grouped in the center of the invagination. Aldehyde fixed, stained en bloc with uranyl acetate. x20,000. Marker 1.0 nm. appear to have only a single, large invagination into which enter 20 to 40 processes (Plate II, Figs. 11 and 12, Plate HI, Fig. 13). Stout, electron-lucent dendrites arising from external horizontal cells take a vertical course into the cone base where they form a layer that lines the invagination, abutting the cone. Within the invagination, horizontal cell processes divide into several lobes which participate in ribbon synaptic complexes. In aldehyde-fixed tissue, horizontal cell dendrites manifest prominent punctate densities (Plate II, Figs. 11 and 12) and often contain a dense body (Plate III, Fig. 13). In addition, gap junctions are observed between horizontal cell dendrites, just proximal to the cone pedicles, as well as between adjacent horizontal cell bodies within the inner nuclear layer. The irregular processes of Miiller fibers are characterized by relatively clear cytoplasm containing abundant membraneous fragments, possibly of endoplasmic reticulum, as well as numerous mitochondria, but lacking vesicles. Close junctional appositions between the membranes of adjacent Miiller fiber processes were noted. Many thin processes course horizontally within the outer plexiform layer. The cyto- plasm of one group of processes frequently contains microtubules oriented parallel to the long axis of the process; no other prominent cellular inclusions were noted. Collateral processes, lacking microtubules, branch off at right angles to enter the cone invagination (Plate II, Fig. 12), where they terminate as a central element in the ribbon synaptic complex. We presume that such processes are bipolar cell dendrites, since they conform to the orientation of the bipolar cell dendrites seen in Golgi-impregnated specimens. 19 Bipolar cell dendrites were never seen to participate in close junctional appositions. The cytoplasm of the cone pedicle has a

8 Volume 13 Number 12 Teleost retina 1003 Figs. 14 and 15. Photos one and four of a series through a carp cone pedicle. In Fig. 14, three processes having a similar cytoplasmic appearance (arrows) make contact in the region of a cone pedicle. The connection of process No. 1 to the pedicle is shown in Fig. 15. In this section, processes two and three now form a junctional complex. Aldehyde fixed, stained en bloc with uranyl acetate. x24,o00. Marker 1.0 imi. uniform gray appearance and is evenly filled with agranular vesicles 300 to 400 A in diameter. The stout, vertically directed processes of the pedicle enter the external plexiform layer and sometimes abut the distal face of the external horizontal cells. Thin, laterally directed processes of the cone pedicle were followed in serial sections (Plate IV, Figs. 14 and 15). The gray appearance of the cytoplasm was retained in the processes, but the concentration of vesicles diminished sharply within a micron or two from the pedicle. Junctional appositions frequently were observed between cone processes and profiles of other processes having similar dimensions and appearance (Plate III, Fig. 14). In addition, contacts between cone pedicles and other processes running horizontally in the outer plexiform layer were observed. The latter were difficult to trace with certainty in serial sections due to the small dimensions of the telodendria and the fact that they intermingle with many other processes of similar size and appearance. At higher levels of magnification, these unions were characterized by a 140 A width; in some examples, the area of apposition was a

9 1004 Witkovsky, Shakib, and Ripps Inocstigative Ophthalmology December 1974 Fig. 16. A cone outer segment of the catfish retina. Note the openings of the discs to the exterior (arrow) and the radial fins of cytoplasm (F) which extend into the outer segment region. Aldehyde plus osmium fixed, x 14,400. Marker 1.0 tun. Fig. 17. A rod outer segment of the catfish retina. The discs of the rod outer segment swell slightly at their lateral limits, and do not open to the exterior of the cell. Rods also have radial fins. Aldehyde plus osmium fixed. xl4,400. Marker 1.0 /mi. punctate region, in others, it extended up to 1 ju.m. In three series, it was possible to trace the entry of a cone process into the synaptic invagination of a neighboring cone (Plate II, Fig. 11). The process did not appear to participate directly in the ribbon complex; it occupied a relatively shallow position in the tangle of processes within the invagination, and there was no apparent membrane specialization. Electron microscopy, catfish. We noted previously that rod and cone outer segments are readily identified in light microscopy. This applies equally to their electron microscopic appearance. As shown in Plate IV, Fig. 16, the cone lamellae are an infolding of the outer plasma membrane and are therefore in direct contact with the extracellular fluid. Rod discs, on the other hand, are isolated from each other and from the extracellular matrix except at the proximal ends of the outer segments (Plate IV, Fig. 17). Both rod and cone outer segments are surrounded by an array of radial fins (i.e., calycal processes^) extending distally from the inner segments. The clear distinction between rods and cones seen in their outer segments was not maintained at the level of the receptor nuclei or their synaptic bases. Adomian (personal communication) has found that in aldehyde-fixed phosphate-buffered catfish retina, rod nuclei appear darker than cone nuclei. Unfortunately, we could not reliably apply this criterion to our material; differentia] staining was frequently observed, but both darkly and lightly stained nuclei were found in the distal (presumably cone) and proximal (rod) positions. Nor were there any characteristic differences in the staining properties of receptor bases. Close membrane appositions were observed between catfish photoreceptors both at their terminals and less frequently at the level of their nuclei. Plate IV, Fig. IS shows a junctional contact between the nuclei of adjacent receptors. At this site the glial processes (Miiller fibers) that nor-

10 Volume 13 Number 12 Teleost retina 1005 H Fig. 18. A close junctional apposition between two photoreceptors of catfish retina in the perinuclear region (Nuclens-N). Note also the desmosomal junctions between processes of Muller fibers above (M). Aldehyde plus osmium fixed, stained en bloc with uranyl acetate. x29,500. Marker 0.5 urn. Fig. 19. A presumed cone receptor of catfish retina is penetrated by a dendrite nf an external horizontal cell (H). Note the vertical orientation of the axona! stem. The cone also makes a close junctional apposition with a process An the external plexiform layer (arrow). Aldehyde plus osmium fixed, stained en bloc with uranyl acetate. *10,140. Marker 1,0 t*m. Fig. 20. A group of junctions among receptor basal processes in the external plexiform layer of catfish retina. Aldehyde plus osmium fixed, stained en block with uranyl acetate. x43,000. Marker 0.5 ^m. mally intervene between cells are excluded. So-called desmosomal contacts between Muller fibers are also illustrated. Junctional contacts between terminals were seen principally between telodendritic extensions from the photoreceptor bases (Plate IV, Figs. 19 and 20), which in favorable cases could be traced to the bases themselves (Plate V, Fig. 21). Due to the absence of identifying features in the terminals, we were unable in most instances to ascertain the identity of the junctional elements. However, in the case of Plate IV, Fig. 19, the vertical orientation of the stem connecting the perinuclear region of the receptor with its base, and the fact that this base receives a. dendritic process from the external horizontal cell suggests that it is a

11 1006 Witkovsky, Shakih, and Ripps Investigative Ophthalmology December 1974 Fig, 21. Close junctional contacts between photoreceptor bases of catfish retina. Aldehyde plus osmium fixed, stained en bloc with uranyl acetate, xl8,600. Marker 1.0 pm. cone terminal. In a recent correlative study of the physiology and morphology of neurons in the catfish retina it was shown that the external horizontal cell is cone connected.- 4 At higher magnifications, the junctions occurring both in the region of the terminals (Plate V, Fig. 22) and at the nuclear level (Plate V, Fig. 24) have a sevenlayered appearance consisting of two-membrane pairs separated by a 20 to 30 A gap; the total width of the junctional complex is 140 to 150 A. The cytoplasm adjacent to the junctions contains an accumulation of fuzzy material adhering to the membranes in the region of the junction. The lattice work apparent in an obliquely sectioned junction (Plate V, Fig. 23) probably represents the network of intercellular bridges. 14 Discussion The view has long been held that vertebrate photoreceptors are functionally independent units whose receptive fields are no greater than the physical dimensions of their pigment-bearing outer segments. However, recent electrophysiological studies have demonstrated conclusively that the light-evoked responses of both rods and cones can be influenced by illumination of surrounding elements.- 5 " 30 Two kinds of pathways for information flow have emerged from these studies: an inhibitory feedback from neurons lying postsynaptic to the receptors, and a facilitatory effect of one photoreceptor upon its neighbor. In turtle retina, the coupling between neighboring cones as revealed by current injection techniques, 2 "' so together with the presence of gap junctions between their terminals, 1 - JS is consistent with the hypothesis that the facilitatory effect is mediated electrotonically. The several recent studies of the outer plexiform layer in retinas of submammalian vertebrates clearly indicate a great diversity of junctional contacts among photoreceptors and between them and horizontal or bipolar cells. It is apparent that the anatomical circuits which subserve particular physiological interactions will have to be established independently for each species. Although the relevant physiological studies

12 Volume 13 Number 12 Teleost retina 1007 Figs. 22 through 24. Junctional contacts between receptors of catfish retina, Fig. 22, junction at level of receptor bases. Fig, 23, an oblique section through a junction in which an alternating pattern of high and low electron densities is seen suggesting cytoplasmic channels between the receptors. Fig. 24, junction at the level of receptor nuclei. The junctions arc seven-layered, with a 20 to 40 A gap between membrane leaflets. A prominent cytoplasmic fuzz adjacent to the union is noted. Aldehyde plus osmium fixed, stained en bloc with uranyl acetate. x350,000. Marker 500 A. have yet to be performed, our anatomical data clearly support the supposition that electrical coupling exists among photoreceptors of catfish retina. Gap junctions with all the structural characteristics of those known to mediate electronic coupling (e.g., mesencephalic root of fifth nerve"' 31 are found both at the photoreceptor terminals and between membranes in the outer nuclear layer. The latter, observed also in the human retina 32 are seen relatively infrequently, but this may be a consequence of the plane of sectioning usually parallel to the long axis of the receptors. At the terminals, on the other hand, interreceptoral contacts are seen in virtually every randomly sampled section. The frequency with which these contacts occur suggests that neighboring photoreceptors are joined via a network of gap junctions. Due to the similarity of all photoreceptor terminals in this retina, we were unable to determine whether cones or rods or both participated in particular gap junctions. Close appositions resembling gap junctions in several respects were found between adjacent rods and between neighboring cones in the carp retina. However, a clear picture of a seven-layered junction, by which Brightman and Reese 1 ' 1 characterize the gap junction, was not obtained in carp retina, although the 140 A diameter of

13 1008 Witkovsky, Shafob, and Ripps stinalioc Ophthalmology December 1974 some of the junctional complexes between carp photoreceptors is consistent with the dimensions of the gap junction. Raviola and Gilula 15 report both punctate appositions and typical gap junctions among photoreceptors in several vertebrate species. The carp rod-rod union resembles the punctate junctions between rods and cones of the monkey, 15 whereas the cone-cone junctions in carp typically have a broader area of close apposition. Light microscopic observations indicate the existence in carp retina of rod-cone junctions similar to those reported by Parthe'" in the fish Eugenes. However, we were unsuccessful in locating these contacts in the electron microscope. An additional pathway for information flow between photoreceptors in carp retina is suggested by the penetration of a basal process of one cone into the invagination of another. The shallow position occupied by the basal process within the invagination indicates that it is not directly involved in a ribbon synapse, as seen in salamander retina ; it does, however, resemble the end contact of a basal process of one cone with the pedicle of another as described by Lasansky 11 in the turtle retina (cf. his Fig. 1, No. 8). In this paper we express our appreciation of the late Professor G. K. Smelser whose broad interests encompassed all areas of ocular research. In addition, P. W. thanks him for 10 years of unstinting encouragement and intellectual stimulation. We thank Drs. W. K. Stell and A. I. Cohen for helpful criticism of the manuscript, and Jane Zakevicius, E. Douglas MacDonald, and Emilia Corpus for excellent technical assistance. REFERENCES 1. Yamada, E., and Ishikawa, T.: Fine structure of the horizontal cells in some vertebrate retinas, Cold Spring Harbor Symposium Quantitative Biology 30: 383, Witkovsky, P., and Dowling, J. E.: Synaptic relationships in the plexiform layers of carp retina, Z. Zellforsch. Mikroskop. Anat. 100: 60, Witkovsky, P., and Stell, W. K.: Retinal structure in the smooth dogfish Mustelus canis: electron microscopy of serially sectioned bipolar cell synaptic terminals, J. Comp. Neurol. 150: 147, Kaneko, A.: Electrical connections between horizontal cells in the dogfish retina, J. Physiol. 213: 95, Sjostrand, F. S.: Ultrastructure of retinal rod synapses of the guinea-pig eye as revealed by three dimensional reconstructions from serial sections, J. Ultrastruct. Res. 2: 122, Dowling, J. E.: Structure and function in the all-cone retina of the ground squirrel. In: Conference on the Physiological Basis for Form Discrimination, Providence, 1964, Brown University, pp Nilsson, S. E. G.: Interreceptor contacts in the retina of the frog (Rana pipiens), J. Ultrastruct. Res. 11: 147, Cohen, A. I.: Some electron microscopic observations on interreceptor contacts in the human and macaque retinae, J. Anat. 99: 595, Lasansky, A.: Synaptic organization of cone cells in the turtle retina, Proc. Roy. Soc. B 262: 365, Dowling, J. E., and Boycott, B. B.: Organization of the primate retina: electron microscopy, Proc. Roy. Soc. B 166: 80, Custcr, N.: Structurally specialized contacts between the photoreceptors of the retina of the axolotl, J. Comp. Neurol. 151: 35, Lasansky, A.: Cell junctions at the outer synaptic layer of the retina, INVEST. OPH- THALMOL. 11: 265, Revel, J. P., and Karnovsky, M. J.: Hexagonal array of subunits in intercellular junctions of the mouse heart and liver, J. Cell Biol. 33: C7, Brightman, M. W., and Reese, T. S.: Junctions between intimately apposed cell membranes in the vertebrate brain, J. Cell Biol. 40: 648, Raviola, E., and Gilula, N. B.: Gap junctions between photoreceptor cells in the vertebrate retina, Proc. Nat. Acad. Sci. 70: 1677, Witkovsky, P.: Synapses made by myelinated fibers running to teleost and elasmobranch retinas, J. Comp. Neurol. 142: 205, Stell, W. K., and Witkovsky, P.: Retinal structure in the smooth dogfish, Mustelus canis: general description and light microscopy of giant ganglion cells, J. Comp. Neurol. 148: 1, Schultze, M.: Zur Anatomie und Physiologie der Retina, Arch. Mikroskop. Anat. 2: 175, Cajal, S. R. y: La retine des Vertebres, La Cellule 9: 121, Parthe, V.: Horizontal, bipolar and oligopolar

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