Cell Tiss. Res. 177, 181-193 (1977) Cell and Tissue Research �9 by Springer-Verlag 1977

The Accessory Outer Segment of Rods and Cones in the Retina of the Guppy, Poecilia reticulata P. (Teleostei)

An Electron Microscopical Study

Anwar Yacob, Christina Wise and Yvette W. Kunz

Department of Zoology, University College, Dublin, Ireland

Summary. The ultrastructure of the accessory outer segment (AOS) - a cilium- like structure emanating from the inner segment and running alongside the outer segment of photoreceptors - i s described. The AOS occurs in both rods and cones of Poecilia reticulata. Its ultrastructure, including the arrangement of microtubules, which originate from the ciliary stalk, is the same in rods and cones. The cone-AOS is connected with the outer segment by a thin plasmabridge, whereas the rod-AOS lies embedded within the outer segment. The outer segment of the cone, in contrast to that of the rod, is separated from the pigment epithelium by a large extracellular space. An intimate contact, however, is secured by the AOS; its membrane is closely appositioned to the pigment epithelium membrane. The functional significance of the AOS and its possible occurrence in other vertebrate classes, are discussed.

Key words: Accessory outer segment - Photoreceptors - Poecilia reticulata P. - Ultrastructure - Microtubules.

Introduction

Electron microscopical investigations have shown that the cones of certain fish possess a cilium-like structure emanating from the inner segment and running alongside the outer segment sensu stricto. This structure is named "accessory outer segment" by Engstr6m (1963) and referred to as "lateral sac" by Fineran and Nicol (1974). The term "accessory outer segment" (AOS) is used in this study.

Engstr6m (1963) describes the ultrastructure of the AOS in the wrasses (Labridae) as showing a fine reticular cytoplasm with no definite specialization apart from a few fine tubules. The AOS is thought to have a ciliary origin and is found to be connected with the outer segment proper by a short plasma bridge.

S e n d o f f p r i n t reques t s to ." Dr. Yvette W. Kunz, Department of Zoology, University College, Belfield, Dublin 4, Ireland

182 A. Yacob et al.

In the rod "the shape of the accessory outer segment is not sufficiently revealed". Similarly, an electron microscopical study of the eyes of various parrot fishes (Labridae) by Fineran and Nicol (1974) mentions that the outer segment of the rod does not contain an AOS. The cytoplasm of the AOS of catfish cones is reported to contain filamentous material (Arnott et al., 1974).

The present study describes the ultrastructure of the AOS in the cones and rods of Poecilia reticulata, and reveals a distinctive microtubular organization in both. The photoreceptors of the guppy have been extensively studied at the light microscopical level. The cones are arranged in a mosaic with the rods ran- domly interspersed. Moreover, the cones are arranged in tiers and are named inner, middle and outer cones in a vitreal towards scleral direction (Mfiller, 1952). The inner and middle cones are single, and the outer cones are twin cones, with regard to their joint retinomotor movements, but double cones with regard to differences in structure (Kunz and Wise, 1973).

Material and Methods

The light-adapted fish were decapitated, the eyes enucleated and the lens and sclera removed with watchmaker's forceps. The eyes were fixed in 0.075 M phosphate buffered 2~ glutaraldehyde, pH 7.2 (osmolality: 380 mOsm), for one hour at 4 ~ C. After rinsing in phosphate buffer, the eyes were postfixed in phosphate buffered 1~o osmium tetroxide, pH 7.2, for ll/2h at 4~ Dehydration was carried out through a graded ethanol series followed by propylene oxide. The eyes were embedded in Araldite (CY212) via propylene oxide. Ultrathin sections were cut on a LKB III ultrotome, mounted on Formvar-coated copper grids, stained with uranylacetate, followed by lead citrate and examined with a Philips 201 C electron microscope.

Results

In electron micrographs rods and cones of P. reticulata are easily identified in both longitudinal and transverse sections. In longitudinal sections through the inner segment of a rod, the mitochondria are of relatively equal size and are elongated in outline. They have a smooth, well defined outer membrane and cristae. The matrix is finely granular and contains numerous mitochondrial granules. In the inner segment of the middle and outer cones the mitochondria show a gradual increase in size along the vitreal-to-scleral axis. The cristae become sclerad progressively more convoluted and the matrix more electron dense. In the outer cones, one member contains a giant modified mitochondrion of either the cristate or matrix type, located at the scleral end of the inner segment (Berger, 1965; Kunz and Wise, 1973) (Figs. 1-3). The inner cones are not dealt with in this study.

In transverse section the rod outer segment shows an irregular outline, while the cone outer segments appear nearly circular in shape (Figs. 1 a-c , 4, 5). In longitudinal section the rod outer segments are identified as having floating bimembraneous discs (saccules), i.e. discs isolated from the cell membrane, as found in vertebrate rods generally. The discs of the cone outer segments are continuous with the plasma membrane (Figs. 1-3, 6, 7). As seen in cross-sections, the area of continuity extends to approximately 5/6th of the perimeter; in 1/6th

Accessory Outer Segment in Rods and Cones of a Teleost 183

> \ SCLERAD

A \ ?os

b----~___________ ~ J I . . . . . . . . ~ LMT

C O N E

~-AOS - _ _ 0S

R O D

1 c

~ ~ ~ ~ ,

.%

P b

)M T

A B

~ T

ABC

Fig. I a-c. Schematic drawing of cone and rod with cross sections at three levels (a-c). AOS accessory outer segment; CP calycal process; CS ciliary stalk; IS inner segment; MI mitochondrion; MTmicro- tubule; OS outer segment

184 A. Yacob et at.

Accessory Outer Segment in Rods and Cones of a Teleost 185

Fig. 6. Longitudinally sectioned rod. Ciliary stalk (CS) extends into outer segment (OS) as accessory outer segment (AOS). x 37,500

Fig. 7. Longitudinally sectioned cone showing accessory outer segment (AOS), closely appositioned to pigment epithelium (PE). • 20,000

Figs. 2-5. CP calycal process; IS inner segment; MG mitochondrial granule; MI mitochondrion; OS outer segment; PE pigment epithelium

Fig. 2. Longitudinal section of rod. Intimate relationship of outer segment with pigment epithelium. x 20,000

Fig. 3. Longitudinal section of cone. "Empty space" between outer segment and pigment epithelium. x 17,600

Fig. 4, Transverse section of rod outer segments. Note irregular perimeter and close association with pigment epithelium. • 17,500

Fig. 5. Transverse section of cone outer segment. Note circular perimeter and surrounding "empty space", x 22,500

186 A. Yacob et al.

of the perimeter the discs are open to the extracellular space (Figs. 1 a-b , 5, 14). It is not known if the area of continuity between plasma- and disc membrane is in register along the length of the whole outer segment as serial sections are not available.

The Accessory Outer Segment (AOS)

In the cone the ciliary stalk arises from the basal body located in the peripheral part of the inner segment close to its scleral end. It extends alongside the outer segment proper into the AOS, which remains connected to the outer segment by a thin plasma bridge. The limiting membrane of the AOS is, therefore, continuous with that of the outer segment, and the AOS lies opposite the area of the open discs (Figs. 1, 7). The AOS becomes wider towards its scleral end but does not extend to the tip of the outer segment (Fig. 5).

The ciliary stalk possesses nine sets of microtubules, evenly spaced in the form of a circle. At the basal extremity of the stalk each set consists of triplets of microtubules embedded in a dense amorphous matrix. The tubular arrangement is shown diagrammatically in Figure 1. The three microtubules which make up each "axial filament" in the basal part are called A, B, C, similar to the nomen- clature for basal bodies in motile cilia (Rhodin, 1974). They each share portions of their adjoining microtubular walls (Fig. 1 b, c, centre). As the ciliary stalk extends sclerally to become the AOS, the triplet (A, B, C) arrangement of tubules becomes a double one (A, B). More sclerally, the sets lose another member and only consist of single tubules (A). The circular arrangement found at the base of the ciliary stalk is now totally lost. Three of the nine sets of microtubules are found in the cytoplasmic bridge connecting the AOS with the outer segment. The cytoplasm of the AOS is finely granular, and, apart from microtubules no discs, mitochondria or other organelles have been observed (Figs. 9, 11, 13, 14).

The origin of the ciliary stalk in the rod is the same as in the cone. However, it extends as AOS directly into the outer segment and courses eccentrically within its limiting membrane. The AOS is now so embedded that it causes, at most, a peripheral bulge of the outer segment. Either the presence of the AOS in the outer segment causes the indentations in the free-floating discs, or the AOS itself fits into the indendations already formed (Figs. 1, 8, 10, 12).

Fig. 8. Rod, cross-sectioned through base of ciliary stalk (rectangle in Fig. 1 c). Microtubules in triplets (arrows). x 90,000

Fig. 9. Cone, cross-sectioned through base of ciliary stalk (rectangle in Fig. 1 c). Microtubules in triplets (arrows). x 90,000

Fig. 10. Rod, cross-sectioned through outer segment (rectangle in Fig. 1 b). Microtubules in doublets (arrows). x 90,000. AOS accessory outer segment; OS outer segment

Fig. 11. Cone, cross-sectioned through outer segment (rectangle in Fig. I b). Microtubules in doublets (arrows). x 60,000. OS outer segment

Accessory Outer Segment in Rods and Cones of a Teleost 187

188 A. Yacob et al.

Accessory Outer Segment in Rods and Cones of a Teleost 189

Fig. 14. Paired cone, cross-sectioned through scleral part of outer segment (OS), Note close apposition between accessory outer segments (AOS) and pigment epithelium. Accessory outer segments never lie between paired outer segments, x 25,000

The organisa t ion of the microtubules in the rod is the same as in the cone, with triple sets becoming double sets and finally single tubules towards the scleral end. Of the double sets and of the single microtubules , one always faces the inden ta t ion (Figs. 1, 10, 12).

Dur ing light adap ta t ion rods move sclerad and cones vitread. The rods become embedded in the processes of the p igment epithel ium. These extend as far as the

Fig. 12. Rod, cross-sectioned through outer segment (OS) and accessory outer segment (AOS). Rectangle in Fig. 1 a). Microtubules single (arrows). x 75,000

Fig. 13. Cone, cross-sectioned through outer segment (OS) and accessory outer segment (AOS). (Rectangle in Fig. 1 a). Microtubules single (arrows). x 45,000. PE pigment epithelium

190 A. Yacob et al.

scleral part of the inner segment of the outer cones; they reach to half the length of the middle cone outer segments and surround the tips of the inner cones. At the electron microscopical level distinct differences between the relationship of pigment-epithelial processes and rod and cone outer segments are observed. The rod outer segments, especially in their scleral region, are closely apposed to the pigment epithelium (Fig. 4), while no intimate association between pigment epithelium and cone outer segment proper has been found (Fig. 5). However, there is a close anatomical association provided by the AOS (Figs. 7, 13, 14).

Di~uss ion

The results show that the cones and rods of P. reticulata possess an AOS, which runs alongside the conventional outer segment. Only serial sections of whole outer segments would establish whether the AOS runs parallel the outer segment or wraps around it. The AOS in both rods and cones show the same ultrastructure, and especially the same arrangement of microtubules. In the cone the AOS is set off from the outer segment by a thin plasma bridge. In the rod the AOS fits into, or causes, the indentations of the discs and thereby is contained within the peri- meter of the outer segment (embedded AOS). This explains why in the isolated retinal cell preparations of Anctil, et al. (1973) the cone-AOS of various fish were strikingly evident, while the rod-AOS were not detectable.

Using the same method, the above authors observed AOS also in amphibian cones (Rana pipiens and Rana catesbeiana). While AOS have not been described ultrastructurally in amphibia, an electron microscopical study of the photo- receptors in Rana pipiens mentions and illustrates a "ridge along one side of the cone outer segment" (Nilsson, 1965).

There is as yet no mention of AOS in electron microscopical studies of mam- malian photoreceptors. However, the rod outer segment of guinea pig, mouse, rat and flying squirrel, when viewed in cross section, looks structurally similar to that of P. reticulata. It also shows an incision (cleft) facing a bundle of nine microtubules (Cohen, 1972). In rods with multiple clefts (man, cat) one or two microtubules face individual clefts. It seems, therefore, that these structures described in mammalian rods may be homologous with the (embedded) AOS in the rods of the guppy. So far cones of two mammalian species (cat and ground squirrel) have been found to possess "discs with clefts containing microtubules" (Steinberg and Wood, 1974). These might represent embedded AOS in cones, similar to the ones observed in rods.

The early anatomists described a thread-shaped element which may correspond to the AOS observed at the electron microscopical level (Ftirst, 1904; Held, 1904; Kolmer, 1904, 1914; Retzius. 1905). Walls (1942) describes this structure as a long filament, connected just within the inner segment with a pair of granules and run- ning axially or peripherally in the outer segment of (presumably) every visual cell. Later, this structure was more extensively studied, with the light microscope, by Engstr6m (1961) in cyprinids, gadids and labrids, and by Munk and Andersen (1962) in Homo, two deep sea fish and the goose, Anser anser L. It seems likely, therefore, that the AOS is found throughout the vertebrate phylum, maybe with an evolutionary sequence resulting in an embedded AOS.

Accessory Outer Segment in Rods and Cones of a Teleost 191

Various suggestions may be put forward as to the functional significance of the AOS. The microtubular arrangement of the ciliary stalk and the AOS in the guppy photoreceptors is that of a classical cilium (Rhodin, 1974). Although it contains the normal nine peripheral sets, the central pair is lacking. This has been found in other photoreceptor cilia, and is a known characteristic of non- motile cilia, as found e.g. in the crown cells of the fish brain (Satir, 1961). How- ever, high voltage electron microscopy of thick sections, stained with ruthenium red, revealed two single microtubules in the core of the connecting cilium of rat photoreceptors (Matsusaka, 1975). At the recently held conference on "The Biology of Cytoplasmic Microtubules" (Soifer, 1975), the microtubules in the ciliary stalk of photoreceptors were not included.

One of the functions of the AOS could be supportive. Its incorporation into the outer segment proper of the rod holds the extremely long segment in position. In the cone, provided the outer segments are divided into packages, as Grtin (1975) suggests (and there seems to be some evidence that this occurs in P. reti- culata), the AOS could serve to hold the packages together. The AOS, as well as the ciliary stalk, being the connecting structures between outer and inner segment and having a confluent plasmamembrane, must also be engaged in the conduction of visual impulses.

The ciliary stalk, and with it the AOS, are also the only pathway for the transport of material between inner and outer segment. Studies of the renewal of protein within vertebrate photoreceptors, combining electron microscopy with autoradiography, have shown that new proteins are synthesized in the myoid region and reach the outer segment through the ciliary stalk. In rods the labelled protein accumulates in the continuously forming discs at the base of the outer segment, while some of it spreads diffusely through the outer segment. In the cone, all of the labelled protein is diffusely spread throughout the outer segment (Young, 1970). The route taken by these newly formed proteins has not been established ultrastructurally. The AOS being in cytoplasmic continuity with the outer segment proper, is a likely channel. The microtubules could act either as "stationary or moving tracks", involving either transport of attached components along the microtubular surface, or simultaneous biterminal assembly and dis- assembly of microtubular subunits, resulting in a net transport of the attached components (Smith et al., 1975).

The association between photoreceptor outer segments and pigment epi- thelium, ranging from close apposition to fusion of the two membranes, and from simple microvilli to modifications such as leaf-like sheaths of the pigment epithelium, has been described for various vertebrates (reviewed by Steinberg and Wood, 1974). In the light adapted retina of P. reticulata, the entire rod outer segment is embedded in the pigment epithelium. Electron micrographs demon- strate a particularly close association between the scleral part of the outer segment- and the pigment epithelium membranes. The cone outer segment, however, is surrounded by an "empty space", which, in view of histochemical findings on other retinae, is probably filled with mucopolysaccharides synthesized in the myoid region (Ocumpaugh and Young, 1966). The close association between cone and pigment epithelium is secured by the AOS. It has been known for a long time that the viability of the photoreceptors is dependent on the presence of the pigment epithelium (reviewed by Wald, 1958; O'Brien, 1976). The close

192 A. Yacob et al.

apposition of photoreceptor and pigment epithelium membranes reflects, there- fore, a physiological relationship (transfer of Vitamin A, metabolites, O2). But there might be an additional functional aspect in the fish (and maybe amphibia), where the intimate morphological contact might be essential for the retinomotor activity. It was suggested that the movements of the visual cells in fish are passive, and depend on the expansion and retraction of the pigment epithelial processes (Anctil et al., 1973; Couillard, 1975). The anchoring of the cone AOS in the pig- ment epithelium, as demonstrated in P. reticulata, could be, therefore, instru- mental in preserving the spatial (mosaic + tiered) arrangement of the various cones during the repeated sclerad and vitread movements from light to dark adaptation.

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Accepted October 21, 1976