J. Cell Set. 22, 597-606 (1976) Printed in Great Britain
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GAP JUNCTIONS IN THE DIFFERENTIATED NEURAL RETINAE OF NEWLY HATCHED CHICKENS H. FUJISAWA*, H. MORIOKAf, H. NAKAMURA* AND K. WATANABE* The Department of Anatomy*, and tlie Electron Microscope Laboratory^, Kyoto Prefectural University of Medicine, Kaivaramachi Hirokoji, Kamikyoku, Kyoto, 602, Japan
SUMMARY
Gap junctions in the neural retinae of newly hatched chickens were examined in thin section and by freeze cleaving. Unusual gap junctions containing linear arrays of intramembrane particles are found between principal and accessory cones which form a double cone at the region of the outer limiting membrane. These unusual gap junctions are often continuous with macular aggregates of hexagonally packed intramembrane particles which are characteristic of a typical gap junction. Typical gap junctions are also found in both the outer and the inner plexiform layers and in the outer nuclear layer, but are not so abundant as in the outer limiting membrane region. The sizes of intramembrane particles and their centre-to-centre spacing within the macular aggregate of a gap junction in differentiated neural retinae are slightly larger than those in undifferentiated neural retinae. Tight junctions are not found in differentiated neural retinae.
INTRODUCTION
In a previous paper (Fujisawa, Morioka, Watanabe & Nakamura, 1976), it was shown that gap junctions were found only between differentiated cells with mitotic potential in the neural retinae of chick embryos, and not between the presumptive neurons or photoreceptors which were postmitotic. On the other hand, it is well known that gap junctions are present in the adult neural retinae of some kinds of vertebrates (Witkovsky & Dowling, 1969; Kaneko, 1971 ; Witkovsky & Stell, 1973 ; Raviola & Gilula, 1973). Thus, the following questions arise : (1) Is the gap junction also found in some of the functionally differentiated cells of chick neural retinae ? (2) If it is, do the gap junctions in differentiated neural retinae differ from the ones in undifferentiated neural retinae in their morphology and function ? The present studies have been made in order to answer these questions through electron-microscopic observations of thin-sectioned and freeze-cleaved neural retinae of newly hatched chickens, in which the neural retina is already morphologically and functionally differentiated.
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MATERIALS AND METHODS Only the posterior poles of the neural retinae of newly hatched chickens were prepared for electron microscopy. The procedure for preparation of tissues for thin section and freeze cleaving was the same as that given in the previous paper (Fujisawa et al. 1976). In thin section, a fixative prepared in Hanks' balanced salt solution (BSS) was more suitable than that prepared in cacodylate buffer to preserve the membranous structures of the neural retina of the present material. Also, no difference in the morphology of gap junctions in freeze-cleaved materials was found between any of the buffer solutions used in tissue preparation. Thus, the figures presented in this paper were obtained from the material prepared with the fixative in Hanks's BSS.
OBSERVATIONS
Gap junctions at the outer limiting membrane region
In the neural retinae of chickens, 5 types of photoreceptor have been distinguished ; i.e. rods, double cones which comprise a principal and an accessory cone, and 2 types of single cones (Morris & Shorey, 1967). In the present observations, the 5 types of photoreceptor are also identified in the neural retinae of newly hatched chickens. In a nearly tangential section made through the outer limiting membrane of the neural retina, photoreceptors are separated from each other by Miiller cells, except for the members of a double cone (Fig. 1). Between each photoreceptor and Miiller cell and also between Miiller cells, continuous junctional structures of zonula adhaerens type (Farquhar & Palade, 1963) are formed (Fig. 1). In a radial section made along the long axis of the neural retinal cells, the junctions of zonula adhaerens type extend 0-5 /tm in depth from the apices of Miiller cells (Fig. 2). On the cytoplasmic side of each zonula adhaerens is an amorphous electron-dense layer, and the apposed plasma membranes are generally separated by a gap about 25 nm wide (Fig. 3). Among the junctions of zonula adhaerens type, some focal close appositions of plasma membranes can be seen (Fig. 3 : indicated by arrows). In freeze-cleaved preparations, the junctional region of zonula adhaerens type can be easily identified as a zone in which intramembrane particles are scanty on A fracture faces of photoreceptors (Fig. 4) and of Miiller cells. The zone is expanded about Figs. 1-3. Outer limiting membrane region on thin-sectioned neural retina. Fig. 1. A nearly tangential section made through outer limiting membrane. Various types of photoreceptors (r) are separated by Miiller cells (m) except principal (pc) and accessory (ac) cones. Arrows, junctions of zonula adhaerens type between photoreceptor and Miiller cell and between Muller cells, x 10000. Fig. 2. Radial section. Junctions of zonula adhaerens type (arrows) are formed between photoreceptors (r) and Miiller cells (m). m», microvilli belonging to Muller cells, x 23000. Fig. 3. Focal close appositions of plasma membranes (arrows) among the junction of zonula adhaerens type ; m, Muller cells, x 250000. Fig. 4. Freeze-cleaved replica of outer limiting membrane region. Particle-reduced zone enclosed with dotted line is found on A fracture face of photoreceptor (r). m, Muller cells ; mv, microvilli belonging to Miiller cells. Arrow in circle in this and subsequent micrographs indicates direction of shadow, x 28000.
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0-5 /im depth from the apex of the Miiller cell, and situated vertically to the long axis of neural retinal cells. However, the special junctional structure which should correspond to the focal close appositions of plasma membranes found in the zonula adhaerens in thin-sectioned material is not observed in these zones. Only a few single intramembrane particles are found (Fig. 4). On the other hand, at the region of the outer limiting membrane, a principal and an accessory cone of a double cone are not separated by Miiller cells, and are directly in contact with each other (Fig. 1). In a tangential section made through the outer limiting membrane, an amorphous electron-dense layer is found on the cytoplasmic side of this direct contact region (Fig. 5). However, the intercellular gap of about 25 nm width which is usually found in the junction of zonula adhaerens type, is not observed. The plasma membranes of cone cells are nearly parallel and are sometimes closely apposed, with an intercellular gap 2-3 nm in width, which is characteristic of a typical gap junction (Fig. 6). Such a close apposition of plasma membranes of the members of a double cone is not restricted at the outer limiting membrane region. Similar structures are also found between the plasma membranes of cone cells slightly outside (Fig. 7) and inside (Fig. 8) the outer limiting membrane. In freeze-cleaved replicas of the contact region between principal and accessory cones, many macular aggregates and linear arrays of intramembrane particles are always found on the A fracture face (Fig. 9), and some of them are often situated outside or inside the outer limiting membrane level (Fig. 10). Within the macular aggregates of intramembrane particles on the A fracture face, the particles of 10—20 nm diameter are hexagonally packed with a centre-to-centre spacing of 12-15 nm (Figs. 11, 12). The size of the intramembrane particles and their centre-to-centre distance are slightly larger than those of the gap junctions found in the undifferentiated neural retinae, in which the gap junctions consist of 8-9-nm intramembrane particles arranged with 9 nm centre-to-centre distance (Fujisawa et al. 1976). The subgroup of intramembrane particles characteristic of the gap junction of the undifferentiated neural retina (Fujisawa et al. 1976) is not found in the interreceptor gap junction of the differentiated neural retinae. On the other hand, each linear array consists of 3-100 intramembrane particles arranged usually in a single row. These arrays are mostly randomly arranged ; some arrays are parallel, and others are oblique to the long axis of the cells (Figs. 9, 10). The rows of intramembrane particles are straight, curved, angular, and, sometimes, circular (Figs. 9, 10, 13). The intramembrane particles in a row are about 10-12 nm in diameter, and their centre-to-centre spacings are about 12-15 nm. On B fracture faces of the principal or the accessory cones, the complementary depressions of the linearly arrayed particles are observed (Fig. 14; arrows). Some of the linear arrays of intramembrane particles are comprised of more than one closely adjacent row of particles (Figs. 11, 12; arrows), and are continued with the macular aggregates of intramembrane particles (Figs. 11, 12). Such linear arrangements of intramembrane particles have been also found in some other tissues (Friend & Gilula, 1972 ; Raviola & Gilula, 1973; Decker & Friend, 1974; Benedetti, Dunia & Bloemendal, 1974), in what have generally been considered to be gap junctions.
Gap junctions in neural retina
Figs. 5—8. Interreceptor contacts between principal (pc) and accessory (ac) cones. Fig. 5. Contact (asterisk) at the outer limiting membrane region; m, MOller cells; arrows, junctions of zonula adliaerens type, x 18000. Fig. 6. High magnification of interreceptor contact; arrows, gap junctions of a 7-layered appearance, x 250000. Fig. 7. Contact (asterisk) just outside the outer limiting membrane level, x 18000. Fig. 8. Contact (asterisk) just inside the outer limiting membrane level; m, Miiller cells ; arrows, junctions of zonula adliaerens type, x 18000.
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Gap junctions in the other regions of the neural retinae
In the present observations, the typical gap junctions of macular aggregates of hexagonally arranged intramembrane particles and their complementary depressions are sometimes found in both the outer and the inner plexiform layers (Figs. 15, 16), and also in the outer nuclear layer of the neural retina in freeze-cleaved materials. The morphology of the gap junctions in freeze cleaving is similar to that of the interreceptor gap junctions found in the outer limiting membrane region, and is different from the gap junctions found in the undifferentiated neural retinae (Fujisawa et al. 1976). However, the frequency of gap junctions in freeze-cleaved materials is too low to demonstrate this type of junction in thin-sectioned materials. Thus, it is still difficult to decide the cell types between which gap junctions are formed. No gap junctions are detectable in the inner nuclear layer or in the ganglion cell layer. Tight junctions are not found at all in the neural retinae of newly hatched chickens. DISCUSSION Morphology of gap junctions
The present observations of freeze-cleaved replicas show that gap junctions in the neural retinae of newly hatched chickens exhibit 2 different appearances, i.e. macular aggregates and linear arrays of intramembrane particles. Though gap junctions consisting of the macular aggregates of intramembrane particles were also found in the undifferentiated neural retinae (Fujisawa et al. 1976), some morphological differences were found between differentiated and undifferentiated neural retinae. The size of intramembrane particles and their centre-to-centre spacings within a macular aggregate of differentiated neural retinae were slightly larger than those of undifferentiated neural retinae. In addition, subgroups of intramembrane particles which were found in gap junctions of undifferentiated neural retinae were not observed in gap junctions of differentiated neural retinae. Since it has been revealed that the gap junctions all disappeared at 9 days of development (Fujisawa et al. 1976), the new gap junctions must be produced during the processes of morphological and functional differentiation of neural retinae at the later stages of embryonic life. Thus,
Figs. 9-14. Freeze-cleaved replicas of the contact regions between the members of a double cone at the outer limiting membrane level. Fig. 9. Linear arrays and macular aggregates (arrows) of intramembrane particles on A fracture face ; ac, accessory cone ; pc, principal cone ; m, Miiller cells; mv, microvilli of Miiller cells, x 28000. Fig. 10. Linear arrays or macular aggregates of intramembrane particles located outside or inside the outer limiting membrane level. Two lines exhibit a region which should be corresponded to the outer limiting membrane, ac, accessory cone ; pc, principal cone ; m, Miiller cells ; mv, microvilli of Miiller cells, x 29000. Figs, i i , 12. Linear arrays and continuous macular aggregates of intramembrane particles on A fracture faces ; small arrows, linear arrays comprised of more than one row of intramembrane particles, x 63000.
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it may be speculated that such differences in morphology of gap junctions between undifferentiated and differentiated neural retinae are related to different functions. Besides typical gap junctions with macular aggregates of intramembrane particles, linear arrays of intramembrane particles were always found between the members of a double cone at the outer limiting membrane region and its neighbourhood of the neural retinae of newly hatched chickens. Similar linear arrangement of intramembrane particles has been shown in the process of assembling intramembrane particles into a gap junction during amphibian neurulation (Decker & Friend, 1974), and during differentiation of lens fibres (Benedetti et al. 1974). Thus, it has been generally considered that the presence of linear arrays of intramembrane particles may represent one aspect of gap junction membrane differentiation (Decker & Friend, 1974). Another consideration is also possible to explain the linear arrangement of intramembrane particles. As shown in the previous paper (Fujisawa et al. 1976), no linear arrays of intramembrane particles were found between undifferentiated cells of neural retinae of 3- to 6-day-old chick embryos, in which the expansion of gap junctions was remarkable. This finding may present a possibility that the linear arrays of intramembrane particles do not represent a transitional feature of intramembrane particles into a gap junction, but reflect the existence of another type of gap junction which is also different in function. Possible role of gap junction in the differentiated neural retinae
It has been reported that in the outer plexiform layer of the retinae of monkeys, rabbits and turtles, gap junctions of 2 different appearances, i.e. macular aggregates and linear arrays of intramembrane particles, are formed between the synaptic endings of photoreceptors (Raviola & Gilula, 1973). It has been speculated that these interreceptor gap junctions mediate electrotonic coupling between neighbouring photoreceptors (Raviola & Gilula, 1973). On the other hand, in the neural retinae of newly hatched chickens, these 2 types of gap junctions were present only between principal and accessory cones which form a double cone, at the outer limiting membrane region. Only a few gap junctions were observed at the synaptic ending of photoreceptors or in the outer plexiform layer. These findings permit us to speculate on the several other functions that might be subserved by these junctional structures. The first is a mechanical structure anchoring 2 members of a double cone. A similar possible function for the gap junction was also suggested for that found between neural retina and pigmented epithelium of developing chick embryos (Fujisawa et al. 1976). Secondly, because a paraboloid which contains a large quantity of glycogen granules is found at the inner segment of the
Fig. 13. Linear arrays of intramembrane particles of various configurations, x 60000. Fig. 14. Complementary depressions (arrows) of linear arrays and continuous macular aggregates of intramembrane particles on B fracture face, x 90000. Figs. 15, 16. Gap junctions (arrows) in outer and inner plexiform layers, respectively. Both figures x 45 000.
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accessory cone, but not in the principal cone (Morris & Shorey, 1967), the gap junction may be a site mediating some metabolic substances such as glycogen. Finally, because it has been generally considered that the members of a double cone may act as a functional unit in vision (Cohen, 1963 ; Morris & Shorey, 1967), the gap junction may be the pathway of visual information between the members of a double cone. The authors wish to express their gratitude to Professor T. S. Okada (Kyoto University, Japan), to Dr D. A. Ede (University of Glasgow, Scotland), and to Dr R. B. Kemp (The University College of Wales, Aberystwyth, Wales) for critical reading of the manuscript. Thanks are also due to Dr G. Eguchi (Kyoto University, Japan) for valuable guidance in the techniques of electron microscopy. This work was supported in part by a Research Grant (no. 054216) from the Japan Ministry of Education.
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