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Some observations on the patency in the outer segments of photoreceptors of the nocturnal gecko
SOME OBSERVATIONS ON THE PATENCY IN THE OUTER SEGMENTS OF PHOTORECEPTORS OF THE NOCTURNAL GECKO’ MASAO
Yoskm~
Institute of Biology. Oita University. Oita. Japan 870-I (Receiced
I
18 March 1977)
Abstract-The retinas of nocturnal geckos are of particular morphological interest. The photoreceptors are represented by rods only. They are peculiar in that they possess numerous invaginations of their plasma membrane which are irregularly arranged at the proximal area of the outer segments. These openings can be seen in both embryonic and adult photoreceptors. Key Words-gecko
retina: photoreceptor: adult: embryo: infolding; rod-like photoreceptor.
The majority of geckos are nocturnal or crepuscular in habit and commonly regarded to possess a pure rod retina (Underwood, 1954). The rods of nocturnal geckos have been suspected of being derived by transmutation from ancestral cones. The transmutation theory of Walls (1934, 1942) was confirmed by further findings of morphologically intermediate types of photoreceptors among several species of geckos (Underwood. 1951). and of intermediate photopigpositioned ments which are, spectroscopically, between iodopsin and classical rhodopsin (Crescitelli. 1958). The shift in absorbance to the longer wavelength was assumed to be due to the specific nature of the opsin. not due to the occurrence of 3-dehydroretinal as the prosthetic group (Crescitelli, 1972). The outer segments of photoreceptors in the retinas of vertebrates are composed of a stack of transversely oriented double membrane discs. The double membrane discs of cones in lower vertebrates are formed by infolding of the plasma membrane throughout the entire length of the outer segments (Sjiistrand, 1959, 1961; Moody and Robertson, 1960; Yoshida, 1961; Brown. Gibbons and Wald, 1963). Likewise, in mammalian cones, the double membrane discs are open to the extracellular space at the base of the outer segment. However. a stack of these discs is enclosed by the plasma membrane at the outer portion of the outer segments (Cohen, 1961; Hollenberg and Bernstein, 1966). In contrast, the double membrane discs of rods appear to be isolated, and the great majority of the discs are enclosed by plasma membrane, except for a very limited portion at the proximal end of the outer segment, where a few pairs of double membrane discs are continuous with th the plasma membrane (Moody and Robertson. 1960). These openings in the rods and cones of the retinas of vertebrates were confirmed by infiltration studies with ferritin and lanthanum tracers (Cohen. 1968, 1970). The fine structure of photoreceptors in vertebrate retinas was reviewed recently in relation to their physiological significance (Cohen, 1972).
Two types of photoreceptors, the single and double rods, were described in Gecko s~+honis (Detwiler. 1923). in the banded gecko. Coleon~x cariegatus (Walls. 1934). and in Tarentola mauriranica (Carasso, 1956). Photoreceptors of geckos were classified into class A single, class B double, and class C double (Underwood, 1951). Electron microscopy of the retinal photoreceptors in Co[eotix: carirgartrs has revealed the presence of four addttional photoreceptors, and a new designation for photoreceptor cells was proposed (Dunn, .l966). The fine structure of the retinas of diurnal and nocturnal geckos has been studied by several authors (Carasso. 1956; Pedler and Tansley, 1963; Pedler and Tilly, 1964: Tansley, 1964; Dunn, 1966, 1973; Ishikawa and Yamada 1969; Yoshida, 1975). Little has been known about the fine structure of the outer segments of photoreceptors of geckos, although the lamellae were described as atypical whorls (Pedler and Tansley, 1963: Pedler and Tilly, 1964). The continuity of the double membrane discs with the plasma membrane was mentioned briefly to occur at the proximal portion of the outer segments of photoreceptors in Coleon_r.x rariegatus (Dunn, 1966) and in Gekko gekko (Cohen, 1970). There are at least two cytoarchitectural types of double membrane discs. First, the disc formmg by infolding of the plasma membrane as commonly found in cones; second, the rod-type disc which ends with a button-like structure at the periphery. The question of whether each type of the double membrane discs is responsible for retaining specific different photopigments or not is still open. The present paper deals with some preliminary observations illustrating the presence of unusual types of openings found in the outer segments of rod-like photoreceptors of the adult and embryonic nocturnal gecko. Additional information on the fine structure of the connecting cilium is also given.
part of this work was presented at the Third International Symposium on The Structures of The Eye (Japan.
Eyes from adults and embryos of nocturnal geckos, Gekko japonicus, were used. Embryos were at a stage 22 days before hatching. Eyes of embryos at this stage
‘A
1975). 137
RESULTS ASD DISCUSSION
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that oi adults. Eyeballs removed from the adult and embryonic geckos were transected and their posterior hemispheres were fixed in 3.j”0 glutaraldshyds in 4lillonig’s phosphate butTer IpH 7.1). folloued with postfixation in I”., osmium tetroxide in !+tillonig‘s butTer (pH 7.-l) for 3 hr. Retinal tissues were embedded in low-viscosity epoxy resin (Spurr. 1969). Thin sections. cut with glass knives on a Porter-Blum microtome and stained with uranyl acetate and citrate. were examined with a Hitachi HU-11 electron microscope. at 75 k\‘. Observations were made mostly on longitudinal sections. since the present study was aimed to ascertain whether the double membrane discs are open or not at the edge or the basal outer segments of photoreceptors. Ltght microscopy has revealed that the outer segments of photoreceptors are larger and cylindrical in shape as was described in other nocturnal adult geckos (Detwiler. 1923). Fat globules or droplets. which disappeared in successive stages after the hatching. were usually found in the ellipsoidal region of the inner segments of embryonic retinas. Types of photoreceptors of both adult embryonic retinas were easily identified by comparing the inner structure of mitochondria in the inner segments o’oshida. 1975). .41so. the central mass or clusters of glycogen particles in the paraboloid (Dunn. 1966). and the rudimentary cristae of the mitochondria in the ellipsoid are characteristics of the accessory member of the D, double photoreceptor (class B double). .-\s to the connecting cilium of the gecko retina. the scalloped appearance of the connecting cilium in cross-section on the retina of the adult banded gecko (Dunn. 1966) and the rudimentary ciliated structure in the retina of diurnal geckos have been described (Pedler and Tansley. 1963). The fibers of the connecting cilium extend their finger-like projections into the cytoplasmic matrix of the outer segments (Figs. I and 7). The stalk of the connecting cilium is extremely short. The cross-section shows that the connecting cilium consists of nine pairs of double filaments arranged radially and enclosed by plasma membrane. The junction between the inner and outer segments of the photoreceptor cells of the gecko is structurally similar to that found in other vertebrate retinas. It is of interest that the plasma membrane of the stalk of the connecting cilium is surrounded by amorphous dense materials in muffler-like profile (Figs. 1 and 7). According to Dunn (1966). the outer segment discs are five-layered under the preparative condition described. and they are surrounded by the plasma membrane. It has been mentioned that there is a continuity of the double membrane discs with the plasma membrane: however. the openings are located in the narrowed basal zone in the outer segment. It has been noted that in the rod of nocturnal geckos, Gekko grkko. there is “a fairly extensive basal zone of open rod saccules” (Cohen. 1970). The present results demonstrate that. as in other vertebrate rods. the stack of double membrane discs of the outer segments of the photoreceptor cells in the retina of adult geckos is enclosed by the plasma membrane. except for the proximal zone of the outer segment. where more than
30 iniolding
st’ :hr
to
double
the
iorm
th:
plasm>
membrsntt
msmbrane
discs
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proximal zone ot’ the outer segmsnts IFis 21. !y the gecko. the infoldings are wider than those in other vertebrate species. The great majority oi the daub!? membrane discs ot‘ the photoreceptors euamlned JX usually enclosed by the plasma membrane (Ftg. 4) Also. the inscisure vvhtch is common in rods is mostiy found at the apical area of the outer segment (Fis. 3). as was described by Dunn (1366). .Another type of opening of the double membrane discs vans found to be irregularly spaced along the edge of the plasm:! membrane (Fig. 6). The architectural plan of such scattered openings in the adult photoreceptors mght be elucidated by studying the outer segments of rhrt embryonic retinas (Fig. 5). Some double membrandiscs already established at thn stage are continuous with the plasma membrane (Fig. S). Also of particuiar interest are some broad saccules whtch contain t\\ri, or three rod-type discs which end in button-like structures or hairpin loops at the periphery of the outer segments. The plasma membrane of the broad saccule was found to join with that of adjacent broad saccules. thus forming the opening of the disc membrane between two broad saccules. A similar structure 01’ the outer segment was described in the cone outer segment of the adult ground squirrel (Hollenberg and Bernstein, 1966). During development. both rods and cones of frog retinas are formed by a process of repeated infolding of the plasma membrane at the base of growing outer segments. In the cones. the double dtsc membranes, once formed. remain continuous with the plasma membrane. while the prear majority of the plasma membrane in the rod outer segment was thought to become pinched off (?Jilsson. 1964). Therefore. it is possible that the disc membrane at the periphery showing the infolding of the plasma membrane which separates two broad saccules may be pinched off: or may not be pinched off because of the presence of several scattered openings (Fig. 6). Autoradiographic studies have revealed that the rod basal lamellae are renewed successively and mlgrate toward the pigment epithelium (Young and Droz. 1968: Young. 1969: Young, 1974). Thus, there is a clear distinction between the rods and cones due to the differences in turnover or mode of incorporation of labeled precursors to opsin in the outer segments (Young. 1971: Ditto. 1975). A striking confirmation to support the autoradiographic evidence of rod outer segment turnover was provided by Procion Yellow infiltration in Gekko gekko (Laties. Bok and Liebman. 1976). The broad zone showing the repeated infoldmgs observed at the base of the photoreceptor outer segment (Fig. 3) might be related to the site of renewal of the double membrane discs. Phagosomes in the pigment epithelium. containing detached fragments of outer segment. have been observed (unpublished data). Retinal extracts of Grkko gekko v:ere found to contain two retinal photopigments: one absorbing maximally at about 521 nm. the second. present in smaller quantities in the extracts. absorbing maximally in the region of 475 nm. and the anatomical allocation For two photopigments in each member of the pair of
Fig. I. Electron micrograph of the junction between inner and outer segments of the class .4 single in the retina of an adult gecko. showing the connecting ciiium. x 25.000. Bar in al! micrographs indicates 500 nm. Fig. I!. Electron micrograph showing the proximal part of the outer segment of an accessory member of the class B double of an adult retina. The extensive basal zone of the outer segment is occupied by a series of openings of the double membrane discs (at arrow). x 25.000. Fig. 3. Electron micrograph
showing the inscisure found in the apical region of the outer se_mnent. x 41.500.
Fie. 1. Electron ret&a.
micrograph
showing a part of the outer segment of the class A single of the adult membrane discs is enclosed by a plasma membrane. .Arrou mdicate~ of the double membrane disc. A peripheral portion of the ellipsoid of the accessor> member of the class B double containing mitochondria is seen tlefti. * 3S.0Wo.
.A stack of the double
an opening
Fjg. 5. Electron micrograph showing the outer segment of a ciass A single iri the retina of an embr>o Numerous openings of the double membrane discs are seen. Some of the double membrane discs at both sides of the outer segment are wsicular. * 42.gC)cI. Fig. 6. Electron micrograph shosing the basal zone of the outer segment of rhe accessor) member of class B double of the adult retina. Arrows indicate the openings of the double membrane discs. .A carycal process is seen along the periphery of the outer segment. x 4300.
I’ig. 8. Electron rnicrogruph showing the junction of inner and outer segment of an accessory member of the class 13 double from the embryonic retina. The connecting cilium is not demonstrated. Note two types of openings of the double mcmbrllnr discs (see text, p. 138). Mitochondria with a few cristae are densely stained. x 50,100.
Fig. 7. Electron micrograph of lhe junclion belween inner and outer segments of a class A single of the embryonic retina, showing the connecting ciliurn and basal bodies. Filaments of the connecting cilium run along the cytoplasmic matrix of the otuer segment. Mitochondrin with well-developed crigae and ribosomes are seen in the inner segment. x 50,100. Insert: :I cross-section of 11 comwting cilium showing the nine pairs of double filaments arranged radially. x 37,500.
Patency of the outer segment in nocturnal gecko retina the double cells was suggested (Crescitelli, 1963). It
was demonstrated in Gekko gekko that the majority of single and double rods contain photopi~ents absorbing maximally at 518 nm: the smaller member of the ciass B double absorbed maximally at 467 nm and the larger member of the double cell at 518 nm (Liebman, 1972). These results obtained by microspectrophotometry show spectrophotic similarity between the single and the accessory member of the class B double. However, the ordinary inner structure of the mitochondria of the ellipsoids in nocturnal geckos was observed to be identical in the sin&e photoreceptor and the principal member of the class B double (Yoshida 1975). In reviewing the spectrophotometrically defined photopi~ents in the retina of several gecko species (Crescitelli, 1958, 1963, 1972). one may assume that the differences are explicable on the basis of minor species differences due to the retaining of some of the structural characteristics of ancestral cones.
Ac~nowledgem~nrs-The technical assistance of Miss M. Ono is gratefully acknowledged. The author wishes to express his deep appreciation to Professor W. J. Hild and to the technical staff of the Department of Anatomy at the University of Texas Medical Branch. Texas, U.S.A., for their aid in the preparation of the manuscript and for their hospitality.
REFERENCES
Brown P. K., Gibbons I. R. and Wald G. (1963) The visual cells and visual pigment of the mudpuppy. Necrurus. J. Celf Bioi. 19, 79-106. Carasso N. (1956) Mise en tvidence de prolongements cytoplasmiques infra-microscopiques au niveau du segment interne des cellules visualles du Gecko (Reptile). C.r. hebd. SPanc. Acad. Sci., Paris 242, 2988-2991. Cohen A. I. (1961) Some preliminary electron microscopic observations of the outer segments of the retina of the Macacu rhesus. In The Structure of the Eye (Edited by Smelser G. K.) pp. 151-I 58. Academic Press. New York. Cohen A. I. (1968) New evidence supporting the linkage to extracellular space of outer segment saccules of frog cones but not rods. J. Cell Biol. 37, 424-444. Cohen A. I. (1970) Further studies on the question of the patency of saccules in outer segments of vertebrate photoreceptors. Vision Res. 10, 445d54. Cohen A. 1. (19721 Rods and cones. In Handbook ofSensor_v Ph~siolag~, Vol. VIf/2. Phys~l~y of Photorecepror Organs (Edited by Fuortes M. G. F.). pp. 63-1 IO. Spring, Berlin. Crescitelli F. (1958) The natural history of visual pigments. Ann. N.Y. Acad. Sci. 74, 230-255.
Crescitelli F. (1963) The photosensitive retinal pigment system of Gekko gekko. J. gen. Physiol. 47, 33-47. Crescitelli F. (1972) The visual cells and visual oiwents of the vertebrate eye. In Handbook of Sensory Physiology, Vol. VII/l. Photochemisrry of Won (Edited by Dartnatl H. J. A.), pp. 245-363. Spring, Berlin. Detwiler .a. S. R. (1923) . Studies .- on the retina an experimental stucly 01 me gecito retma. J. camp. Neural. 36, 12>141. .l
113
Ditto M. (1975) A difference between developing rods and cones in the formation of outer segment membranes. Vision Res. IS. 535-536. Dunn R. F. (19661 Studies on the retina of the gecko CO/eongx cariegatus. I. The visual cell classification. J. ultrastruct. Res. 16, 651-671.
Dunn R. F. (1973) The ultrastructure of the vertebrate retina. In The Ultrastructure of Sensory Organ (Edited by Friedmann I.). pp. 1.53-265. North-Holland. Amsterdam. Hollenberg M. J. and Bernstein M. H. (1966) Fine structure of the photoreceptor cells of the ground squirrel (Citellu~ tridecemlineatus tridecem/ineatus). Am. J. Anat. 118, 359-373. lshikawa T. and Yamada E. (1969) Atypical mitochondria in the ellipsoid of the photoreceptor cells of vertebrate retinas. Incesr. Ophthal. 8, 302-316. Laties A. M.. Bok D. and Liebman P. (1976) Procion yellow: a marker dye for outer segment disc patency and for rod renewal. Expf Eye Res. 23, 139-148. Liebman P. A. (1972) Microspectrophotometry of photoreceptors. In Handbook of Sensory Ph,vsidog_v. Vol. VIII/‘I. Photochemistry of Vision (Edited by Dartnall H. J. A.), pp. 482-528. Spring, Berlin. Moody M. F. and Roberrson J. D. (19601The fine structure of some retinal photoreceptors. J. Biophys. Biochem. C.vtoi. 7, 87-92.
Nilsson S. E. G. (1964) Receptor cell outer segment development and ultrastructure of the disk membranes in the retina of the tadpole (Rana pipiens). J. uhrasrruct. Rrs. 11, 581620. Pedler C. and Tansley K. (1963) The fine structure of the cone of a diurnal gecko (Phe~s~ inunguis). Expt E_ve Res. 2, 39-49. Pedler C. and Tilly R. (1964) The nature of the gecko visual cell, a light and electron microscopic study. Vision Rex 4, 499-510. Sjiistrand F. S. (1959) Fine structure of cytoplasm: the organization of membranous layers. Rev. mod. Phys. 31, 301-318. SjGstrand F. S. (1961) EIectron microscopy of the retina. in The Structure ofrhe Eye {Edited by Smelser G. K.). pp. l-28. Acadeinic Press, New York. Spirr A. R. (1969) A low-viscosity epoxy resin embedding medium for electron microscopy. J. ultrastrucr. Res. 26, 31-43. Tansley K. (1964) The gecko retina. Vision Res. 4, 33-37. Underwood G. (1951) Reptilian retinas. Nature, Land. 167, 183-185.
Underwood G. (1954) On the classification and evolution of geckos. Proc. zool. Sot. Lond. 124, 469492. Walls G. L. (1934) The reptilian retina. I. A new concept of visual-cell evolution. Am. J. Ophthal. 17, 892-915. Walls G. L. (1942) The Vertebrate Eye and irs Adaptice Radiation. Bloomfield Hills, Cranbrook, Institute of Science, Michigan. Yoshida M. (1961) The fine structure of the ohotorecentors in the newt retina. Anat. Rec. 139, 289. ’ ’ Yoshida M. (1975) The fine structure of the photoreceptor of a nocturnal gecko. J. Electron Microsc. 24. 132. Young R. W. (1969) A difference between rods and cones in the renewal of outer segment protein. Incest. Ophthal. 8, 222-231.
Young R. W. (1971) An hypothesis to account for a basic dist&tion between rod; and cones. Vision Res. 11, 1-5. Young R. W. (1974) Biogenesis and renewal of visual cell outer segment membranes. Expl Eye Res. 18, 215-223. Young R. W. and Droz B. (1968) The renewal of protein in retinal rods and cones. J. Cell BioI. 39, 169-184.
Yoskm~
Institute of Biology. Oita University. Oita. Japan 870-I (Receiced
I
18 March 1977)
Abstract-The retinas of nocturnal geckos are of particular morphological interest. The photoreceptors are represented by rods only. They are peculiar in that they possess numerous invaginations of their plasma membrane which are irregularly arranged at the proximal area of the outer segments. These openings can be seen in both embryonic and adult photoreceptors. Key Words-gecko
retina: photoreceptor: adult: embryo: infolding; rod-like photoreceptor.
The majority of geckos are nocturnal or crepuscular in habit and commonly regarded to possess a pure rod retina (Underwood, 1954). The rods of nocturnal geckos have been suspected of being derived by transmutation from ancestral cones. The transmutation theory of Walls (1934, 1942) was confirmed by further findings of morphologically intermediate types of photoreceptors among several species of geckos (Underwood. 1951). and of intermediate photopigpositioned ments which are, spectroscopically, between iodopsin and classical rhodopsin (Crescitelli. 1958). The shift in absorbance to the longer wavelength was assumed to be due to the specific nature of the opsin. not due to the occurrence of 3-dehydroretinal as the prosthetic group (Crescitelli, 1972). The outer segments of photoreceptors in the retinas of vertebrates are composed of a stack of transversely oriented double membrane discs. The double membrane discs of cones in lower vertebrates are formed by infolding of the plasma membrane throughout the entire length of the outer segments (Sjiistrand, 1959, 1961; Moody and Robertson, 1960; Yoshida, 1961; Brown. Gibbons and Wald, 1963). Likewise, in mammalian cones, the double membrane discs are open to the extracellular space at the base of the outer segment. However. a stack of these discs is enclosed by the plasma membrane at the outer portion of the outer segments (Cohen, 1961; Hollenberg and Bernstein, 1966). In contrast, the double membrane discs of rods appear to be isolated, and the great majority of the discs are enclosed by plasma membrane, except for a very limited portion at the proximal end of the outer segment, where a few pairs of double membrane discs are continuous with th the plasma membrane (Moody and Robertson. 1960). These openings in the rods and cones of the retinas of vertebrates were confirmed by infiltration studies with ferritin and lanthanum tracers (Cohen. 1968, 1970). The fine structure of photoreceptors in vertebrate retinas was reviewed recently in relation to their physiological significance (Cohen, 1972).
Two types of photoreceptors, the single and double rods, were described in Gecko s~+honis (Detwiler. 1923). in the banded gecko. Coleon~x cariegatus (Walls. 1934). and in Tarentola mauriranica (Carasso, 1956). Photoreceptors of geckos were classified into class A single, class B double, and class C double (Underwood, 1951). Electron microscopy of the retinal photoreceptors in Co[eotix: carirgartrs has revealed the presence of four addttional photoreceptors, and a new designation for photoreceptor cells was proposed (Dunn, .l966). The fine structure of the retinas of diurnal and nocturnal geckos has been studied by several authors (Carasso. 1956; Pedler and Tansley, 1963; Pedler and Tilly, 1964: Tansley, 1964; Dunn, 1966, 1973; Ishikawa and Yamada 1969; Yoshida, 1975). Little has been known about the fine structure of the outer segments of photoreceptors of geckos, although the lamellae were described as atypical whorls (Pedler and Tansley, 1963: Pedler and Tilly, 1964). The continuity of the double membrane discs with the plasma membrane was mentioned briefly to occur at the proximal portion of the outer segments of photoreceptors in Coleon_r.x rariegatus (Dunn, 1966) and in Gekko gekko (Cohen, 1970). There are at least two cytoarchitectural types of double membrane discs. First, the disc formmg by infolding of the plasma membrane as commonly found in cones; second, the rod-type disc which ends with a button-like structure at the periphery. The question of whether each type of the double membrane discs is responsible for retaining specific different photopigments or not is still open. The present paper deals with some preliminary observations illustrating the presence of unusual types of openings found in the outer segments of rod-like photoreceptors of the adult and embryonic nocturnal gecko. Additional information on the fine structure of the connecting cilium is also given.
part of this work was presented at the Third International Symposium on The Structures of The Eye (Japan.
Eyes from adults and embryos of nocturnal geckos, Gekko japonicus, were used. Embryos were at a stage 22 days before hatching. Eyes of embryos at this stage
‘A
1975). 137
RESULTS ASD DISCUSSION
poj:jesjed
ths \ertlcal
FCpil SimiiJr
:o
s11t with
xrrA.tsd
mxgin
of the
that oi adults. Eyeballs removed from the adult and embryonic geckos were transected and their posterior hemispheres were fixed in 3.j”0 glutaraldshyds in 4lillonig’s phosphate butTer IpH 7.1). folloued with postfixation in I”., osmium tetroxide in !+tillonig‘s butTer (pH 7.-l) for 3 hr. Retinal tissues were embedded in low-viscosity epoxy resin (Spurr. 1969). Thin sections. cut with glass knives on a Porter-Blum microtome and stained with uranyl acetate and citrate. were examined with a Hitachi HU-11 electron microscope. at 75 k\‘. Observations were made mostly on longitudinal sections. since the present study was aimed to ascertain whether the double membrane discs are open or not at the edge or the basal outer segments of photoreceptors. Ltght microscopy has revealed that the outer segments of photoreceptors are larger and cylindrical in shape as was described in other nocturnal adult geckos (Detwiler. 1923). Fat globules or droplets. which disappeared in successive stages after the hatching. were usually found in the ellipsoidal region of the inner segments of embryonic retinas. Types of photoreceptors of both adult embryonic retinas were easily identified by comparing the inner structure of mitochondria in the inner segments o’oshida. 1975). .41so. the central mass or clusters of glycogen particles in the paraboloid (Dunn. 1966). and the rudimentary cristae of the mitochondria in the ellipsoid are characteristics of the accessory member of the D, double photoreceptor (class B double). .-\s to the connecting cilium of the gecko retina. the scalloped appearance of the connecting cilium in cross-section on the retina of the adult banded gecko (Dunn. 1966) and the rudimentary ciliated structure in the retina of diurnal geckos have been described (Pedler and Tansley. 1963). The fibers of the connecting cilium extend their finger-like projections into the cytoplasmic matrix of the outer segments (Figs. I and 7). The stalk of the connecting cilium is extremely short. The cross-section shows that the connecting cilium consists of nine pairs of double filaments arranged radially and enclosed by plasma membrane. The junction between the inner and outer segments of the photoreceptor cells of the gecko is structurally similar to that found in other vertebrate retinas. It is of interest that the plasma membrane of the stalk of the connecting cilium is surrounded by amorphous dense materials in muffler-like profile (Figs. 1 and 7). According to Dunn (1966). the outer segment discs are five-layered under the preparative condition described. and they are surrounded by the plasma membrane. It has been mentioned that there is a continuity of the double membrane discs with the plasma membrane: however. the openings are located in the narrowed basal zone in the outer segment. It has been noted that in the rod of nocturnal geckos, Gekko grkko. there is “a fairly extensive basal zone of open rod saccules” (Cohen. 1970). The present results demonstrate that. as in other vertebrate rods. the stack of double membrane discs of the outer segments of the photoreceptor cells in the retina of adult geckos is enclosed by the plasma membrane. except for the proximal zone of the outer segment. where more than
30 iniolding
st’ :hr
to
double
the
iorm
th:
plasm>
membrsntt
msmbrane
discs
ticre uhich
,T?jcr,.:.: ICL’;~>
proximal zone ot’ the outer segmsnts IFis 21. !y the gecko. the infoldings are wider than those in other vertebrate species. The great majority oi the daub!? membrane discs ot‘ the photoreceptors euamlned JX usually enclosed by the plasma membrane (Ftg. 4) Also. the inscisure vvhtch is common in rods is mostiy found at the apical area of the outer segment (Fis. 3). as was described by Dunn (1366). .Another type of opening of the double membrane discs vans found to be irregularly spaced along the edge of the plasm:! membrane (Fig. 6). The architectural plan of such scattered openings in the adult photoreceptors mght be elucidated by studying the outer segments of rhrt embryonic retinas (Fig. 5). Some double membrandiscs already established at thn stage are continuous with the plasma membrane (Fig. S). Also of particuiar interest are some broad saccules whtch contain t\\ri, or three rod-type discs which end in button-like structures or hairpin loops at the periphery of the outer segments. The plasma membrane of the broad saccule was found to join with that of adjacent broad saccules. thus forming the opening of the disc membrane between two broad saccules. A similar structure 01’ the outer segment was described in the cone outer segment of the adult ground squirrel (Hollenberg and Bernstein, 1966). During development. both rods and cones of frog retinas are formed by a process of repeated infolding of the plasma membrane at the base of growing outer segments. In the cones. the double dtsc membranes, once formed. remain continuous with the plasma membrane. while the prear majority of the plasma membrane in the rod outer segment was thought to become pinched off (?Jilsson. 1964). Therefore. it is possible that the disc membrane at the periphery showing the infolding of the plasma membrane which separates two broad saccules may be pinched off: or may not be pinched off because of the presence of several scattered openings (Fig. 6). Autoradiographic studies have revealed that the rod basal lamellae are renewed successively and mlgrate toward the pigment epithelium (Young and Droz. 1968: Young. 1969: Young, 1974). Thus, there is a clear distinction between the rods and cones due to the differences in turnover or mode of incorporation of labeled precursors to opsin in the outer segments (Young. 1971: Ditto. 1975). A striking confirmation to support the autoradiographic evidence of rod outer segment turnover was provided by Procion Yellow infiltration in Gekko gekko (Laties. Bok and Liebman. 1976). The broad zone showing the repeated infoldmgs observed at the base of the photoreceptor outer segment (Fig. 3) might be related to the site of renewal of the double membrane discs. Phagosomes in the pigment epithelium. containing detached fragments of outer segment. have been observed (unpublished data). Retinal extracts of Grkko gekko v:ere found to contain two retinal photopigments: one absorbing maximally at about 521 nm. the second. present in smaller quantities in the extracts. absorbing maximally in the region of 475 nm. and the anatomical allocation For two photopigments in each member of the pair of
Fig. I. Electron micrograph of the junction between inner and outer segments of the class .4 single in the retina of an adult gecko. showing the connecting ciiium. x 25.000. Bar in al! micrographs indicates 500 nm. Fig. I!. Electron micrograph showing the proximal part of the outer segment of an accessory member of the class B double of an adult retina. The extensive basal zone of the outer segment is occupied by a series of openings of the double membrane discs (at arrow). x 25.000. Fig. 3. Electron micrograph
showing the inscisure found in the apical region of the outer se_mnent. x 41.500.
Fie. 1. Electron ret&a.
micrograph
showing a part of the outer segment of the class A single of the adult membrane discs is enclosed by a plasma membrane. .Arrou mdicate~ of the double membrane disc. A peripheral portion of the ellipsoid of the accessor> member of the class B double containing mitochondria is seen tlefti. * 3S.0Wo.
.A stack of the double
an opening
Fjg. 5. Electron micrograph showing the outer segment of a ciass A single iri the retina of an embr>o Numerous openings of the double membrane discs are seen. Some of the double membrane discs at both sides of the outer segment are wsicular. * 42.gC)cI. Fig. 6. Electron micrograph shosing the basal zone of the outer segment of rhe accessor) member of class B double of the adult retina. Arrows indicate the openings of the double membrane discs. .A carycal process is seen along the periphery of the outer segment. x 4300.
I’ig. 8. Electron rnicrogruph showing the junction of inner and outer segment of an accessory member of the class 13 double from the embryonic retina. The connecting cilium is not demonstrated. Note two types of openings of the double mcmbrllnr discs (see text, p. 138). Mitochondria with a few cristae are densely stained. x 50,100.
Fig. 7. Electron micrograph of lhe junclion belween inner and outer segments of a class A single of the embryonic retina, showing the connecting ciliurn and basal bodies. Filaments of the connecting cilium run along the cytoplasmic matrix of the otuer segment. Mitochondrin with well-developed crigae and ribosomes are seen in the inner segment. x 50,100. Insert: :I cross-section of 11 comwting cilium showing the nine pairs of double filaments arranged radially. x 37,500.
Patency of the outer segment in nocturnal gecko retina the double cells was suggested (Crescitelli, 1963). It
was demonstrated in Gekko gekko that the majority of single and double rods contain photopi~ents absorbing maximally at 518 nm: the smaller member of the ciass B double absorbed maximally at 467 nm and the larger member of the double cell at 518 nm (Liebman, 1972). These results obtained by microspectrophotometry show spectrophotic similarity between the single and the accessory member of the class B double. However, the ordinary inner structure of the mitochondria of the ellipsoids in nocturnal geckos was observed to be identical in the sin&e photoreceptor and the principal member of the class B double (Yoshida 1975). In reviewing the spectrophotometrically defined photopi~ents in the retina of several gecko species (Crescitelli, 1958, 1963, 1972). one may assume that the differences are explicable on the basis of minor species differences due to the retaining of some of the structural characteristics of ancestral cones.
Ac~nowledgem~nrs-The technical assistance of Miss M. Ono is gratefully acknowledged. The author wishes to express his deep appreciation to Professor W. J. Hild and to the technical staff of the Department of Anatomy at the University of Texas Medical Branch. Texas, U.S.A., for their aid in the preparation of the manuscript and for their hospitality.
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