The nature of the gecko visual cell

Vision Res. Vol. 4, pp. 499-510. Pergamon Press 1964. Printed in Great Britain.

T H E N A T U R E OF T H E G E C K O V I S U A L C E L L A LIGHT

AND ELECTRON

MICROSCOPIC

STUDY

CHRISTOPHER PEDLER and RITA TILLY Anatomy Department, Institute of Ophthalmology, University of London

(Received 13 December 1963) Abstract--In view of the conflicting evidence which now exists regarding the nature of the gecko photo-receptor a combined light and electron microscopic study has been made of two diurnal and several nocturnal species. Fine structural evidence is presented to suggest that instead of the morphologically distinguishable rod and cone populations found in other vertebrate species, the geckoes have evolved changes in the intracellular components of one basic cell variety to meet the demands of sensitivity and acuity. THE first recorded study of the Gecko retina is attributed by Cajal to Ranvier, who apparently found horizontal neurones similar to those described in the lizard retina by CAJAL (1933) (RANVIER, 1889). The visual cells of a nocturnal species, Tarentola mauretanica, are referred to as rods (ROCHON-DUVIGNEAUD, 1943) and the retinae of other species have also been described as pure rod in nature (DETWILER, 1923; VERRIER, 1935). The retina is also alleged to contain "visual purple" (DETWILER, 1923). WALLS (1934, 1942) describes "rods" in nocturnal species and "cones" in diurnal species and concludes that the archetypal gecko visual cell was the cone of a diurnal lizard and that in the course of evolution, transmutation occurred to a rod form, going on to suggest that in Phelsuma and Lygodactylus cones have re-appeared by transmutation from the secondarily nocturnal rod forms. He also suggested that a fovea is absent from all diurnal species. But the presence of a fovea has been described in Sphaerodactylus argus and Sphaerodactylus parkeri (UNDERWOOD, 1951) and unmistakable evidence o f a fovea is shown in the diurnal Phelsuma madagascariensis longinsulae (TANSLEY, 1961). CROZIER and WOLF (1939) found that the flicker response contour for an allegedly rodcontaining gecko, Sphaerodactylus inaguae (Noble), agreed in all essential respects of intensity, range and shape with that of a turtle, Pseudemys, the retina of which is alleged to contain a large predominance of cones. Both sets of experiments were performed under similar conditions. The authors conclude that their results are opposed to the correlation of duplex retinal phenomena with specific morphological visual cell types. WALLS (1942) uses these results to suggest that colour vision may not necessarily have been lost in the transmutation from cone to rod. In an extensive work on the classification and evolution of Geckoes, UNDERWOOD (1954) states from general zoological evidence that geckoes were originally animals with pure cone retinae and that transmutation into rods occurred separately in the three separate families: geckonoidiae, eublepharidae and sterodactyli. The same author has also studied the retina of many gecko species and suggested the existence of three rod-like visual cell types; a single (Class A single), and two kinds o f double, one which he called the Class B double in which two members are of unequal size and another, called the Class C double, in which 499

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CHRISTOPHER PEDLER AND RITA TILLY

they are equal. The same author also describes alternate rows of single and double visual cells in flat section (UNDERWOOD, 1951). DENTON (1956), in a study of the pupil responses of the nocturnal Gecko gecko, found an unusual spectral response with its maximum displaced between 20 and 30 m/~ towards the red end of the spectrum as compared with rhodopsin. He also found that responses continued even in an illuminating field corresponding to 106 quanta/sec per retinal rod and that considerable spatial summation was present. An excellent agreement is evident when these results are compared with those of CRESCITELLI (1956), who examined the absorption spectra of retinal extracts made from a closely related Australian species Phyllurus milii. When the absorption spectra of these abstracts are compared with the results from Gecko gecko plotted on a quantum basis the maxima are almost identical. The author goes on to remark that this unusual pigment may be relevant to the special phylogenetic position of the geckoes as nocturnal animals which have retained some of the characteristics of their diurnal ancestors. He also considers that a pigment intermediate in spectral position between iodopsin and rhodopsin provides support for the transmutation theory of Walls, but concludes that too great an emphasis is placed on the duplicity theory. In a later work (CREsCITELLI, 1958) the same author gives the wavelength maxima of 18 gecko species, all of which lie between 516 and 530 m/z, again concluding that the gecko retina is in a state of intermediacy and agreeing with WALLS (1942) that the diversity encountered among different gecko species is probably the result of independent evolution in different gecko stocks. The absorption maxima listed by Crescitelli in two species are in fair agreement with the spectral sensitivity data obtained by DODT and WALTHER (1958) for Tarentola mauretanica and Hemidactylus turcicus. A positive off-effect has also been found in Sphaerodactylus muralis, another presumptively rod-containing species (DODT and HECK, 1954), a result usually obtained only from cone-predominant eyes. By studying the effect of light- and dark-adaptation on the electrical activity it was found that the electroretinogram of T. mauretanica changes from the scotopic type in the dark-adapted state to the photopic type after strong light-adaptation (DODT and JESSEN, 1961). Since the two species used in these experiments have one morphological type of receptor only, at the light-microscopic level, these experimental results are clearly contrary to the hypothesis that scotopic and photopic processes are mediated by rods and cones respectively. The authors concluded that it is not the receptors but the nature of the retina which is most closely related to the duplex responses of the retina. The light-microscopic structure of the retinae of Hemidactylus turcicus and Tarentola mauretanica has recently been described (TANSLEV, 1959), and the author shows that there is no structural basis for summation and that if the assumption is made that the gecko rod resembles the human in being able to respond to one quantum of light energy, then the increase in cross-section of the outer segment over that of the diurnal forms would not necessarily confer any significant increase in sensitivity. It is concluded that the relatively high in situ density of visual pigment as found by D~NTON (1956) would be sufficient to ensure a twofold increase in sensitivity over the individual mammalian rod. The light microscopy of a diurnal gecko Phelsuma madagascariensis longinsulae has also been studied (TANSLEY, 1961) and compared with Hemidactylus turcicus. It is shown that there is the conventional difference between the relative cell layer thicknesses in the two species consistent with diurnal and nocturnal habit and that the overall size of the diurnal receptors is smaller and that they are more closely packed together. The author considers that the features of the Phelsuma retina support Walls' transmutation theory but admits that the visual cells of the two species do not show all the differences between rods and cones.

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501

The free structure of the visual ceils of a diurnal gecko, Phelsuma inunguis, has recently been studied with the electron microscope (PEDLER and TANSLEY, 1962) and the three basic cell varieties proposed by Underwood have been confirmed. The two classes of double cells have been shown to be pairs of intact single cells with their plasma membranes in close apposition. No clearly defined cilial structures were encountered and the oil droplet is shown to be intimately connected to the cristae of the mitochondria in the inner segment. The synaptic pedicle was found to be unusually large and components from four ceils were shown to be in close relationship to its proximal end: the horizontal cells, the bipolar cells, the radial fibres and probably processes from adjacent receptors. The electron microscopy of the radial fibre material in the vicinity of the visual cell in the reptilian retina is also of considerable interest in relation to the mechanism of receptor action. This has now been studied in Hemidactylus turcicus, Phelsuma inunguis, Hemidactylus flaviviridis, Gecko gecko and the lizard, Lacerta muralis (PEDLER, 1963). In view of conflicting evidence which now exists pertaining to the nature of the gecko visual cell, we thought it would be useful to compare and contrast the electron microscopic appearances of two diurnal and several nocturnal species to see if any conclusions could be reached. MATERIALS AND METHODS Retinae from four Phelsuma inunguis, one Lygodactylus coloratus, one Hemidactylus turcicus, one Tarentola mauretanica and six Gecko gecko were examined after preparation in the following manner. After decapitation and pithing of the head, the eyes were removed and bisected in the coronal plane. The retina was then dissected from the posterior half in one piece and the peripheral third trimmed off with scissors and discarded. The remainder was immediately immersed in a cold (+4°C) 1 ~o solution ofveronal acetate buffered osmium tetroxide and cut into pieces approximately 1 mm × 1 mm. After a maximum of 3 hr fixation, the tissues were dehydrated in alcohols and embedded in "Araldite" (CIBA). Electron contrast was assisted in all cases by the addition of 1 ~o phosphotungstic acid to the final alcohol bath before immersion in "Araldite" and sections were mounted on Celloidin-carbon or pure carbon films. All micrographs reproduced in this article were taken with an A.E.I. Em6 microscope at 50kV at an objective aperture of 25/t. Magnifications are given as a product of the figure attained in the microscope and the secondary optical enlargement. RESULTS The micrographs reproduced in this article are of the nocturnal species mentioned above. Those of the diurnal Phelsuma are in a previous publication (PEDLEr. and TANSLEY, 1962). The other diurnal species Lygodactylus coloratus is indistinguishable morphologically from

Phelsuma. Outer Segment The most obvious difference between the outer segments of the diurnal and nocturnal species studied is the overall shape and size, which is best shown by light microscopy (Figs. 1 and 2). The outer segment of the diurnal Phelsuma is much smaller, tortuous, conical in outline and extremely fragile. It is, in fact, difficult to find specimens in which the outer segments can be seen at all, since in most eases they are firmly embedded in a closely applied

502

CHRISTOPHER PEDLER AND RITA TILLY

pigment epithelial surround and are invisible unless the retina has detached during preparation. The outer segments of the nocturnal species (Fig. 1) are larger and cylindrical in profile, and are also surrounded by processes of the pigment epithelium, but not so closely. At the resolutions employed, no difference was found between the spacing of the larnellae of the outer segments. Atypical whorls of lamellae which have already been reported (PEDLER and TANSLEY, 1962) in the diurnal Phelsuma are also present in all the nocturnal species examined, and no significant differences were seen between them and the diurnal forms. The nocturnal and diurnal cilial apparatus at the junction between the outer and inner segments is poorly developed and only occasionally seen and the lamellae of which the outer segment is composed are sometimes split transversely.

Inner Segment The inner segments of nocturnal and diurnal species are also of different shape and size. The nocturnal are broader, larger and more barrel-shaped (Fig. 1), particularly in the larger member of the Class B double pair, whereas the diurnal are more closely packed and cylindrical. Fins are present on the outer surface of both forms, and differ only in detail: in the diurnal form, these number between thirty and thirty-five per single visual cell, whereas in the nocturnal form (Fig. 3), between forty-four and fifty-five are usually counted. This difference is probably not of great significance, since the less numerous fins of the diurnal species are larger in relation to the total cell area in transverse section, so that approximately the same surface area is present in the two types of cell. In addition, the surrounding processes of the radial fibre complex are considerably finer and more numerous in the nocturnal species (Figs. 3 and 8). At the level of the outer limiting membrane (Fig. 5) all visual cells in both species contain attachment plaques or desmosomes, formed by an aggregation of tono-fibrils which are directly related to the terminal bars formed by the radial fibres of which the greater part of the outer limiting membrane is composed. In some specimens, the radial fibre material in the region of the visual cells was occasionally found to be shrunken (Fig. 5). We have as yet been unable to establish a cause for this phenomenon. Preliminary experiments indicate that it is not solely a consequence of light- or dark-adaptation and neither can it be produced by differences of ambient temperature. It is, however, convenient since it separates each separate process of the radial fibres. Most of the inner segments in both diurnal and nocturnal species contain three specialized groups of structures, the paraboloid, the ellipsoid and the Golgi apparatus. Paraboloid. A marked difference was encountered between the structure of the paraboloids - - i n the diurnal forms it is limited by a distinct rind of dense material with a core of regularly arranged glycogen granules, and its external aspect is clearly demarcated from the surrounding cytoplasm. In the nocturnal species, however, the rim of the paraboloid is much less evident and merges gradually into the surrounding cytoplasm (Fig. 6). The centre is often vacuolated and the groups of granules are not so evenly arranged. A paraboloid was not found in all the visual cell types either in the nocturnal or the diurnal species. However, there is no particular distribution of this structure in any of the Class A single, or Class B or C double cells. The paraboloids of the nocturnal species are not usually visible in the light microscope. The Ellipsoid. The ellipsoid of the diurnal species Phelsuma inunguis is composed of a tightly packed mass of unusually dense mitochondria. Three main mitochondrial types were

F m s . 1 and 2. Two photo-micrographs at the same magnification ( × 1875) of a vertical section of a nocturnal Gecko (Geckogecko), Fig. 1, and a diurnal species (Phelsumainunguis), Fig. 2. The nocturnal receptors are considerably larger and the aggregations of transmuted mitochondria packing the inner segment of the main member of the nocturnal Class B double cells (clear arrows) are well seen. The nocturnal outer segments (black arrows) are smaller and fragmented. The oval visual cell nuclei (V) of the diurnal species are closely packed together and the horizontal cells (H) in the neighbourhood of the receptor nuclei are also present in both sections. The paraboloid (P) is only seen in the diurnal species in the light microscope and occurs just sclerad to the outer limiting membrane (dotted line). In the diurnal species, the synaptic pedMes (S) can be seen in the outer plexiform layer. Stain: Mallory Phosphotungstic acid haematoxylin.

FIG. 3. A transverse section cut at a level just sclerad to the outer limiting membrane. Two members of a class C pair are shown (1 and 2) surrounded by a mass of radial fibre prolongations (3). The close association of the two cells can be seen and towards the centre an interdigitation between adjacent cell membranes is present (4); this is shown at higher power in Fig. 7. The cytoplasmic density of the visual cells varies strikingly; for example in cell no. 5 the cytoplasm is of greater density than the others. The nucleus of an adjacent receptor is shown (6) and Golgi apparatus (7) is present in both members of the double pair ( × 7500).

FIG. 4. The junction between the inner and outer segments of a nocturnal specimen (Gecko gecko). The large arrow indicates the long axis of the cell and three mitochondria (M) of the inner segment are shown ( x 32,000).

FIG. 5. Longitudinal section of a single nocturnal visual cell (Hemidactylusflaviviridis) at the level of the outer limiting membrane (arrows), which is formed by thickenings in the downward prolongations of the radial fibre processes together with desmosomes in the visual cell cytoplasm (D) with convergent tono-fibrils ( × 15,000).

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FIG. 10. A lower power view ( × 6250) of the main member of a nocturnal Class B double seen in longitudinal section (Gecko gecko). Part of the accessory member (A) is wrapped around the main member and the junction between the two is indicated by the white arrows. A paraboloid (P) is present and is indented on its sclerad aspect by the ellipsoid. The latter is composed of a highly dense central mass of transmuted mitochondria (TM) some of which contain less dense centres, together with closely packed mitochondria of more usual appearance around the periphery (dark arrows). Forms intermediate between these two extremes are often found. Parts of two Class A single cells (S) are also present. These contain mitochondria which are morphologically similar to one of the varieties encountered in the diurnal Phelsuma.

The Nature of the Gecko Visual Cell

503

noted: the first, which is small, has a smoothly rounded external membrane and only a few cristae. The second is a larger variety with dense cristae occupying only a part of the total internal space; and a third, apparently highly specialized type, has a large number of cristae aligned to form a central and regularly spaced lamellar system. The latter variety, with specialized cristae, was only found adjacent to the junction between the inner and outer segments, and the remaining two showed an unequal distribution between the individual members of both classes of double cell. In the most peripheral part o f the mitochondrial mass forming the ellipsoid, in the accessory member of the Class B doubles, an oil droplet, composed of an amorphous material, is present (PEDLERand TANSLEY, 1962). In the nocturnal species, a considerable variety of structures were found in the inner segment. In some cells (Fig. 1l) there are dense aggregations of identifiable mitochondria packed between the myoid region and the junction of the inner and the outer segments, in others there are groups of greatly elongated mitochondria (Fig. 12) packed into the most peripheral region of the inner segment. In other inner segments, an entirely new structure was found, composed of a large number of closely packed aggregations of moderately dense and slightly granular material (Figs. 10 and 13). Many of these showed complicated inter-digitations and some are found in the midst of otherwise recognizable mitochondrial masses. It appears that these structures are altered mitochondria which are devoid of all internal structure. Their texture resembles that of the oil droplets of the diurnal species. In transverse sections it can be seen that the remaining material of the inner segment is pushed out towards the periphery by these large bodies. These transmuted mitochondria are mainly found in the main member of the Class B double cells but are also occasionally found in the other two basic cell varieties. In yet other forms, more conventional aggregations of mitochondria were seen alone forming the ellipsoid (Fig. 11). Finally, in occasional cells the aligned mitochondria, the transmuted mitochondria and conventional mitochondria were all found in separate groups in a single inner segment. Golgi apparatus was seen in the myoid regions in both nocturnal and diurnal forms. The nuclei of both the diurnal and nocturnal species are unremarkable, nucleoli are common, and in double cell pairs a different chromatin pattern is seen in the two adjacent nuclei (Fig. 9). In the nocturnal forms the adjacent plasma membranes of double cell pairs are frequently interdigitated (Figs. 3 and 7).

Synaptic Pedicle. The synaptic pedicles in both varieties of animal are connected by a short, thick conducting fibre (Fig. 15) to the nuclear region of the cell, which occasionally contains axially aligned fibrils. One of the most remarkable facts about the synaptic pedicles in all the species studied, whether diurnal or nocturnal, Js the extreme variability of their shape, size and direction. In general, the synaptic surface of the pedicle faces towards the vitreous, but this may face in any direction from this position to one turned through 90 °, or even pointing, in some cases, towards the outer limiting membrane. In all the species studied no fundamental differences were found between any of the pedicles; in most cases, the pedicle outline is one of a smoothly rounded hemispherical object with a highly convoluted internal surface pointing towards the vitreous. Again, considerable variations in this basic shape were found, in some cases for unknown reasons the pedicle is entirely flattened and shows a highly convoluted external p r o n e (Fig. 16), each convolution containing an intrusion of a radial fibre material. The most highly convoluted pedicles were most commonly seen in the nocturnal species. Only detailed differences were found between nocturnal and diurnal pedicles. In the diurnal, more of the large synaptic vacuoles were seen

504

CHRISTOPHER PEDLER AND RITA TILLY

in the body of the pedicle and in the nocturnal more frequent and extensive radial fibre processes were found projecting into the body of the pedicle. The other main difference is that in the diurnal species the pedicles are much more often seen singly, without close contact with an adjacent member. In the nocturnal species, it is the rule to find that the pedicles are in groups of either two, three or four, and that their adjacent surfaces are interdigitated (Fig. 17). Similarly, the leashes of neurites from the outer plexiform layer in the diurnal species are more frequently found separate from one another, whereas in the nocturnal species the groups of pedicles are intimately surrounded by radial fibre material In addition to this intimate contact with radial fibre material, there are separate radial fibre processes arising from the main trunks and protruding into the mass of neurites in the outer plexiform layer and eventually coming into close apposition with the synaptic surface of the pedicle. These are only rarely traced to the synaptic surface of the pedicle and then usually at the extreme periphery. They are not associated with pre-synaptic thickening, synaptic bars or with any particular aggregation of synaptic vesicles. The general structure of a typical pedicle is depicted in Figs. 14-17, and in the schematic diagram. A very large number of processes from the outer plexiform layer make contact with the convoluted surface of the pedicle. The dendritic end-bulbs which enter the body of the pedMe come from the periphery of the leash of fibrils arising from the outer plexiform layer. Finally, the number of individual neurites making synaptic contact with the surface of the pedicle is approximately similar in both diurnal and nocturnal species. This is in marked contrast to the number of contacts which would be expected if the nocturnal pedicles were true rod pedicles. For example, the bat rod synapse shown in Fig. 18 is a typical "rod" pedicle and as can be seen there are only one or two dendritic processes inserted into it.

Nuclear Counts In Tables 1 and 2 the results of twenty central and twenty peripheral nuclear counts from two geckoes are shown. Table 1 is from the diurnal P. inunguis and Table 2 from the nocturnal G. gecko. All eighty fields were scanned at × 400 and no counts were made in areas showing any obvious histological artefacts such as splits in the section or shrinkage. The term "central" is used to indicate the area in a given section with the greatest number of ganglion cell layers and the word "peripheral" to indicate the part adjacent to the point where the retina thins out to become continuous with the epithelium of the Pars plana. It will be noted that there is a surplus of ganglion cells in the central region of both species, indicating that there are more than enough neurone pathways available for a one-toone relationship to exist. In the periphery of both species there is a convergence which, as might be expected, is more marked in the nocturnal G. gecko. In both species, centrally and peripherally, there is a relative surplus of cells in the inner nuclear layer, which is more evident in the diurnal P. inunguis. For the purposes of counting, the fovea of Phelsuma was avoided. DISCUSSION When an animal changes from diurnal to nocturnal habit, its photoreceptors need not undergo an overall change from one cell type to another. Each time the words "rod" or "cone" are applied to a particular gecko visual cell, for example, a specific morphology and function is implied for the whole cell, precluding, by definition, an analysis of the real nature of the receptors. If the receptors are examined according to their component parts, however, and if the hypothesis that different parts of the cell can evolve separately is advanced, then

FIG. 11. A longitudinal section of a nocturnal class A single cell (Hemidactylus flaviviridis) showing the myoid region (M) without a paraboloid and the ellipsoid (E). The mitochondria nearest to the myoid region are recognizable and have clear internal cristae; the mitochondria towards the centre of the ellipsoid are darker and less distinct, and probably represent an intermediate stage between the normal mitochondria of the diurnal species and the fully transmuted variety shown in Fig. 10 ( × 15,000).

FIG. 12. Longitudinal section of a nocturnal (Gecko gecko) inner segment to show the aligned mitochondria indicated in position 7 on the schematic diagram ( × 36,000). [facingpage 5041

FIG. 13. Transverse section of the main member of a nocturnal class B double (Hemidactylus turcicus). Four of the transmuted mitochondria occupying the centre of the ellipsoid are shown (1 to 4). These have extended to a sufficient size to compress the remaining components of the ellipsoid towards the exterior of the cell (arrow) ( x 24,000).

FIG. 14. A longitudinal section of a nocturnal retina (Gecko gecko) in the region of the outer plexiform layer showing four adjacent synaptic pedicles (1 to 4). Radial fibres invest all these structures and surround a part of another visual cell in which the nucleus (N) can be seen. The aggregated pedicles shown here are much more common in nocturnal species; diurnal pedicles in general occur singly ( x 8000).

FIG. 15. A longitudinal section of a nocturnal visual cell (Geckogecko) to show the nucleus (N), the short conducting fibre (C) containing large vesicles and characteristic crossed fibrils, the synaptic pedicle (P) and the surrounding radial fibre material (R). This pedicle shows none of the characteristic synaptic vacuoles found so frequently in the diurnal species Phelsuma ( × 7000).

FIGS. 16 and 17. Both micrographs are transverse sections of a pair of nocturnal synaptic pedicles (Geckogecko) (P1, P2). The plane of Fig. 16 is more sclerad than that of Fig. 17. Both pedicles are surrounded by radial fibre material (R) and in Fig. 16 the interdigitated and closely associated plasma membrane of the two cells can be seen (arrows); in Fig. 17 this junction is not so apparent because the centre of the conjoined pedicles is full of a complex but common mass of neurites from the second neurone (Fig. 16 x 4000) (Fig. 17 x 12,000).

FIG. 18. A longitudinal section of the conducting fibre (C) and synaptic pedicle (P) of a bat. This is a typical rod pedicle and is included for comparison to show the much simpler arrangement of the synapse. Dendrites from elements of the second neurone are shown (D), surrounded by synaptic vesicles and characteristic ribbons. Dendritic protofibrils are also shown. If this micrograph is compared with the pedicles shown in Fig. 11, which also belong to a cell which is normally termed a rod, it will be noted that the complexity of the former is considerably greater, particularly in relation to the elements of the second neurone on the vitread aspect. This rod pedicle is, in addition, much smaller ( x 20,000).

The Nature of the Gecko Visual Cell

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TABLE 1. Phelsuma inunguis Central Ganglion cells/ hundred visual cells Inner nuclear layer

Mean

114 145 129 40 135 120 118 113 100 117 105 100 120 106 109 106 90 102 118 126

720 665 594 672 640 616 530 583 590 756 672 597 605 832 812 720 620 694 915 671

111

675

Peripheral Visual cells/ hundred Ganglion cells Inner nuclear layer

Mean

95 129 88 107 121 91 81 110 143 137 130 103 118 145 138 146 89 128 119 101

455 402 312 318 385 343 368 208 300 260 348 294 212 378 348 300 240 413 385 265

116

328

TABLE 2. Gecko gecko Central Ganglion cells/ hundred visual cells Inner nuclear layer

Mean

136 125 104 104 115 127 110 101 127 117 116 123 105 96 134 130 104 106 141 120

323 269 338 320 306 287 280 246 307 249 204 300 220 291 244 255 340 247 298 288

117

281

Peripheral Visual cells/ hundred Ganglion cells Inner nuclear layer

Mean

183 150 157 167 231 141 150 127 121 193 205 220 104 168 227 163 141 165 181 165

203 176 230 199 243 249 180 198 170 168 190 159 172 186 169 205 200 161 177 168

168

190 II

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FIG. 19. A schematic diagram to illustrate the main difference between the nocturnal (left) and diurnal (right) receptors studied. Synaptic vacuoles (1) are more common in the diurnal species, and the pedicles (2) of the diurnal forms are separated from each other, whereas the nocturnal variety are usually found in closely associated groups of two, three or four. The nuclei (3) of the diurnal species are elongated in a radial direction and the nocturnal paraboloid (4) is compressed and less regularly organised than its diurnal counterpart. The mitochondria forming the ellipsoid (5) show several differences--the diurnal variety have a highly organized internal structure and are of even size, whereas the nocturnal variety show less well organized internal structure, and surround central masses of large, amorphous material separated by membranes which are probably transmuted mitochondria (6). The distal mitochondria of the nocturnal cells are axially aligned (7), whereas in this position, in some of the diurnal cells there is an oil droplet (8). The outer segments (9) show only the difference in shape and size depicted. The dotted line indicates the level of the outer limiting membrane.

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507

the problem is more comprehensible. Thus, environmental needs can be met with minimum change, and instead of assuming the transmutation of an entire receptor into another, changes in the outer segment, the inner segment, the synapse and the intracellular organelles can be considered separately. It is thus possible to envisage cells that in sensitivity resemble those of a rod retina but in acuity those of a cone retina. Such ceils in one set of ambient conditions could take part in a one-to-one information pathway in the retina, and in another, participate in a convergent transfer of information. Further, there is no law demanding that the rest of the retinae follow suit when a receptor transmutes into a particular form. Thus there may be rod-like cells in a cone-like retina, as well as cone-type ceils in a rod-type retina. The dearest morphological difference between the nocturnal and diurnal visual cells is their overall size and the relative sizes of the inner and outer segments. The nocturnal outer segments are larger and cylindrical and the diurnal are smaller and conical, a difference that is probably related solely to sensitivity since the only known function of the outer segment is the transduction of light. Thus in nocturnal forms the chances of light quanta being absorbed are increased by evolving an outer segment of greater length and cross-sectional area. In the diurnal forms, on the other hand, chances of an adequate number of quanta being absorbed are clearly far higher. The gecko also has a higher in situ density visual pigment than the cat, given a correction for the presence of a tapetum in the latter; DENTON (1956) gives the pigment density in two gecko species as 0.5, whereas WEALE(1955) gives a figure for the cat of 0.16. By the use of these figures and from other evidence, TANSLEY (1959) shows that the individual gecko rod may have an effective sensitivity about twice that of the individual mammalian rod. Photopigment density then is certainly relevant to sensitivity, as is the comparative retinal illumination which, in the geckoes, is probably higher than other species (TANSLEY, 1959). Neither of these possibilities, however, is in conflict with an increase in cross-section and length of the outer segment as a means of increasing sensitivity. Neither is the fact that there are enough physical pathways available for an effective one-to-one pathway between each receptor and ganglion cell in the central retina. Electron microscopy of the outer plexiform layer shows that there are more than sufficient neurites available, particularly in the plexiform layers for each visual cell, either to discharge information along discrete routes or to take part in a process involving summation or spatial convergence. The differences between the inner segments are more difficult to assess since there is no apparent visual cause for some of the morphological features. The main difference between the paraboloids is that the diurnal form is more regular and more precisely demarcated, whereas the nocturnal variety is vacuolated and irregular and merges gradually into the surrounding cytoplasm. This, considered with the finding that the mitochondria of the ellipsoids are so different suggests the possibility that a change in emphasis may have taken place in the balance between the local metabolic demands of the visual cell and the need for the condensation of light on to the outer segment. In the diurnal form there is a welldemarcated paraboloid partially enclosed by the closely packed mitochondria of the ellipsoid. WALLS(1942) suggested that this arrangement, as he saw it in the light microscope, resembles an achromatic lens system. If this is the case, the nocturnal forms would presumably have less need for colour correction and may have failed to retain this particular feature. Conversely, the local energy turnover in a given time is likely to be higher in the diurnal species and so, presumably, the need for highly organised mitochondria dominates the need for the condensation of light on to the outer segment. This assumes that the more

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CHRISTOPHER PEDLER AND RITA TILLY

cristae a mitochondrion possesses, the higher its activity. Relevant to this context is the finding that there is a positive correlation between respiratory activity and profuseness of cristae in many tissues and further that cristae as well as the continuous inner layer of surrounding membrane contain most, if not all, of the respiratory enzymes (PALADE, 1956). In the nocturnal forms, however, the need for colour correction may have disappeared but condensation of light on to the outer segment presumably dominated the local need for high energy storage and turnover, thus the highly organized cristae of the mitochondria transmuted into the aggregations of amorphous material shown in Figs. 7 and 10. Of interest in this context is that light transmission experiments with fresh retina of Tropidonotus natrix natrix show that fight is condensed on the outer segment by the inner segment (TANSLEY and JOHNSON, 1956). We have recently found that electron microscopy of the inner segments of T. natrix shows structures similar in almost every way to those of the nocturnal gecko inner segment, with the inference that in the grass snake, although it is diurnal in habit, the need for a light condensing system has over-ridden the need for a high energy turnover, particularly since the inner segment of the grass snake is very large relative to the outer segment. The vertically aligned mitochondria shown in Fig. 12 also probably functions as a light-condensing system. Finally, it is necessary to account for the varied shapes of the inner segment and the occurrence of multiple cell forms. These features are common to a number of species; they are, for example, present in teleosts, amphibians, reptiles, birds and marsupials (DraKE ELDER, 1958); various theories have been expressed to account for this peculiar configuration (KOGANEI, 1884; CAMERON,1911 ; FRANZ, 1913; DETWILER and LAURENS, 1921 ; SAXEN, 1956). It is known that the pedicles from the two members of an associated pair may terminate at different levels in the outer plexiform layer (CAJAL, 1933). This may well be of visual significance. On the other hand the fact that two inner segments are so closely associated may only indicate an adaptation for the purposes of metabolic economy. Another possibility is that the multiple association of visual cells is the most economical way of packing the more voluminous inner segments together, so that the outer segments can be more closely associated to increase retinal resolving power. Whatever the final answer to this particular aspect of the problem, there are no significant differences between the distribution and type of the double cells in the species studied. It is clear from the work already referred to in the introduction that geckoes have a highly atypical retina and that from the general zoological standpoint they are probably transitional in their present form. What then is the nature of their receptors ? A comparison of the difference between the nocturnal and diurnal forms studied shows that there are insufficiently distinct structural criteria for the cells as a whole to divide them into nocturnal rods and diurnal cones; neither is there any advantage to be gained from using the concept of rods and cones at all. If, instead, it is assumed that intracellular components can alter without involving a transmutation of the whole cell, then the overlap of fine structure can be more readily understood. It explains, for example, the finding that except for the minor changes already referred to, the synaptic pedicles and the number of synaptic contacts they make are entirely similar. Reference to the nuclear ratios in the tables also indicates that there are more than enough cells available in the central area of both types, for a one-to-one relationship between the receptors, the bi-polars, and the Ganglion cells, implying that a proportion of the bi-polars, at least equal to the number of receptors, are probably of the "midget" variety (POLYAK, 1955). This, in turn, suggests that the majority of the dendrites of one bipolar are probably inserted into a single receptor pedicle in both nocturnal and diurnal forms, since the "midget" or one-to-one bipolars also have a large number of dendrites

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509

( C M A I , 1933). I t is not surprising therefore t h a t in b o t h cases the structure o f the pedicles a n d their relationship to the c o m p o n e n t s o f the second n e u r o n e are similar. N o t h i n g further can be said at present a b o u t the surplus o f ganglion cells in the central r e g i o n ; this is a n interesting a n d p o t e n t i a l l y p r o d u c t i v e finding for which a n u m b e r o f solutions are feasible, b u t will have to wait for i n t e r p r e t a t i o n until m o r e is k n o w n a b o u t the m o r p h o l o g i c a l a n d physiological extent o f each ganglion cell d o m a i n . W e therefore have, at least in G e c k o gecko, a retina where a one-to-one r o u t e is structurally feasible, b u t which has been shown b y DENTON (1956) to exhibit considerable spatial s u m m a t i o n . The same m u s t be said o f the oil d r o p l e t in the accessory m e m b e r o f the Class B double, which is f o u n d only at the outer end o f the inner segment in Phelsuma. W h y it is f o u n d only here is uncertain, b u t again it m a y be concerned with the necessity for o p t i m u m spatial display o f the various receptor components. I n conclusion, the G e c k o e s a p p e a r to have achieved a highly e c o n o m i c a l solution to the p r o b l e m o f evolving effective p h o t o p i c a n d scotopic a p p a r a t u s , for instead o f developing two separate types o f receptor, changes in intracellular c o m p o n e n t s have evolved, to meet the d e m a n d s o f sensitivity a n d acuity using the facilities o f one basic cell variety. REFERENCES

CAJAL, S. R. (1933). La r6tine des vert6br6s. X I V Concil. ophthal. Madrid. CAMERON,J. (1911). The lamina terminalis and its relation to the fornix system. J. anat., Lond. 45, 211-224. CRESClTELLI, F. (1956). The nature of the gecko visual pigments. J. geE. Physiol. 40, 217. CRESCtTELLI, F. (1958). The natural history of visual pigments. Ann. N. Y. Acad. Sci. 74, 230. CROZIER, W. J. and WOLF, E. (1939). The flicker response contour for the gecko (rod retina). J. geE. PhysioL 22, 555-566. DENTON, E. J. (1956). The responses of the pupil of gecko gecko to external light stimulus. J. geE. Physiol. 40, 201. DETWILER,S. and LAURENS,H. (1921). Studies on the retina. Histogenesis of the visual cells in amblystoma. J. comp. Neurol. 33, 493-508. DETWILER, S. (1923). Studies on the retina. An experimental study on the gecko retina. J. comp. Neurol. 36, 125-141. DODT, E. and HECK, J. (1954). Retinal potentials from the pure-rod eye of the gecko (Sphaerodactylus muralis). Pflug. Arch. ges. Physiol. 259, 226-230. DODT, E. and WALTrIER, J. B. (1958). Electroretinographic evaluation of the gecko's visibility function. Proc. XV. Internat. Cong. Zool., p. 541. DODT, E. and JrssErq, K. H. (1960). The duplex nature of the nocturnal gecko as reflected in the electroretinogram. J. geE. Physiol. 44, 1143-1158. DUKE-ELDER, S. (1958). System of ophthalmology. Vol. 1. Kimpton, London. FRANZ, V. (1913). Oppel's Lehrb. vergl, mikr. Anat. Wirbeltiere. Vol. 7. G. Fischer, Jena. KO~ANEI, J. (1884). Untersuchungen uber die Histiogenese dee Retina. Arch. mikr. Anat. 23, 335. PALADE,G. E. (1956). Enzymes: units of biological structure and function, p. 185. Academic Press, New York. PEDLER, C. and TANSLEV,K. (1962). The fine structure of the cone of a diurnal gecko (Phelsuma inunguis). Exp. Eye Res. 2, 39. PEDLER, C. (1963). The fine structure of the radial fibres in the reptile retina. Exp. Eye Res. 2, 296. POLYAK, S. (1955). Vertebrate visual system. Univ. of Chicago Press. RANVIER, L. (1889). Traitdtechnique d'histologie. Saw, Paris. ROCI-IoN-DuvIGNEAUD, A. (1943). Les yeux et la vision des vertdbrds. Masson, Paris. SAXEN, L. (1956). The initial formation and subsequent development of the double visual ceils in amphibia. J. embryol, exp. Morphol. 4, 57. TANSLEV, K. and JomqsoN, B. K. (1956). The cones of the grass snake's eye. Nature, Lond. 178, 1285. TANSLEY, K. (1959). The retina of two nocturnal geckos, Hemidactylus turcicus and Tarentola mauritanica. Pflug. Arch. ges. Physiol. 286, 213. TANSLEY, K. (1961). The retina of the diurnal gecko. Pflug. Arch. ges. Physiol. 272, 269. TANSLE¥, K. (1961). The fine structure of the cone of the diurnal gecko, ensis longisulae. Pflug. Arch. ges. Physiol. 272, 262. UNDERWOOD, G. (1951). Reptilian retinas. Nature, Lond. 167, 183. UNOERWOOD, G. (1954). On the classification and evolution of geckos. Proc. zool. Soc. Lond. 123, 469.

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VERRIER, M. I. (1935). Recherches sur l'histophysiologie de la r6tine des vert6br6s et les probl6mes qu'elle sotfl~ve. Bull. biol. 20, 140. WALLS, G. (1934). The reptilian retina. Amer. J. OphthaL 17, 892. WALLS, G. (1942). The vertebrate eye and its adaptive radiation. Cranbrook Institute of Science, Michigan. WEALE, R. (1955). Bleaching experiments on the eyes of living grey squirrels (Sciurus carolinensis leucotis). J. PhysioL 127, 587.

R6sum6---En vue d'61ucider les contradictions actuelles sur la nature du photo-r6cepteur du gecko, on a 6tudi6 ~ la fois aux microscopes optique et 61ectronique deux esp~ces diurnes et plusieurs nocturnes. La structure fine obtenue sugg~re qu'an lieu des populations morphologiquement distinctes de bg~tonnets et de cbnes observ6es chez d'autres vert6br6s, il existerait chez le gecko des modifications intracellulaires d'une seule vari6t6 de r6cepteurs permettant de r6pondre aux exigences de sensibilit6 et d'acuit6. Zusammenfassung--Angesichts des widersprechenden Materials, das bisher fiber die Natur der Photorezeptoren des Geckos existiert, wurde eine kombinierte Licht- und elektronenmikroskopische Untersuchung yon zwei Tag- und mehreren Nachtgeckoarten durchgefiihrt. Die struktureUen Ergebnisse weisen daraufhin, dass anstelle der morphologisch unterscheidbaren St~ibchen und Zapfenverteilungen, die bei anderen Wirbeltierarten gefunden werden, die Geckos Anderungen der innerzellularen Komponenten einer einzigen Grundzellart entwickelt haben, urn den Anforderungen an Empfindlichkeit und Sch~irfe zu geniigen. Pe3IoMe---B BH~¢ TOrO, qTO ce~'mc HMetOTC~t npOTI4BOpeqnabIe ~aHHbIe O npHpo~e peuertTOpOB reKKoHa, 6bInO Npon3Be~eHo cpaBnHTe~t,Hoe KOM6naapoBaHHoe n3y,~eHrle ~ByX ~HeBHIaIX H HeCKO~IbKHX HOtIHbIX BH~OB ~THX )IKHBOTHbIX, C IIOMOmbIO O~BIHHO~ CBeTOBO~ MIIKpocKoHHH H 3reKTpOHHO~ MHKpocKonI4tt. Hpe~cTanyleHHt,ie ;zanHbIe 3aCTaBJDItOT ~/yMaTb, HTO BMeCTO pa3BHTH~I MOpqboJIOrHqeCKH Hepa3.rlll~IHMbIX rIa~IoqeK H Ko~t60"-IeK, Haxo;IHMblX y ~pyrrlx BH~OB HO3BOHOHHBIX~ y FeKKOHOB pa3BHYlHCB i,i3MeHeHt/a B HHTpaHeYI~IIOYI~IpHBIX KOMrlOHeHTaX B BH~e BapHaHTOB O~HO~I OCHOBHOH KJIeTIGI, rOTOpbIe COOTBeTCTBytOT Tpe6OBaHH~IM Hpe~a~lBJIfleMblM K qbOTO1-IH~IeCKOMy II CKOTOIIHqeCKOMy 3peHHIO.