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The retina of a fruit bat (Pteropus giganteus Bru¨nnich)
Vision Res.
Vol. 9, pp. 909-922.
THE RETINA
Pcrgamon
Press
1969.
Printed
in Great
OF A FRUIT C. PEDLER
Britain.
BAT (Pteropus giganteus Briinnich) and R. TILLEY
Department of Anatomy, Institute of Ophthalmology, Judd Street, London, W.C.1. {Received 14 Januury 1969)
THE UNIQUE structure of the macro-chiropteran retina was first reported by KOLMER(1910). A year later, the same author published an exhaustive account of a macro~~ropteran eye (Prer0pu.r m&us) which included an analysis of the dioptric apparatus and a minute description of both choroidal and retinal histology. It was shown that the choroid is raised into a series of papillae that project into the external aspect of the retina: each papilla contains both choroidal and retinal pigment together with an external and internal leash of vessels derived from the choriocapillaris and posterior ciliary arteries. It was the author’s view that the vessels penetrate the outer limiting membrane and enter the retinal substance to end in recurring loops at the level of the ganglion cells. No other intra-retinal vasculature was reported, neither was the equivalent of a pecten or a conus papillaris found. The retina was described as pure-rod and although “small granules” were reported between the inner segments of some rods, the author found no cones (KOLMER, 1911). Almost simultaneously, FRITSCH (1911) described the retina of another species Pteropus c~~du~e~~isPeters and, though differing caustically from Kolmer in a number of details, also reported the retina as pure-rod. The disc?epancies between the observations of these two workers were then shown to be due to species difference in a further communication by KoImer who finally summarised his findings on both micro- and macro-chiropteran retinae (KOLMRR, 1926). The retina of another species (EpomophoruswahlbergihaldemaniHalowell) was found to be similar to that studied by Kolmer; again no cones were reported (GERARD, ROCHONDUVIGNEAUD, 1930). In a study, mainly of the choroido-retinal va~ula~~tion, the possibility was raised that the choroidal vessels enter the retina surrounded by a thinned layer of retinal pigment epithelium (ROCHON-DUVIGNEAUD, 1943). We have been unable to find any record of fundus photography but the ophthalmoscopic appearances of one species were recorded in a classic series of paintings of vertebrate fundi (LINDSAY-JOANN, 1927). All the workers mentioned made attempts to correlate the choroidal papillae with both visual and metabolic functions. These are referred to in the discussion. This paper reports light and electron microscopic observations on the retinae of two members of one species (Pteropus giganteus Br~nich). MATERIALS AND METHODS The animals were anaesthetised with intra-peritoneal Nembutal and the eyes removed ante-mortem. Three eyes were then bisected coronally and the vitreous removed. The posterior half of the first eye was placed in 2% Verona1 buffered osmium tetroxide for 2 hr. The second posterior half was placed in 25% phosphate buffered gluteraldehyde f%BATINl, 1963) for 4 hr., washed in sucrose phosphate buffer and then post fixed in 2% Verona1 buffered osmium tetroxide for 1 hr. The posterior half of the third eye was also immersed in 2.5% gluteraldehyde and post fixed in buffered osmium dichromate mixture, but this eye was then subjected to Golgi impregnation. 909
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The three posterior halves were dehydrated in graded ethanol series, the retinae being removed and diced, half way through the series and embedded via immersion in epoxy-propane in Araldite (CIBA). Ultrathin sections were cut on a Reichert ultramicrotome, stained with lead citrate (REYNOLDS,1963) and examined with an AEI EM6 electron microscope at an accelerating voltage of 75kV. l-lop sections were cut from tissue from the third eye which had been subjected to the Golgi technique. These together with thick sections from the first and second eye were stained with toluidine blue, and examined by light microscopy. The fourth eye was processed whole for conventional light microscopy.
RESULTS
‘These can be seen with the opthalmoscope in the living animal as a regular pattern of dark patches with a tendency to form short linear arrays. They were recorded, in the region of the optic disc, by fundus photography (Fig. 3) but it was not possible to see or photograph their arrangement peripherally, in the live animal, since we were not able to achieve precise focus on the retinal surface in this region even after pupillary dilation with Homat~opine, Optical microscopy of thick (1 p) sections in both vertical and tangential planes confirms the regular arrangements of the papillae (Figs. 4,7,8,10) and shows them to be of two main varieties : one that always contains a pair of central penetrating vessels, and is long and thin and penetrates deeply into the retina, and another, sessile variety, penetrating as far as the outer limiting membrane but not always enclosing vessels. In spite of the complex interdigitations of the retina and choroid, the retina separates quite readily in the freshly removed eye, leaving the heavily pigmented, conical pegs of the choroid exposed. Closer examination of the pegs shows that, in some areas, the pairs of penetrating vessels that pass into the retina from the chorio-capillaries are pulled out of the retina during separation and can be seen as small colourless threads protruding from the tips of the choroidal pegs. This appearance was confirmed by light microscopic examination of sections from separated retinae, where it could be seen that many of the intra-retinal vessels were missing although some were still present, having broken off at the apex of the peg. The thin layer of pigment epithelium, which in the electron microscope can be seen to surround the innermost extent of these vessels, could not be resolved at this magnification. Thus, the normal place of cleavage between choroid and retina (in conventional retinae) appears to be preserved in this species, even though there is deep indentation of the retina by choroidal structures. All papillae of both types show a similar internal construction. This is summarised in Fig. 18. There is an outer single layer of pigment epithelium backed by Bruch’s membrane which lies on a conical mass of tissue composed of elongated processes of pigmented choroidal cells and penetrating vessels arising from the chorio-capillaris. The borders of the choroidal cells are not in exact membrane apposition and the spaces between them are occupied by loosely bound collagen. Similar aggregates of collagen are also found between Bruch’s membrane, the choroidal cells and the basement membranes of the vessels. Biuch’s membrane shows a well marked double density, the denser part lying next to the convoluted brush border of the pigment epithelium. The unusually thick basement membrane of the vessels in the choroid and the papillae is ~ontinuo~ with Brueh’s membrane. This is seen most clearly towards the apex of the papilla where Bruch’s membrane is thinner and loses its double density. The vascular supply to the papillae is most easily understood from tangential sections (Fig. 7,8,10,12). At the base, there are two central and between seven and twelve peripheral vessels (Fig. 10). Nearer to the vitreous, there are fewer peripheral vessels, and the centre
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The Retina of a Fruit Bat FIG. 1. One of the two specimens studied, in normal inverted attitude.
Frc;. 2. The “eyeshine” recorded by flash photography after pupillary dilation by Homatropine. Frc. 3. Fundus photograph showing the spotted appearance due to the choroidal papillae. Ftc;. 4. A vertical section cut through the apex of a choroidal papilla. The long axis of the papilla is indicated by the two white arrows and it can be seen that the outer segments of the photo-receptors lie approximately parallel to this axis. The apex of the papilla (dark arrow) penetrates the inner nuclear layer. carrying with it prolongations of the outer nuclear layer. Opposite to the apex is an aggregation of radial fibre material (clear arrow) which also forms an almost complete iamina (r) next to the inner limiting membrane. This layer fills in the gaps left by the small number of ganglion cells (g). A diagrammatic summary of this micrograph is shown in text Fig. 1. The pigment epithetium in this specimen contains pigment and not tapetal spheres. (Osmium, gluteraldehyde, toluidine blue). FOG.5. Vertical section from a specimen impregnated by the Golgi method showing the inner extent of a rod-like photo-receptor. (arrow). An extended myoid leads up to an expanded nuclear region. which is connected to a small synaptic pedicle by a long thin conducting fibre.
FIG. 6. Vertical section from a specimen impregnated by the Golgi method showing a cone-like photoreceptor (arrow). The cell is at the base of a papilla and the expanded nuclear region is adjacent to the outer limiting membrane. A broad straight conducting fibre terminates in a large triangular synaptic pedicle, which gives rise to a number of extensive basal filaments spread out in the curved plane of the outer plexiform layer. FIG. 7. Flat section through the inner nuclear layer. The pale nuclei are mainly those of the bipolar cells. Protruding into this layer are localised aggregations of more darkly staining photo-receptor nuclei. These surround pairs of penetrating vessels. The clear annular zones surrounding the photoreceptor nuclei are what remains of the outer plexiform layer at this level. (Osmium, gluteraldehyde, Toluidine blue). FIG. 8. Flat section through the choroidal papillae showing rings of transversely cut outer segments surrounding the pigment and vessels of the papillae. Islands of photo-receptor nuclei fill the remaining space. Compare with text Fig. 2. (Osmium, gluteraldehyde, Toluidine blue). FIG. 9. Vertical section off-centre to the long axis of a papilla in a Go@ impregnated specimen. There is an apparent concentration of bipolar and horizontal cell processes (arrows) around the innermost part of the papilla. FIG. 10. High power light micrograph of a flat-sectioned choroidal papilla. The pigment epithelium (p.e.) in this region is packed with spherical bodies and surrounds a ring of seven penetrating vessels (white arrow) on the outer surface of the densely pigmented choroidal papilla. At the centre of the papilla are two nuclei belonging to the endothelium of the central penetrating vessels. FIG. 1I. Vertical section of the outer plexiform layer showing four entire rod-like synaptic pedicles (sp.). These contain invaginated processes from other neurones which are double membrane bound. (dark arrow). These also contain synaptic vesicles. Some of these invagination also appear to contain further double membrane bound zones. These probably originate in the manner shown by the configuration marked by the clear arrow. The “ribbons” in this species are particularly long and are surrounded by ordered ag~egations of synaptic vesicles. The remaining space in this preparation is occupied by radial fibre material (r) surrounding aggregations of other neurites some of which are in direct contact.
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FIG. 12. Transverse section of a single choroidal papilla. These vessels (v), two of which will penetrate the retina, are embedded among five processes of pigmented choroidal cells. This group is surrounded hy three cells ofthe pigment epithelium which are densely packed with spherical bodies. One intercellular junction is marked by opposing arrows. Arranged loosely around the outer surface of the pigment epithelium are the transversely sectioned outer segments, and between these and the pigment epithelial surface are small circular bodies. These are the transversely sectioned processes which arise from the inner surface of pigmentepithelium to enclose the outermost part of the outer segments. Transversely sectioned inner segments also appear in the plane ol‘ section (I .S). The insert showIs a single outer segment in transverse section. A main incisurc is present (clear arrow), there are also three accessory incisures.
FIG. 13. High power view of the spherical bodies which fill some pigment epithelial cells. In the plane of section, the larger the diameter of the body, the smaller the thickness of the electron-dense material around the edge. This suggests that the bodies are indeed spherical and lined by an even layer of dense material.
FIG. 14. Transverse section of the outer nuclear layer showing parts of five photo-receptor bodies surrounding a closely packed group of conducting fihrcs. Some of these fibres arc in direct contact, others are separated by material arising from the radial fihre system.
FIG. 15. Vertical section of the outer plexiform layer showing a typical cone-like synaptic pedicle (C) surrounded by a number of rod-like pedicles (r). The cone-like pedicle shows a broad conducting fibre (f) containing characteristic dendritic filaments and is connected to a large number of processes on its synaptic surface. The remaining space in this layer is occupied by radial fibrc material (r a) and processes from other neurones.
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pair are more clearly seen. Sections of the long papillae close to the level of the outer limiting membrane contain only the two central vessels surrounded by a thin layer of pigment epithelium. The two central vessels continue through the retinal substance to end in a loop either in the inner plexiform layer or adjacent to the ganglion cells and nerve frbres. The general arrangement is shown in Figs. 16-18, and electron microscopy confirms that the penetrating vessels are separated from the neurones and radial fibres of the retina by a very thin covering of pigment epithelium.
FIG. 16. Schematic diagram to illustrate the dislocation of the retinal layers, A central pair of vessels penetrates the retina carrying with it a thin covering of pigment epithelium (P.E.). The vessels reach almost as far as the ganglion cell layer(G) where they are capped by a thin rind of pigment epithelium (arrow). Pegs formed by the pigmented choroidal cells also occur closely related to the penetrating vessels (black areas). The cells of the outer nuclear layer (O.N.) are swept inwards by the vessels and displace the cells of the inner nuclear layer (I.N.). Thus, in some planes of flat or tangential section, cell bodies from both inner and outer nuclear layers can be seen in the same specimen, Arising from the inner limiting membrane there is usually an enlarged radial fibre in line with the tip of the penetrating vessels.
FIG. 17. Schematic diagram to illustrate the relative relationships of the cell layers in a flat plane passing through the choroidal papillae. The outer segments (1) are arranged axially to the incident light path in the form of inter-connected annular areas: they are therefore closely related to the covering of pigment epithelium surrounding the pegs of choroidal pigment (dark circles). The zones containing the inner segments of the photo-receptors (2) surround separated islands of photoreceptor nuclei which together form the outer nuclear layer.
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The Retina of a Fruit Bat
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FIG. 18. Schematic drawing to illustrate the principal membrane relationships seen in the electron microscope. The drawing represents half of one papilla, the apex in the top, left-hand corner. The pigmented choroidal ceils (A) form into a cone or peg of tissue separated by locdiised areas of loosely bound collagen and enclosing vessels from the cho~~apilla~s (B). At the apex of the papilla the choroidal vessels give rise to a pair of penetrating vessels one of which is shown (C). The pigment epithelium (E) has a well marked brush-border in contact with Bruch’s membrane (D) which shows a layer of higher density next to the brush-border. At the apex of the papilla, the pigment epithelium is drawn out into a thin layer, (L) covering the penetrating vessels (C) and passing through the outer limiting membrane (M). The outer segments (F) of the receptors are in loose contact with processes arising from the inner surface of the pigment epithe~ium and are, in general, arranged axially to the incident light path. Thus the inner segments towards the apex of the papilla are bent through a sharp angle as they pass through the outer limiting membrane. (I) (see text) and arranged The receptor cell bodies are both “rod-like” (H) and “cone-like” in columns. The radial fibres of Muller (G) separate the columns of receptor cells and also partly enclose closely related groups of conducting fibres from the receptor cells. At the level of the outer limiting membrane the radial fibres give rise to short processes which partly enclose the outer surface of some inner segments. In the outer plexiform layer, both “cone-like” (K) and “rod-like” (J) synaptic pedicles are found.
The pigment
epithehn
This is a single layer of large cuboidal cells, lying in a complex plane co-extensive with the outer aspect of the retina. The plasma membrane on the scleral surface is raised in the form of a well marked brush border lying against the denser layer of Bruch’s membrane, but the small convolutions of the cell border are not followed precisely by the plane of the membrane. The inner or vitreal surface of each cell is raised into long thin processes that loosely surround the surfaces of the photo-receptor outer segments. The cytoplasmic structure of this layer varies in different parts of the retina. Ophthalmoscopic examination of the live animal, reveals that pigment is visible only in the inferior half of the fundus whereas the upper half exhibits a reflecting quality. Examination of the whole, freshly removed retina by low power light microscopy shows that the parts of the pigment epithelium that are still adherent to the outer retinal surface exhibit a faint yellowish reflection. If the specimen is then moved about in the field of view, the reflecting surface appears to glitter unevenly, even within the pits left by the removal of the choroidal pegs. It must be emphasised, however, that the reflection is extremely faint and does not compare in intensity with the tapetal shine of other species we have studied in this laboratory, such as the cat and the bush-baby (DARTNALL et al., 1965; PEDLER, 1963). The most probable structural basis for this refractile property can be seen in both optical and electron microscopes. Cells from the lower half of the fundus are filled by dense aggregations of large pigment granules, whereas cells from the upper half are filled by closely packed approximately spherical bodies. These vary in diameter and show a dense periphery and a lighter centre. They are embedded in closely packed endoplasmic reticulum (Figs. 12, 13). The pigment granules are considerably larger than those in the choroidal cells and occasionally show a faintly lamellated structure. In the superior retina where the cells contain the maximum number of spherical bodies, occasional pigment granules are also found. The cells nearest to the apex of a papilla contain practically no spherical bodies or pigment granules. Where the pigment epithelium actually penetrates the retina it contains densely packed endoplasmic reticulum and localised groups of mitochondria.
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The ~?hoto-receptors Although cones have not yet been reported in any macro-chiropte~dn retina, we found clear evidence of two receptor types in this species. Most of the receptors fit the classical pattern ofthe rod but approximately one in every two hundred and fifty shows a nucleus with tigroid chromatin pattern close to the outer limiting membrane, a broad straight conducting fibre and a typical, complex synaptic pedicle (Fig. 15). The inner segment is slightly broader than that of the rod-like receptors and contains a greater number of larger mitochondria. The outer segment of this type is of the same length and general shape as that of the rod-like cells but contains irregular groups of lamellae which are not necessarily arranged normal to the long axis of the cell. This second cone-like cell was found only at the base of the papiliae. None were seen near the apex. The question of what to call this receptor variety now arises. Disregarding the outer segment, the remainder of the cell fits the type A classification, which is the same as the classical cone. But since there is a long approximately cylindrical outer segment connected to a cone-like remainder this must be regarded as a type B receptor (PEDLER,1965a, b 1969). The sclerad quarter of the outer segments are loosely embedded in processes arising from the pigment epithelium and the inner or ~itrad ends are joined to the inner segments by a narrowed area containing typical cilia1 apparatus. No paraboloids or oil droplets were seen in either variety of receptor. The cell bodies of the receptors are arranged in columns and the extended myoids and conducting hbres lie in closely bound groups between them (Figs. 4 and 14). Some of these groups are penetrated by elements from the main radial fibre system and others are arranged in membrane-to-memb~ne contact, surrounded by radial fibre material. The general mo~hoIogy of both the rod-like and the cone-like synaptic pedicles is generally similar to those of other vertebrate retinae which we have studied. But the cone pedicles have particularly extensive basal filaments (Fig. 6) which show in Golgi preparations as a profuse knot of processes arising from a wedge-shaped pedicle. Both varieties of pedicle also show complex, deeply infolded connections with elements of other neurones, which are unusual in that many of these contain synaptic vesicles on what would be conventionally regarded as the post-synaptic side of the cleft (Fig. 11). Since the outer nuclear layer is folded into the inner nuclear, we examined the inner plexiform layer to see whether any photo-receptor synapses could be found. There were in fact, occasional, simple, or rod-like synapses, but none of the complex or cone variety were found, Thus, this retina again appears to be unique in that photo-receptor synapses occur in the same plane as bipolar-ganglion cell connections. Also, due to the folded layer of receptor cell bodies the remaining parts of the outer plexiform layer are divided up into islands of tissue separated by the papillae. It can be seen in Fig. 11 that the “ribbons” in the rod-like synapses are particularly kmg. They are, in fact, sufficiently large just to resolve in the light microscope. The horizontal cells These are found in the outermost zone of the inner nuclear layer and their cell bodies are most easily distinguishable electron microscopically after fixation in gluteraldehyde and post-fixation by osmium tetroxide. After this treatment, the cytoplasm appears clear and relatively empty, as compared with that of the bipolar cells where there is a narrower region of much denser cytoplasm surrounding the nucleus. The processes of the horizontal cells are unusually profuse and can be recognised in
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electron micrographs by their large size, relative to what are, presumably, bipolar cell processes in the outer plexiform layer. The cytoplasm of these processes is similar to that of the cell bodies and contains groups of mitochondria. In specimens fixed as for electron microscopy in gluteraldehyde and osmium tetroxide and then subjected to Golgi imprecation and examined in the light microscope, the lateral extent and density of the processes is unusual. Collectively, they form a densely interwoven network of terminally branching processes each one ending in one or a number of slightly expanded bulbs. The overlap of adjacent dendritic fields is large, and in some cases appears to be as much as eighty per cent when viewed in vertical section. The most interesting finding, however, is that the densest aggregations of horizontal cell processes are found extending over and around the apices of the papillae (Fig. 9). Aggregations do occur in the region between the papillae, but these are less dense and do not show such a pronounced degree of dendritic overlap, In addition to the dendritic complexes mentioned, the horizontal cells also give rise to ‘“axons” with terminal arborisations extending tangentially for as far as 4&t. The arborisations also show terminal bulbs. Since receptor synapses occur in the inner plexiform layer one might expect to find horizontal cell processes there as well. This probably does occur, but we were unable positively to identify the processes from these cells because the processes of other cells in this region show an almost identical appearance to those of the horizontal ceils. In one section, a synapse between a horizontal cell body and a horizontal cell process was found. Ribbon synapses are usual in the pedicles of the photo-receptors and have been reported in the inner plexiform layer (KIDD, 1962), but this is the first we have seen between horizontal cells. There is thickening and increased density of both membranes, well marked periodic structures in the inter-synaptic cleft and a localised aggregation of vesicles around the double ribbons. No similar specialisation was found elsewhere in the two cells. The bipolar cells
These are few in number compared with the photo-receptors, but similar in structure to those of other species. They are arranged as a thin closely packed layer fenestrated by the apical projections of the outer nuclear layer (Fig. 7). The cell bodies are frequently in membrane-to-membrane contact, but where they are not, they are separated by unusually thin tangential processes arising from the radial fibres. In other retinae, it is common to find the axons and dendrites of the bipolar cells enclosed by a cuff of radial fibre material, but in this species, the processes of the bipolar cells lie in indentations or partly closed tunnels formed in the surface of other bipolar cell bodies. Specimens impregnated by the Golgi technique show that the axons develop typical multi-~yered arborisations and clubs extending tangentially at different levels of the inner plexiform layer. The amacrine cells, the ganglion cells and the radial.fibres
Very few amacrine cells were identified with certainty. None were recognised by the light-microscope, either in toluidine blue or Golgi stained preparations. However, occasional cells were seen on the inner aspect of the inner nuclear layer, giving rise to broad single processes extending into the inner plexiform layer. These cells possess more cytoplasm than the bipolars, and also contain a number of mitochond~a and large vesicles in the region of the cell next to the origin of the main process. No terminal arborisations were identified el~tron-microscopically.
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<'.fkDt,Eit AND R. TILL.EY
The ganglion cells are again relatively less frequent than the bipolar cells. For example, in a single vertical section, a light microscopic field including three papillae may show four to six ganglion cells only. The cellbodies are large and unusually close to the inner limiting membrane from which they are separated by broad plaques of radial fibre material (Fig. 4) which fill in the space left by the small numbers of ganglion cells and also enclose localised groups Of very small unmyelinated nerve fibres lying between the ganglion cell bodies and the inner limiting membrane. The normally straight course of the radial hbres between inner and outer limiting membranes is not significantly altered by the presence of the papillae. In the outer nuclear layer, the main trunks of the fibres are expanded to enclose the groups of conducting fibres referred to above (Fig, 14) and also to divide the cell bodies of the photo-receptors into columns (Fig. 4). In this region also, the radial fibres contain a high concentration of mito~hondria as compared to other species we have studied. Arising from the inner limiting membrane, there are unusually dense aggregations of prolonged radial fibre trunks close to the innermost extent of the penetrating choroidal vessels. In the light microscope, the radial fibre material adjacent to the inner limiting membrane appears almost as a separate layer, except for the prolongation near the vessels. The nuclei of the radial fibres lie in triangular expansions of the main trunks on the innermost side of the inner nuclear layer (Fig. 4). This species has two varieties of photo-receptor which, considered together, are considerably more numerous than the bipolar cells. Similarly there is a refative surplus of bipolar cells when compared with the sparsely populated ganglion cell layer. Thus the retina is convergent in general arrangement, which is consistent with the predominantl~~ nocturnal habit of the animal. But we have also found cells possessing many of the structural features of cones. These are found only at the base of the papillae and are very uncommon. The estimate, mentioned above, of one in every two hundred and fifty is only approximate, since the folding of the receptor layer makes it difficult to measure accurately the relative cell counts in a single light microscopic field. However, the fact that this second receptor type is uncommon, suggests that it may not play a very significant part in the total visual capacity of the animal. The profusion of basal filaments arising from the synaptic pedicle of this receptor variety is, however, unusual. But, this cannot be related to the function of the cell, until more is known about the connectivity of the filaments. The apparently uneven distribution of the horizontal cell groups in Colgi preparations is of interest and this may well indicate functional differences between the apical and basal regions of the papillae. But this possibility must also be considered with caution, because of the capricious effects of the Golgi method. Since this is well known to pick out localised groups of cells in other, normally arranged, retinas for no defined reason, the apparent concentration of the horizontal cells and other processes over the apex of some papillae could be due to this peculiarity of the silver impregnation method rather than any essential functional differences. Considerable attention has been given to the papillae and their possible significance to the vision of the animal. KOLMER (1911) discusses this point at length and points out that none of the species he examined (not including the one we were able to study) showed any sign of accommodative musculature, and therefore the eye could be compared to a “snapshot” camera containing a lens of fixed focal length. He goes on to suggest that the papillae could be a means whereby a sharp image is focussed on the outer segments of the photo-
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receptors, which are arranged so that the sharpness could register at a number of different Iev& in the retina, Although he m$kes the point that he is suspicious of such teleological argument, he concludes that apart from the metabolic significance of the penetrating vessels that this is the only visually significant reason for the presence of the papillae. This conchsion is supported by Walls, who in comparing the arrangement in the fruit bat with the ramp retina of a ray (~uja batis), agrees that the papillae could provide a broad region of focus for a fixed lens (WALLS, 1963). ROCHON-DUVIGNEAUD (1943) however, showed that in one genus, (Epomoghorus), measurements taken from whole eye sections revealed a hypermetropia of approximately 15 dioptres, a figure which he stated is modified to take account of possible posterior lens dislocation and changes in shape during histological preparation. If this is the general case among the macrochiropterans, then as Rochon-Duvigneaud points out, it is difficult to accept the hypothesis erected by Kolmer, and supported by Walls. For there would be no sharp image in the plane of the receptors at all. There could well be blurred images of contrast and colour difference, but throughout the extent of the papillae, there would be no plane of sharp focus, It is therefore possible to conclude that the papillae would not provide a real alternative to accommodation, provided that the dioptric measurements made by Rochon-Duvigneaud are generally true for other species. A further hypothesis was put forward and then rejected by Rochon-Duvigneaud. He suggested that the choroidal papillae would have the effect of increasing the total number of visual cells per unit retinal area since the total, corrugated, length of the outer limiting membrane would allow more receptors to be packed into a particular region. But he then goes on to say that, as a result of his observations in E~~~o~~~r~s,if the number of receptors around a particular papilla are considered as lying flat over the area covered by the base of the papilla they would fit into the area without overlap. He concludes therefore that this ides must be abandoned. In our study, we repeated this observation on material fixed as for electron microscopy, stained with toluidine blue and examined in the light microscope. In several vertical sections cut through the apex of the papillae, we attempted to estimate whether the receptors lying around the papillae would indeed fit over the area occupied by its base. Taking the width of each inner segment as it passes through the outer limiting membrane and counting the number of inner segments around the papillae, we then projected the same number of inner segments on to the choroidal base of the papillae. By this procedure we found that approximately eighty percent of the inner segments would fail to fit into the base. A proportion which would be much larger if the total areas covered by the receptors and the papillae were to be considered. We were therefore able to conclude that, in the species studied, the choroido-retinal architecture would allow a very much larger number of photoreceptors to be packed into a given retinal area. We also noted, in sections of the whole eye that the outer segments all lie in a direction approximately parallel to the long axis of the papillae and that the papillae all point approximately to the posterior nodal point. Thus, it appears that most if not all the outer segments could well be involved in the reception of light. But it is necessary to account for the difference between Rochon-Duvigneaud’s conclusion and our own. This may well be due to species difference, but, in addition, material fixed for electron microscopy and viewed in the light microscope, offers a much better basis for measurement than does the material prepared in the classical manner. FRITSCH (1911) compared the choroidal array to the pecten of the bird, first from the
920
C. PEDLER AND
R. TILLEY
point of view of its nutritive function and secondly as a means of casting retinal shadows. He pointed out that one hypothesis of pecten function is to act as a sundial or shadowcaster, thus giving the bird a means of continuous attitude discrimination. But he goes on to reject the idea since he also found that the papillae were directed towards the nodal point and thus would not be likely to cast any shadows. There seems little doubt that an important function of the choroidal papillae is to provide retinal nutrition. We found no other evidence of intra-retinal vasculature, nor did we see any structure analogous to the pecten of the bird, or the conus papillaris of the reptile. We were also able to confirm Kolmer’s observations in other species, that there are no central vessels but only a rete of vessels surrounding the periphery of the optic nerve. Thus, the papillae allow a greater concentration of outer segments in a given area, as well as subserving a metabolic nutritive role, but there is one additional finding that complicates the assumption that the papillae are of significance to the animal’s vision. That is the presence and architecture of the retinal pigment epithelium. This layer follows the contour of the penetrating vessels closely even to the innermost point of penetration. But we have also found that it is this layer which provides the “eyeshine” of the animal. It is therefore, in part, a retinal tapetum. So it is necessary to account for a weakly reflecting layer posterior to the outer segments with its surface raised into pointed projections. We have assumed the spherical bodies found in this layer of cells to be the origin of the reflecting property. And we have also shown that the density per cell of these bodies becomes less as the pigment epithelium penetrates the retina. As the inner layers of the retina are reached the spheres are replaced by a gradually increasing concentration of endoplasmic reticulum. Therefore, it is probable that the reflecting properties of this layer diminish as the layct reaches the inner surface of the retina. Nevertheless at the level of the outer segments. ;I high concentration of spheres is still present, and low power microscopy of whole preparations reveals that the tapetum is still refractile at this level. So now we have to account fol densely packed outer segments adjacent to a highly irregular reflecting layer in an eye which is probably hypermetropic. On this basis it is difficult to see how the animal is able to see anything. For presumably light would scatter among the outer segments from the sides of the papillae covered by the reflecting layer of the pigment epithelium. It may be, of course, that the outer segments only admit light in the axial direction, in which case this problem would not arise. There seems to be little evidence to show what the animal “sees”. During the time we kept our two specimens we were gble to make some crude estimates of their visual capability. We found that in daylight conditions in a cage, the animal hanging upside down would follow a dark object (a gloved finger) against a white background. It would first move its eyes in the direction of movement and then the head would move to restore the eyes to a central position in the head. If the moving target was shaken it appeared to follow more readily. It would also follow a pen torch when moved in dim background illumination and would move away if the torch was moved closer to it. When these tests were repeated with the animal held erect it appeared to perform just as well. We are also able to confirm Kolmer’s observation that both pupils remained small even in dim conditions: they did, however, react normally and briskly to light. KOLMER(1911) also reports that fruit bats have been observed to swoop low over Water and catch fish. We have also found accounts of their flying action, which is apparently rapid and effective in the avoidance of objects, so the creature appears to be able to make effective use of iti visual system, for there is no evidence to show that it posesses the echo-location
The Retina of a Fruit Bat
921
capability of some micro~hiropterans. So how does the animal make use of its unique retina and hypermetropic eye in successful aerial navigation? The following hypothesis is possible. &suming that there is some significance in the preponderance of horizontal and bipolar cell connections at the apices of the papillae and that this is not merely a vagary of the Golgi technique, then it could be that the receptors surrounding the papillae are preferentially connected in convergent array to a particular afferent visual path. Given the presence of a refractile spike of tapetum in the centre of the grouped receptors, it would be possible to look at this group of receptors as, perhaps, a giant receptor generating large bursts of activity as out of focus changes in contrast pass over it. Such an arrangement might well give some fairly rapid control of attitude over and above any pattern recognition properties, which the receptor matrix as a whole, might possess. It would be of particular interest to see whether neuro-physiological study of this retina revealed any unusual electrical response to moving stimuli, but until this is done it is perhaps better to conclude by quoting an ancient recipe: “minced bat’s brain and molasses, when taken internally, is good for dim vision”. Acknowledgement-The electron microscope used in this study was provided by the Wellcome Trust. We are also grateful to Miss M. BOYLEfor valuable technical assistance and to the British Museum of Natural History for species identification. REFERENCES DARTNALL,H. J. A., ARDEN,G. B., IKEDA,H., LUCK,C. P., ROSENBERG, M. E., PEDLER,C. M. H. and TANSLEY, K. (1965). Anatomical, eiectrophysiological and pigmentary’aspects of vision in the Bush Baby: an interpretative study. Vision Res. 5,399-424. FRITSCH,G. (1911). Contributions to the histology of the Pteropus eye. Z. Zool. 98, 288. JOHNSON,L. (1927). Contributions to the comparative anatomy of the reptilian and amphibian eye, chiefly based on ophthaimologic~ examination. Phil. Trans. 8.215,31.5. KIDD, M. (1962). Electron microscopy of the inner plexiform layer in the cat and the pigeon. J. Anat. %, 1799187. KOLMER,W. (1910). Contribution to the study of the eye of macrochiroptera. Centrnlbkztt Physiol. 256. KOLMER,W. (191 I). The problem of the anatomy of the eye of macrochiroptera. 2. wiss zool. Bd. 97, Heftl. 91. KOLMER,W. (1926). On the eye of the bat. Verhandl. Zoo/. Bot. Ges. Wien 74,29-31. PEDLER,C. (1963). The fine structure of the Tapetum Cellulosum. Exptl Eye Res. 2, 189-195. PEDLER,C. (1965). Rods and cones, a fresh approach. Proceedings, CIBA Symposium on Physiology and Psychology of Colour Vision, J. and A. Churchill, London. PEDLER, C. (1965a). Rods and cones, a new approach. Biochemistry of the Retina, Academic Press, London and New York. PEDLER,C. (1969). The rod and the cone. fnt. Rev. Zoo/., in press. REYNOLDS, E. C. (1963). The use of lead citrate at high pH as an electron opaque stain. J. Cell Biof. 1?,208-212. ROCHON-DUVIGNEAUD, (1943). The Eyes and the Vision of‘ Vertebrates, p. 563, Masson (Paris) Glover. SABATINI,D. D., BENSCH,K. and BARRNETT,R. J. (1963). The preservation of cellular ultrastructure by aldehyde fixation. J. Cell Biol. 17, 19-58. WALLS,G. (1963). The Vertebrate Eye. Hafner, New York.
Abstract-The light and electron microscope architecture of the retina of a fruit bat (Pteropus gigunteus Brtinnich) is described. The relationship between choroidal vessels penetrating the retina and the retinal substance is also described and related to the uniquely papillated structure of the organ. The functional significance of the papillation is discussed and related to the views of other workers. It is concluded that the papillation has, apart from a metabolic role, a visual function in that it allows a greater number of outer segments to be packed together in a given area of retina.
922
R&un-On decrit, en microscopic optique et electronique, I’architecture de la r&tine de la chauvesouris Pmropus gigunreus Briinnich. On d&it aussi la relation entre les vaisseaux choroidiens qui pen&rent dans la r&tine et la substance r&nienne, en liaison avec la structure papWe trts speciale de cet organe. On discute la signification fonctionnelle de cette structure, en relation avec les vues d’autres auteurs. On con&t que, outre son role metabolique, la papillation posstde une fonction visuelle en permettant d un plus grand nombre de segments externes d’etre accumules dans une aire donnee de la retline.
Zusammenfassung-Es wird die licht- und elektronenmikroskopische
Struktur der Netzhaut des Flederhundes (Pteropus gigunteus Brtinnich) beschrieben. Das Verhaltnis der die Netzhaut durchstossenden Aderhautgefasse zur Netzhaut selbst wird such beschrieben und mit der einzigartigen Papilhetung des Organes verghchen. Die funktionelle Bedeutung der Papillierung wird such insofem sie die Ansichten anderer Forscher anbeiangt besprochen. Es wird geschlossen, dass die Papillierung nicht nur eine N~hrungsrol~e erfiilit sondem such dem Sehen dient, da sic es erlaubt eine grijssere Anzahl Aussenglieder in einer gegebenen Netzhaut~~che unterzubringen.
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Vol. 9, pp. 909-922.
THE RETINA
Pcrgamon
Press
1969.
Printed
in Great
OF A FRUIT C. PEDLER
Britain.
BAT (Pteropus giganteus Briinnich) and R. TILLEY
Department of Anatomy, Institute of Ophthalmology, Judd Street, London, W.C.1. {Received 14 Januury 1969)
THE UNIQUE structure of the macro-chiropteran retina was first reported by KOLMER(1910). A year later, the same author published an exhaustive account of a macro~~ropteran eye (Prer0pu.r m&us) which included an analysis of the dioptric apparatus and a minute description of both choroidal and retinal histology. It was shown that the choroid is raised into a series of papillae that project into the external aspect of the retina: each papilla contains both choroidal and retinal pigment together with an external and internal leash of vessels derived from the choriocapillaris and posterior ciliary arteries. It was the author’s view that the vessels penetrate the outer limiting membrane and enter the retinal substance to end in recurring loops at the level of the ganglion cells. No other intra-retinal vasculature was reported, neither was the equivalent of a pecten or a conus papillaris found. The retina was described as pure-rod and although “small granules” were reported between the inner segments of some rods, the author found no cones (KOLMER, 1911). Almost simultaneously, FRITSCH (1911) described the retina of another species Pteropus c~~du~e~~isPeters and, though differing caustically from Kolmer in a number of details, also reported the retina as pure-rod. The disc?epancies between the observations of these two workers were then shown to be due to species difference in a further communication by KoImer who finally summarised his findings on both micro- and macro-chiropteran retinae (KOLMRR, 1926). The retina of another species (EpomophoruswahlbergihaldemaniHalowell) was found to be similar to that studied by Kolmer; again no cones were reported (GERARD, ROCHONDUVIGNEAUD, 1930). In a study, mainly of the choroido-retinal va~ula~~tion, the possibility was raised that the choroidal vessels enter the retina surrounded by a thinned layer of retinal pigment epithelium (ROCHON-DUVIGNEAUD, 1943). We have been unable to find any record of fundus photography but the ophthalmoscopic appearances of one species were recorded in a classic series of paintings of vertebrate fundi (LINDSAY-JOANN, 1927). All the workers mentioned made attempts to correlate the choroidal papillae with both visual and metabolic functions. These are referred to in the discussion. This paper reports light and electron microscopic observations on the retinae of two members of one species (Pteropus giganteus Br~nich). MATERIALS AND METHODS The animals were anaesthetised with intra-peritoneal Nembutal and the eyes removed ante-mortem. Three eyes were then bisected coronally and the vitreous removed. The posterior half of the first eye was placed in 2% Verona1 buffered osmium tetroxide for 2 hr. The second posterior half was placed in 25% phosphate buffered gluteraldehyde f%BATINl, 1963) for 4 hr., washed in sucrose phosphate buffer and then post fixed in 2% Verona1 buffered osmium tetroxide for 1 hr. The posterior half of the third eye was also immersed in 2.5% gluteraldehyde and post fixed in buffered osmium dichromate mixture, but this eye was then subjected to Golgi impregnation. 909
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The three posterior halves were dehydrated in graded ethanol series, the retinae being removed and diced, half way through the series and embedded via immersion in epoxy-propane in Araldite (CIBA). Ultrathin sections were cut on a Reichert ultramicrotome, stained with lead citrate (REYNOLDS,1963) and examined with an AEI EM6 electron microscope at an accelerating voltage of 75kV. l-lop sections were cut from tissue from the third eye which had been subjected to the Golgi technique. These together with thick sections from the first and second eye were stained with toluidine blue, and examined by light microscopy. The fourth eye was processed whole for conventional light microscopy.
RESULTS
‘These can be seen with the opthalmoscope in the living animal as a regular pattern of dark patches with a tendency to form short linear arrays. They were recorded, in the region of the optic disc, by fundus photography (Fig. 3) but it was not possible to see or photograph their arrangement peripherally, in the live animal, since we were not able to achieve precise focus on the retinal surface in this region even after pupillary dilation with Homat~opine, Optical microscopy of thick (1 p) sections in both vertical and tangential planes confirms the regular arrangements of the papillae (Figs. 4,7,8,10) and shows them to be of two main varieties : one that always contains a pair of central penetrating vessels, and is long and thin and penetrates deeply into the retina, and another, sessile variety, penetrating as far as the outer limiting membrane but not always enclosing vessels. In spite of the complex interdigitations of the retina and choroid, the retina separates quite readily in the freshly removed eye, leaving the heavily pigmented, conical pegs of the choroid exposed. Closer examination of the pegs shows that, in some areas, the pairs of penetrating vessels that pass into the retina from the chorio-capillaries are pulled out of the retina during separation and can be seen as small colourless threads protruding from the tips of the choroidal pegs. This appearance was confirmed by light microscopic examination of sections from separated retinae, where it could be seen that many of the intra-retinal vessels were missing although some were still present, having broken off at the apex of the peg. The thin layer of pigment epithelium, which in the electron microscope can be seen to surround the innermost extent of these vessels, could not be resolved at this magnification. Thus, the normal place of cleavage between choroid and retina (in conventional retinae) appears to be preserved in this species, even though there is deep indentation of the retina by choroidal structures. All papillae of both types show a similar internal construction. This is summarised in Fig. 18. There is an outer single layer of pigment epithelium backed by Bruch’s membrane which lies on a conical mass of tissue composed of elongated processes of pigmented choroidal cells and penetrating vessels arising from the chorio-capillaris. The borders of the choroidal cells are not in exact membrane apposition and the spaces between them are occupied by loosely bound collagen. Similar aggregates of collagen are also found between Bruch’s membrane, the choroidal cells and the basement membranes of the vessels. Biuch’s membrane shows a well marked double density, the denser part lying next to the convoluted brush border of the pigment epithelium. The unusually thick basement membrane of the vessels in the choroid and the papillae is ~ontinuo~ with Brueh’s membrane. This is seen most clearly towards the apex of the papilla where Bruch’s membrane is thinner and loses its double density. The vascular supply to the papillae is most easily understood from tangential sections (Fig. 7,8,10,12). At the base, there are two central and between seven and twelve peripheral vessels (Fig. 10). Nearer to the vitreous, there are fewer peripheral vessels, and the centre
2.
3. [ficing p. 910
Il.
12.
14.
13.
15.
The Retina of a Fruit Bat FIG. 1. One of the two specimens studied, in normal inverted attitude.
Frc;. 2. The “eyeshine” recorded by flash photography after pupillary dilation by Homatropine. Frc. 3. Fundus photograph showing the spotted appearance due to the choroidal papillae. Ftc;. 4. A vertical section cut through the apex of a choroidal papilla. The long axis of the papilla is indicated by the two white arrows and it can be seen that the outer segments of the photo-receptors lie approximately parallel to this axis. The apex of the papilla (dark arrow) penetrates the inner nuclear layer. carrying with it prolongations of the outer nuclear layer. Opposite to the apex is an aggregation of radial fibre material (clear arrow) which also forms an almost complete iamina (r) next to the inner limiting membrane. This layer fills in the gaps left by the small number of ganglion cells (g). A diagrammatic summary of this micrograph is shown in text Fig. 1. The pigment epithetium in this specimen contains pigment and not tapetal spheres. (Osmium, gluteraldehyde, toluidine blue). FOG.5. Vertical section from a specimen impregnated by the Golgi method showing the inner extent of a rod-like photo-receptor. (arrow). An extended myoid leads up to an expanded nuclear region. which is connected to a small synaptic pedicle by a long thin conducting fibre.
FIG. 6. Vertical section from a specimen impregnated by the Golgi method showing a cone-like photoreceptor (arrow). The cell is at the base of a papilla and the expanded nuclear region is adjacent to the outer limiting membrane. A broad straight conducting fibre terminates in a large triangular synaptic pedicle, which gives rise to a number of extensive basal filaments spread out in the curved plane of the outer plexiform layer. FIG. 7. Flat section through the inner nuclear layer. The pale nuclei are mainly those of the bipolar cells. Protruding into this layer are localised aggregations of more darkly staining photo-receptor nuclei. These surround pairs of penetrating vessels. The clear annular zones surrounding the photoreceptor nuclei are what remains of the outer plexiform layer at this level. (Osmium, gluteraldehyde, Toluidine blue). FIG. 8. Flat section through the choroidal papillae showing rings of transversely cut outer segments surrounding the pigment and vessels of the papillae. Islands of photo-receptor nuclei fill the remaining space. Compare with text Fig. 2. (Osmium, gluteraldehyde, Toluidine blue). FIG. 9. Vertical section off-centre to the long axis of a papilla in a Go@ impregnated specimen. There is an apparent concentration of bipolar and horizontal cell processes (arrows) around the innermost part of the papilla. FIG. 10. High power light micrograph of a flat-sectioned choroidal papilla. The pigment epithelium (p.e.) in this region is packed with spherical bodies and surrounds a ring of seven penetrating vessels (white arrow) on the outer surface of the densely pigmented choroidal papilla. At the centre of the papilla are two nuclei belonging to the endothelium of the central penetrating vessels. FIG. 1I. Vertical section of the outer plexiform layer showing four entire rod-like synaptic pedicles (sp.). These contain invaginated processes from other neurones which are double membrane bound. (dark arrow). These also contain synaptic vesicles. Some of these invagination also appear to contain further double membrane bound zones. These probably originate in the manner shown by the configuration marked by the clear arrow. The “ribbons” in this species are particularly long and are surrounded by ordered ag~egations of synaptic vesicles. The remaining space in this preparation is occupied by radial fibre material (r) surrounding aggregations of other neurites some of which are in direct contact.
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PEDLER ANDK. ~rlt,LW
FIG. 12. Transverse section of a single choroidal papilla. These vessels (v), two of which will penetrate the retina, are embedded among five processes of pigmented choroidal cells. This group is surrounded hy three cells ofthe pigment epithelium which are densely packed with spherical bodies. One intercellular junction is marked by opposing arrows. Arranged loosely around the outer surface of the pigment epithelium are the transversely sectioned outer segments, and between these and the pigment epithelial surface are small circular bodies. These are the transversely sectioned processes which arise from the inner surface of pigmentepithelium to enclose the outermost part of the outer segments. Transversely sectioned inner segments also appear in the plane ol‘ section (I .S). The insert showIs a single outer segment in transverse section. A main incisurc is present (clear arrow), there are also three accessory incisures.
FIG. 13. High power view of the spherical bodies which fill some pigment epithelial cells. In the plane of section, the larger the diameter of the body, the smaller the thickness of the electron-dense material around the edge. This suggests that the bodies are indeed spherical and lined by an even layer of dense material.
FIG. 14. Transverse section of the outer nuclear layer showing parts of five photo-receptor bodies surrounding a closely packed group of conducting fihrcs. Some of these fibres arc in direct contact, others are separated by material arising from the radial fihre system.
FIG. 15. Vertical section of the outer plexiform layer showing a typical cone-like synaptic pedicle (C) surrounded by a number of rod-like pedicles (r). The cone-like pedicle shows a broad conducting fibre (f) containing characteristic dendritic filaments and is connected to a large number of processes on its synaptic surface. The remaining space in this layer is occupied by radial fibrc material (r a) and processes from other neurones.
The Retina of a Fruit Bat
913
pair are more clearly seen. Sections of the long papillae close to the level of the outer limiting membrane contain only the two central vessels surrounded by a thin layer of pigment epithelium. The two central vessels continue through the retinal substance to end in a loop either in the inner plexiform layer or adjacent to the ganglion cells and nerve frbres. The general arrangement is shown in Figs. 16-18, and electron microscopy confirms that the penetrating vessels are separated from the neurones and radial fibres of the retina by a very thin covering of pigment epithelium.
FIG. 16. Schematic diagram to illustrate the dislocation of the retinal layers, A central pair of vessels penetrates the retina carrying with it a thin covering of pigment epithelium (P.E.). The vessels reach almost as far as the ganglion cell layer(G) where they are capped by a thin rind of pigment epithelium (arrow). Pegs formed by the pigmented choroidal cells also occur closely related to the penetrating vessels (black areas). The cells of the outer nuclear layer (O.N.) are swept inwards by the vessels and displace the cells of the inner nuclear layer (I.N.). Thus, in some planes of flat or tangential section, cell bodies from both inner and outer nuclear layers can be seen in the same specimen, Arising from the inner limiting membrane there is usually an enlarged radial fibre in line with the tip of the penetrating vessels.
FIG. 17. Schematic diagram to illustrate the relative relationships of the cell layers in a flat plane passing through the choroidal papillae. The outer segments (1) are arranged axially to the incident light path in the form of inter-connected annular areas: they are therefore closely related to the covering of pigment epithelium surrounding the pegs of choroidal pigment (dark circles). The zones containing the inner segments of the photo-receptors (2) surround separated islands of photoreceptor nuclei which together form the outer nuclear layer.
914
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AND
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The Retina of a Fruit Bat
915
FIG. 18. Schematic drawing to illustrate the principal membrane relationships seen in the electron microscope. The drawing represents half of one papilla, the apex in the top, left-hand corner. The pigmented choroidal ceils (A) form into a cone or peg of tissue separated by locdiised areas of loosely bound collagen and enclosing vessels from the cho~~apilla~s (B). At the apex of the papilla the choroidal vessels give rise to a pair of penetrating vessels one of which is shown (C). The pigment epithelium (E) has a well marked brush-border in contact with Bruch’s membrane (D) which shows a layer of higher density next to the brush-border. At the apex of the papilla, the pigment epithelium is drawn out into a thin layer, (L) covering the penetrating vessels (C) and passing through the outer limiting membrane (M). The outer segments (F) of the receptors are in loose contact with processes arising from the inner surface of the pigment epithe~ium and are, in general, arranged axially to the incident light path. Thus the inner segments towards the apex of the papilla are bent through a sharp angle as they pass through the outer limiting membrane. (I) (see text) and arranged The receptor cell bodies are both “rod-like” (H) and “cone-like” in columns. The radial fibres of Muller (G) separate the columns of receptor cells and also partly enclose closely related groups of conducting fibres from the receptor cells. At the level of the outer limiting membrane the radial fibres give rise to short processes which partly enclose the outer surface of some inner segments. In the outer plexiform layer, both “cone-like” (K) and “rod-like” (J) synaptic pedicles are found.
The pigment
epithehn
This is a single layer of large cuboidal cells, lying in a complex plane co-extensive with the outer aspect of the retina. The plasma membrane on the scleral surface is raised in the form of a well marked brush border lying against the denser layer of Bruch’s membrane, but the small convolutions of the cell border are not followed precisely by the plane of the membrane. The inner or vitreal surface of each cell is raised into long thin processes that loosely surround the surfaces of the photo-receptor outer segments. The cytoplasmic structure of this layer varies in different parts of the retina. Ophthalmoscopic examination of the live animal, reveals that pigment is visible only in the inferior half of the fundus whereas the upper half exhibits a reflecting quality. Examination of the whole, freshly removed retina by low power light microscopy shows that the parts of the pigment epithelium that are still adherent to the outer retinal surface exhibit a faint yellowish reflection. If the specimen is then moved about in the field of view, the reflecting surface appears to glitter unevenly, even within the pits left by the removal of the choroidal pegs. It must be emphasised, however, that the reflection is extremely faint and does not compare in intensity with the tapetal shine of other species we have studied in this laboratory, such as the cat and the bush-baby (DARTNALL et al., 1965; PEDLER, 1963). The most probable structural basis for this refractile property can be seen in both optical and electron microscopes. Cells from the lower half of the fundus are filled by dense aggregations of large pigment granules, whereas cells from the upper half are filled by closely packed approximately spherical bodies. These vary in diameter and show a dense periphery and a lighter centre. They are embedded in closely packed endoplasmic reticulum (Figs. 12, 13). The pigment granules are considerably larger than those in the choroidal cells and occasionally show a faintly lamellated structure. In the superior retina where the cells contain the maximum number of spherical bodies, occasional pigment granules are also found. The cells nearest to the apex of a papilla contain practically no spherical bodies or pigment granules. Where the pigment epithelium actually penetrates the retina it contains densely packed endoplasmic reticulum and localised groups of mitochondria.
916
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PEDLER ANDR. T‘lJ.J,iiY
The ~?hoto-receptors Although cones have not yet been reported in any macro-chiropte~dn retina, we found clear evidence of two receptor types in this species. Most of the receptors fit the classical pattern ofthe rod but approximately one in every two hundred and fifty shows a nucleus with tigroid chromatin pattern close to the outer limiting membrane, a broad straight conducting fibre and a typical, complex synaptic pedicle (Fig. 15). The inner segment is slightly broader than that of the rod-like receptors and contains a greater number of larger mitochondria. The outer segment of this type is of the same length and general shape as that of the rod-like cells but contains irregular groups of lamellae which are not necessarily arranged normal to the long axis of the cell. This second cone-like cell was found only at the base of the papiliae. None were seen near the apex. The question of what to call this receptor variety now arises. Disregarding the outer segment, the remainder of the cell fits the type A classification, which is the same as the classical cone. But since there is a long approximately cylindrical outer segment connected to a cone-like remainder this must be regarded as a type B receptor (PEDLER,1965a, b 1969). The sclerad quarter of the outer segments are loosely embedded in processes arising from the pigment epithelium and the inner or ~itrad ends are joined to the inner segments by a narrowed area containing typical cilia1 apparatus. No paraboloids or oil droplets were seen in either variety of receptor. The cell bodies of the receptors are arranged in columns and the extended myoids and conducting hbres lie in closely bound groups between them (Figs. 4 and 14). Some of these groups are penetrated by elements from the main radial fibre system and others are arranged in membrane-to-memb~ne contact, surrounded by radial fibre material. The general mo~hoIogy of both the rod-like and the cone-like synaptic pedicles is generally similar to those of other vertebrate retinae which we have studied. But the cone pedicles have particularly extensive basal filaments (Fig. 6) which show in Golgi preparations as a profuse knot of processes arising from a wedge-shaped pedicle. Both varieties of pedicle also show complex, deeply infolded connections with elements of other neurones, which are unusual in that many of these contain synaptic vesicles on what would be conventionally regarded as the post-synaptic side of the cleft (Fig. 11). Since the outer nuclear layer is folded into the inner nuclear, we examined the inner plexiform layer to see whether any photo-receptor synapses could be found. There were in fact, occasional, simple, or rod-like synapses, but none of the complex or cone variety were found, Thus, this retina again appears to be unique in that photo-receptor synapses occur in the same plane as bipolar-ganglion cell connections. Also, due to the folded layer of receptor cell bodies the remaining parts of the outer plexiform layer are divided up into islands of tissue separated by the papillae. It can be seen in Fig. 11 that the “ribbons” in the rod-like synapses are particularly kmg. They are, in fact, sufficiently large just to resolve in the light microscope. The horizontal cells These are found in the outermost zone of the inner nuclear layer and their cell bodies are most easily distinguishable electron microscopically after fixation in gluteraldehyde and post-fixation by osmium tetroxide. After this treatment, the cytoplasm appears clear and relatively empty, as compared with that of the bipolar cells where there is a narrower region of much denser cytoplasm surrounding the nucleus. The processes of the horizontal cells are unusually profuse and can be recognised in
The Retina of a Fruit Bat
917
electron micrographs by their large size, relative to what are, presumably, bipolar cell processes in the outer plexiform layer. The cytoplasm of these processes is similar to that of the cell bodies and contains groups of mitochondria. In specimens fixed as for electron microscopy in gluteraldehyde and osmium tetroxide and then subjected to Golgi imprecation and examined in the light microscope, the lateral extent and density of the processes is unusual. Collectively, they form a densely interwoven network of terminally branching processes each one ending in one or a number of slightly expanded bulbs. The overlap of adjacent dendritic fields is large, and in some cases appears to be as much as eighty per cent when viewed in vertical section. The most interesting finding, however, is that the densest aggregations of horizontal cell processes are found extending over and around the apices of the papillae (Fig. 9). Aggregations do occur in the region between the papillae, but these are less dense and do not show such a pronounced degree of dendritic overlap, In addition to the dendritic complexes mentioned, the horizontal cells also give rise to ‘“axons” with terminal arborisations extending tangentially for as far as 4&t. The arborisations also show terminal bulbs. Since receptor synapses occur in the inner plexiform layer one might expect to find horizontal cell processes there as well. This probably does occur, but we were unable positively to identify the processes from these cells because the processes of other cells in this region show an almost identical appearance to those of the horizontal ceils. In one section, a synapse between a horizontal cell body and a horizontal cell process was found. Ribbon synapses are usual in the pedicles of the photo-receptors and have been reported in the inner plexiform layer (KIDD, 1962), but this is the first we have seen between horizontal cells. There is thickening and increased density of both membranes, well marked periodic structures in the inter-synaptic cleft and a localised aggregation of vesicles around the double ribbons. No similar specialisation was found elsewhere in the two cells. The bipolar cells
These are few in number compared with the photo-receptors, but similar in structure to those of other species. They are arranged as a thin closely packed layer fenestrated by the apical projections of the outer nuclear layer (Fig. 7). The cell bodies are frequently in membrane-to-membrane contact, but where they are not, they are separated by unusually thin tangential processes arising from the radial fibres. In other retinae, it is common to find the axons and dendrites of the bipolar cells enclosed by a cuff of radial fibre material, but in this species, the processes of the bipolar cells lie in indentations or partly closed tunnels formed in the surface of other bipolar cell bodies. Specimens impregnated by the Golgi technique show that the axons develop typical multi-~yered arborisations and clubs extending tangentially at different levels of the inner plexiform layer. The amacrine cells, the ganglion cells and the radial.fibres
Very few amacrine cells were identified with certainty. None were recognised by the light-microscope, either in toluidine blue or Golgi stained preparations. However, occasional cells were seen on the inner aspect of the inner nuclear layer, giving rise to broad single processes extending into the inner plexiform layer. These cells possess more cytoplasm than the bipolars, and also contain a number of mitochond~a and large vesicles in the region of the cell next to the origin of the main process. No terminal arborisations were identified el~tron-microscopically.
918
<'.fkDt,Eit AND R. TILL.EY
The ganglion cells are again relatively less frequent than the bipolar cells. For example, in a single vertical section, a light microscopic field including three papillae may show four to six ganglion cells only. The cellbodies are large and unusually close to the inner limiting membrane from which they are separated by broad plaques of radial fibre material (Fig. 4) which fill in the space left by the small numbers of ganglion cells and also enclose localised groups Of very small unmyelinated nerve fibres lying between the ganglion cell bodies and the inner limiting membrane. The normally straight course of the radial hbres between inner and outer limiting membranes is not significantly altered by the presence of the papillae. In the outer nuclear layer, the main trunks of the fibres are expanded to enclose the groups of conducting fibres referred to above (Fig, 14) and also to divide the cell bodies of the photo-receptors into columns (Fig. 4). In this region also, the radial fibres contain a high concentration of mito~hondria as compared to other species we have studied. Arising from the inner limiting membrane, there are unusually dense aggregations of prolonged radial fibre trunks close to the innermost extent of the penetrating choroidal vessels. In the light microscope, the radial fibre material adjacent to the inner limiting membrane appears almost as a separate layer, except for the prolongation near the vessels. The nuclei of the radial fibres lie in triangular expansions of the main trunks on the innermost side of the inner nuclear layer (Fig. 4). This species has two varieties of photo-receptor which, considered together, are considerably more numerous than the bipolar cells. Similarly there is a refative surplus of bipolar cells when compared with the sparsely populated ganglion cell layer. Thus the retina is convergent in general arrangement, which is consistent with the predominantl~~ nocturnal habit of the animal. But we have also found cells possessing many of the structural features of cones. These are found only at the base of the papillae and are very uncommon. The estimate, mentioned above, of one in every two hundred and fifty is only approximate, since the folding of the receptor layer makes it difficult to measure accurately the relative cell counts in a single light microscopic field. However, the fact that this second receptor type is uncommon, suggests that it may not play a very significant part in the total visual capacity of the animal. The profusion of basal filaments arising from the synaptic pedicle of this receptor variety is, however, unusual. But, this cannot be related to the function of the cell, until more is known about the connectivity of the filaments. The apparently uneven distribution of the horizontal cell groups in Colgi preparations is of interest and this may well indicate functional differences between the apical and basal regions of the papillae. But this possibility must also be considered with caution, because of the capricious effects of the Golgi method. Since this is well known to pick out localised groups of cells in other, normally arranged, retinas for no defined reason, the apparent concentration of the horizontal cells and other processes over the apex of some papillae could be due to this peculiarity of the silver impregnation method rather than any essential functional differences. Considerable attention has been given to the papillae and their possible significance to the vision of the animal. KOLMER (1911) discusses this point at length and points out that none of the species he examined (not including the one we were able to study) showed any sign of accommodative musculature, and therefore the eye could be compared to a “snapshot” camera containing a lens of fixed focal length. He goes on to suggest that the papillae could be a means whereby a sharp image is focussed on the outer segments of the photo-
The Retina of a Fruit Bat
919
receptors, which are arranged so that the sharpness could register at a number of different Iev& in the retina, Although he m$kes the point that he is suspicious of such teleological argument, he concludes that apart from the metabolic significance of the penetrating vessels that this is the only visually significant reason for the presence of the papillae. This conchsion is supported by Walls, who in comparing the arrangement in the fruit bat with the ramp retina of a ray (~uja batis), agrees that the papillae could provide a broad region of focus for a fixed lens (WALLS, 1963). ROCHON-DUVIGNEAUD (1943) however, showed that in one genus, (Epomoghorus), measurements taken from whole eye sections revealed a hypermetropia of approximately 15 dioptres, a figure which he stated is modified to take account of possible posterior lens dislocation and changes in shape during histological preparation. If this is the general case among the macrochiropterans, then as Rochon-Duvigneaud points out, it is difficult to accept the hypothesis erected by Kolmer, and supported by Walls. For there would be no sharp image in the plane of the receptors at all. There could well be blurred images of contrast and colour difference, but throughout the extent of the papillae, there would be no plane of sharp focus, It is therefore possible to conclude that the papillae would not provide a real alternative to accommodation, provided that the dioptric measurements made by Rochon-Duvigneaud are generally true for other species. A further hypothesis was put forward and then rejected by Rochon-Duvigneaud. He suggested that the choroidal papillae would have the effect of increasing the total number of visual cells per unit retinal area since the total, corrugated, length of the outer limiting membrane would allow more receptors to be packed into a particular region. But he then goes on to say that, as a result of his observations in E~~~o~~~r~s,if the number of receptors around a particular papilla are considered as lying flat over the area covered by the base of the papilla they would fit into the area without overlap. He concludes therefore that this ides must be abandoned. In our study, we repeated this observation on material fixed as for electron microscopy, stained with toluidine blue and examined in the light microscope. In several vertical sections cut through the apex of the papillae, we attempted to estimate whether the receptors lying around the papillae would indeed fit over the area occupied by its base. Taking the width of each inner segment as it passes through the outer limiting membrane and counting the number of inner segments around the papillae, we then projected the same number of inner segments on to the choroidal base of the papillae. By this procedure we found that approximately eighty percent of the inner segments would fail to fit into the base. A proportion which would be much larger if the total areas covered by the receptors and the papillae were to be considered. We were therefore able to conclude that, in the species studied, the choroido-retinal architecture would allow a very much larger number of photoreceptors to be packed into a given retinal area. We also noted, in sections of the whole eye that the outer segments all lie in a direction approximately parallel to the long axis of the papillae and that the papillae all point approximately to the posterior nodal point. Thus, it appears that most if not all the outer segments could well be involved in the reception of light. But it is necessary to account for the difference between Rochon-Duvigneaud’s conclusion and our own. This may well be due to species difference, but, in addition, material fixed for electron microscopy and viewed in the light microscope, offers a much better basis for measurement than does the material prepared in the classical manner. FRITSCH (1911) compared the choroidal array to the pecten of the bird, first from the
920
C. PEDLER AND
R. TILLEY
point of view of its nutritive function and secondly as a means of casting retinal shadows. He pointed out that one hypothesis of pecten function is to act as a sundial or shadowcaster, thus giving the bird a means of continuous attitude discrimination. But he goes on to reject the idea since he also found that the papillae were directed towards the nodal point and thus would not be likely to cast any shadows. There seems little doubt that an important function of the choroidal papillae is to provide retinal nutrition. We found no other evidence of intra-retinal vasculature, nor did we see any structure analogous to the pecten of the bird, or the conus papillaris of the reptile. We were also able to confirm Kolmer’s observations in other species, that there are no central vessels but only a rete of vessels surrounding the periphery of the optic nerve. Thus, the papillae allow a greater concentration of outer segments in a given area, as well as subserving a metabolic nutritive role, but there is one additional finding that complicates the assumption that the papillae are of significance to the animal’s vision. That is the presence and architecture of the retinal pigment epithelium. This layer follows the contour of the penetrating vessels closely even to the innermost point of penetration. But we have also found that it is this layer which provides the “eyeshine” of the animal. It is therefore, in part, a retinal tapetum. So it is necessary to account for a weakly reflecting layer posterior to the outer segments with its surface raised into pointed projections. We have assumed the spherical bodies found in this layer of cells to be the origin of the reflecting property. And we have also shown that the density per cell of these bodies becomes less as the pigment epithelium penetrates the retina. As the inner layers of the retina are reached the spheres are replaced by a gradually increasing concentration of endoplasmic reticulum. Therefore, it is probable that the reflecting properties of this layer diminish as the layct reaches the inner surface of the retina. Nevertheless at the level of the outer segments. ;I high concentration of spheres is still present, and low power microscopy of whole preparations reveals that the tapetum is still refractile at this level. So now we have to account fol densely packed outer segments adjacent to a highly irregular reflecting layer in an eye which is probably hypermetropic. On this basis it is difficult to see how the animal is able to see anything. For presumably light would scatter among the outer segments from the sides of the papillae covered by the reflecting layer of the pigment epithelium. It may be, of course, that the outer segments only admit light in the axial direction, in which case this problem would not arise. There seems to be little evidence to show what the animal “sees”. During the time we kept our two specimens we were gble to make some crude estimates of their visual capability. We found that in daylight conditions in a cage, the animal hanging upside down would follow a dark object (a gloved finger) against a white background. It would first move its eyes in the direction of movement and then the head would move to restore the eyes to a central position in the head. If the moving target was shaken it appeared to follow more readily. It would also follow a pen torch when moved in dim background illumination and would move away if the torch was moved closer to it. When these tests were repeated with the animal held erect it appeared to perform just as well. We are also able to confirm Kolmer’s observation that both pupils remained small even in dim conditions: they did, however, react normally and briskly to light. KOLMER(1911) also reports that fruit bats have been observed to swoop low over Water and catch fish. We have also found accounts of their flying action, which is apparently rapid and effective in the avoidance of objects, so the creature appears to be able to make effective use of iti visual system, for there is no evidence to show that it posesses the echo-location
The Retina of a Fruit Bat
921
capability of some micro~hiropterans. So how does the animal make use of its unique retina and hypermetropic eye in successful aerial navigation? The following hypothesis is possible. &suming that there is some significance in the preponderance of horizontal and bipolar cell connections at the apices of the papillae and that this is not merely a vagary of the Golgi technique, then it could be that the receptors surrounding the papillae are preferentially connected in convergent array to a particular afferent visual path. Given the presence of a refractile spike of tapetum in the centre of the grouped receptors, it would be possible to look at this group of receptors as, perhaps, a giant receptor generating large bursts of activity as out of focus changes in contrast pass over it. Such an arrangement might well give some fairly rapid control of attitude over and above any pattern recognition properties, which the receptor matrix as a whole, might possess. It would be of particular interest to see whether neuro-physiological study of this retina revealed any unusual electrical response to moving stimuli, but until this is done it is perhaps better to conclude by quoting an ancient recipe: “minced bat’s brain and molasses, when taken internally, is good for dim vision”. Acknowledgement-The electron microscope used in this study was provided by the Wellcome Trust. We are also grateful to Miss M. BOYLEfor valuable technical assistance and to the British Museum of Natural History for species identification. REFERENCES DARTNALL,H. J. A., ARDEN,G. B., IKEDA,H., LUCK,C. P., ROSENBERG, M. E., PEDLER,C. M. H. and TANSLEY, K. (1965). Anatomical, eiectrophysiological and pigmentary’aspects of vision in the Bush Baby: an interpretative study. Vision Res. 5,399-424. FRITSCH,G. (1911). Contributions to the histology of the Pteropus eye. Z. Zool. 98, 288. JOHNSON,L. (1927). Contributions to the comparative anatomy of the reptilian and amphibian eye, chiefly based on ophthaimologic~ examination. Phil. Trans. 8.215,31.5. KIDD, M. (1962). Electron microscopy of the inner plexiform layer in the cat and the pigeon. J. Anat. %, 1799187. KOLMER,W. (1910). Contribution to the study of the eye of macrochiroptera. Centrnlbkztt Physiol. 256. KOLMER,W. (191 I). The problem of the anatomy of the eye of macrochiroptera. 2. wiss zool. Bd. 97, Heftl. 91. KOLMER,W. (1926). On the eye of the bat. Verhandl. Zoo/. Bot. Ges. Wien 74,29-31. PEDLER,C. (1963). The fine structure of the Tapetum Cellulosum. Exptl Eye Res. 2, 189-195. PEDLER,C. (1965). Rods and cones, a fresh approach. Proceedings, CIBA Symposium on Physiology and Psychology of Colour Vision, J. and A. Churchill, London. PEDLER, C. (1965a). Rods and cones, a new approach. Biochemistry of the Retina, Academic Press, London and New York. PEDLER,C. (1969). The rod and the cone. fnt. Rev. Zoo/., in press. REYNOLDS, E. C. (1963). The use of lead citrate at high pH as an electron opaque stain. J. Cell Biof. 1?,208-212. ROCHON-DUVIGNEAUD, (1943). The Eyes and the Vision of‘ Vertebrates, p. 563, Masson (Paris) Glover. SABATINI,D. D., BENSCH,K. and BARRNETT,R. J. (1963). The preservation of cellular ultrastructure by aldehyde fixation. J. Cell Biol. 17, 19-58. WALLS,G. (1963). The Vertebrate Eye. Hafner, New York.
Abstract-The light and electron microscope architecture of the retina of a fruit bat (Pteropus gigunteus Brtinnich) is described. The relationship between choroidal vessels penetrating the retina and the retinal substance is also described and related to the uniquely papillated structure of the organ. The functional significance of the papillation is discussed and related to the views of other workers. It is concluded that the papillation has, apart from a metabolic role, a visual function in that it allows a greater number of outer segments to be packed together in a given area of retina.
922
R&un-On decrit, en microscopic optique et electronique, I’architecture de la r&tine de la chauvesouris Pmropus gigunreus Briinnich. On d&it aussi la relation entre les vaisseaux choroidiens qui pen&rent dans la r&tine et la substance r&nienne, en liaison avec la structure papWe trts speciale de cet organe. On discute la signification fonctionnelle de cette structure, en relation avec les vues d’autres auteurs. On con&t que, outre son role metabolique, la papillation posstde une fonction visuelle en permettant d un plus grand nombre de segments externes d’etre accumules dans une aire donnee de la retline.
Zusammenfassung-Es wird die licht- und elektronenmikroskopische
Struktur der Netzhaut des Flederhundes (Pteropus gigunteus Brtinnich) beschrieben. Das Verhaltnis der die Netzhaut durchstossenden Aderhautgefasse zur Netzhaut selbst wird such beschrieben und mit der einzigartigen Papilhetung des Organes verghchen. Die funktionelle Bedeutung der Papillierung wird such insofem sie die Ansichten anderer Forscher anbeiangt besprochen. Es wird geschlossen, dass die Papillierung nicht nur eine N~hrungsrol~e erfiilit sondem such dem Sehen dient, da sic es erlaubt eine grijssere Anzahl Aussenglieder in einer gegebenen Netzhaut~~che unterzubringen.
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