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1 From the Department of Morphology and Cellular Biology, University of Oviedo, Spain; the 2 Department of Biochemistry, NCMLS, University of Nijmegen, The Netherlands; the 3 National Institute of Health and Medical Research, Lyon, France; and the 4 Institute of Evolution, University of Haifa, Israel.
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METHODS. The eyes of eight adult animals were analyzed by light and electron microscopy, using conventional staining and immunolabeling with antibodies against phototransduction proteins and calretinin.
RESULTS. Rods accounted for most of the photoreceptor cells in the Spalax retina, although their morphology is dissimilar to that of sighted mammals, in that they contained only rudimentary outer segments. The latter showed strong rod-opsin and transducin immunoreactions. The phagosomes in the retinal pigmentary epithelium were also rod-opsin positive. Synapses were evident at the photoreceptor cells pedicles. Occasionally, several synaptic active sites were present, suggesting cone cell origin; however, cone-opsin was not immunodetected in the study samples. Synaptic ribbon fields, sometimes distant to the active sites, resembled those found in the vertebrate pineal. The other retinal layers were somewhat less organized than in sighted mammals. Some cells were displaced and the calretinin-positive inner plexiform layer had no sublayers. Calretinin immunolabeling was found in horizontal, amacrine, and ganglion cells. Folding of the retina produced rosette-like images similar to those reported before in the retina of nocturnal mammals and in the avian pineal gland.
CONCLUSIONS. These data suggest that the retina of the mole rat has undergone evolutionary restructuring to a photoreceptive pineal-like organization. This supports the thesis that the photoreceptor cells of this unique organ have been reprogrammed during the subterranean adaptation of Spalax, from their original visual function to mediating photoperiodic regulation.
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Introduction |
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Of all mammals, the subterranean mole rat, Spalax ehrenbergi, is reported to possess the most rudimentary eyes. Although ocular development initially proceeds normally, soon the lens starts to degenerate and the eye remains subcutaneous. Only the retina appears to mature unperturbed.6 In addition to the eyes, the optic nerve regresses.7 Retinal projections to visual nuclei, although present, have been observed to be severely atrophied.8 Nonetheless, preservation of a functional photoreceptor role of the retina is indicated by the persistence of a significant projection from the retina, innervating a well-developed suprachiasmatic nucleus (SCN). In fact, light exposure during the dark phase induces c-fos expression in the ventral region of the SCN, where retinal afferents terminate.9 In agreement with this, entrainment of circadian locomotor and thermoregulatory rhythms by ambient light has also been demonstrated. The removal of the eyes leads to a failure to adapt to these photoperiodic stimuli.10 This suggests that this animal has retained a mechanism for photoperiodic perception and circadian regulation. The genes Clock and MOP3, involved in circadian rhythmicity, were recently cloned and sequenced and their expression studied in Spalax.11 Given the degenerate lens and the location of the eyes, completely buried beneath the skin and embedded within a hypertrophied harderian gland, it is highly unlikely that the eye can still process visual image information. Indeed, morphologic structures involved in image analysis and visually guided behavior are reduced in size by more than 90% and show a poorly differentiated cytoarchitecture.7 Behavioral and electrophysiological studies have confirmed that Spalax has no image-forming ability and is visually blind.12 This species thus constitutes an extremely interesting naturally blind animal model for the study of the input of circadian information into the central nervous system.
Meanwhile, extensive data have become available on the functional properties of Spalax visual opsins13 14 and of the Spalax visual system,7 12 but only scattered data are available on the morphologic aspects of this very interesting retina at an ultrastructural level.6
Herein, we present a comprehensive morphologic study of the Spalax retina, which fully supports a functional photoreceptor role. Furthermore, we compare this unique organ to other vertebrate photosensitive organs in an attempt to correlate its structural organization with its presumed physiological role.
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All experiments were approved by the local committees for Animal Care and Use and conformed to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research.
The immunohistochemical (IHC) study of the samples was performed by both light microscopy (LM) and electron microscopy (EM). The following antisera were used (polyclonal except where indicated): (1) rho 4D2, a monoclonal antibody against purified rod outer segments of rat, which is monospecific for rod photoreceptors in mammalian species16
; (2) CERN-886 and CERN-JS858, which were raised against bovine rod rhodopsin; (3) CERN-906, which was elicited against chicken red and green cone opsins; (4) CERN-9412, which labels the 
-subunit of the bovine rod transducin.
The CERN antibodies were developed in rabbits at the Department of Biochemistry, NCMLS, University of Nijmegen (The Netherlands), and their specificity was demonstrated previously.1 14 17 18 19
Besides the listed antisera against phototransductory proteins, anti-calretinin (SWant Antibodies, Bellinzona, Switzerland), raised against the calcium-binding protein calretinin was used. This antibody has been shown to be a good marker of amacrine cells and their processes, which reach into different areas in the inner plexiform layer (IPL) in the mouse.20 It was used to visualize the distribution of this cell type and also to probe the organization of the IPL.
For the IHC study at the LM level, samples fixed in Bouin fixative or 4% paraformaldehyde for 48 hours at 4°C, were dehydrated in ethanol and embedded in paraffin. Four-micrometer-thick sections were obtained with an ultramicrotome (Ultracut; Reichert-Jung, Vienna, Austria), using glass knives, and collected in double-gelatinized glass slides. After deparaffination in xylene, sections were rehydrated gradually in ethanol and once in distilled water, and single labeling with the antisera (dilution 1:2000–1:3000) was performed, using a peroxidase-conjugated avidin-biotin complex staining kit (Vectastain; Vector Laboratories, Burlingame, CA) as a detection system. Labeled sections were dehydrated in ethanol, cleared in xylene, mounted in a hydrophobic medium, and examined at the LM level.
For the IHC study at the EM level, samples were fixed in a phosphate-buffered (0.1 M, pH 7.3) 4% paraformaldehyde+0.25% glutaraldehyde solution for 2 hours at room temperature, washed in distilled water, dehydrated in ethanol and embedded in acrylic resin (LRWhite; London Resin Co., Basingstoke, UK). Ultrathin (approximately 80-nm thick) middle cross sections of the eyes, were obtained with the ultramicrotome, by using a diamond knife, and mounted on nickel grids. The primary antibodies (final concentration, 1:1000) were CERN-886, CERN-JS858, and CERN-9412. Afterward, grids were processed according to a standard immunogold protocol,21 using 10 nm gold particles bound to rabbit IgG as a secondary antibody, counterstained with uranyl acetate, examined, and photographed in a transmission electron microscope (TEM; model EM 109; Carl Zeiss, Oberkochen, Germany).
To label possible apoptotic nuclei, the in situ terminal transferase-mediated nick end labeling (TUNEL) technique (commercial kit; Roche Molecular Biochemicals, Mannheim, Germany) was used on 4-µm-thick paraffin sections.22 In addition, the semithin and ultrathin sections were screened for the presence of apoptotic nuclei according to standard morphologic criteria.23
For morphologic studies at the LM and EM level, samples were fixed in a phosphate-buffered (0.1 M, pH 7.3) 3% paraformaldehyde+2.5% glutaraldehyde solution for 2 hours at room temperature, postfixed in 1% OsO4 at 4°C for 2 hours, washed in distilled water, dehydrated in acetone, and embedded in an Araldite-based resin (Durcupan; Fluka, Buchs, Switzerland). Semithin (1-µm thick) and ultrathin (approximately 80-nm thick) sections were obtained in the ultramicrotome, using glass and diamond knives, respectively. Semithin sections were stained with toluidine blue solution and examined at the LM level. Ultrathin sections, mounted on copper grids and stained with lead citrate and uranyl acetate, were viewed and photographed in the TEM.
LM images were recorded with a digital camera mounted on a microscope (camera model DMC; Polaroid, Cambridge, MA; microscope model Eclipse E400; Nikon, Tokyo, Japan). TEM photographic negative films were digitized using a scanner (SprintScan 35 Plus; Polaroid). The figures selected were arranged in plates using image management software (Corel Draw ver. 8.0; Corel Corp., Ottawa, Ontario, Canada).
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The layer between the outer limiting membrane (OLM) and the RPE, which we will refer to as the outer neural retina (ONR; Fig. 4 ), showed normal inner segments (IS) of the photoreceptor cells (PCs); however, at the LM level, the outer segments (OS) seemed to be reduced or missing.
In the semithin sections, several different types of nuclei, corresponding to different cell types that will be described later, were distinguished in the inner nuclear layer (INL; Fig. 4 ). Furthermore, a cell type with metachromatic cytoplasm was present throughout the entire retina, but was more abundant in the INL and OPL (Fig. 4) . Sometimes these cells were also observed in the ONL.
At the EM level, the ONR showed signs of abnormal photoreceptor morphology (Fig. 5)
. The OLM was clearly distinguishable, the PCs had well preserved IS (Figs. 5
6
and 7)
, but did not contain normal OS. Only whorls of membranes that resembled rudimentary OS were visible (Fig. 5)
. Occasionally, these rudimentary elements were noted to be connected to the cilia (Fig 6
, c). Rod-opsin–like immunoreactivity (IR) was abundant in these rudimentary OS, but was also detected in the plasma membrane of the cilia and the IS (Fig. 6)
. Whorls of membranes, hereafter referred to as myeloid bodies, also were localized within the IS and within other parts of the photoreceptor cells, but these bodies did not show any rod IR. Cisterns of rough endoplasmic reticulum (rER) and many mitochondria were found in the IS as well (Fig. 7) . These rER cisterns did not show any rod-opsin IR, either (Fig 8)
, whereas such IR was observed throughout the plasma membrane of the PCs (Fig. 9)
. The antibody CERN-9412 (anti-rod transducin -subunit) at the LM level labeled only a narrow area under the RPE, corresponding to the photoreceptor outer segments (not shown). In the EM images, CERN-9412 reactivity was detected in the whorls of membranes of the rudimentary OS (Fig. 10)
, but labeling of the plasma membrane of the PC was not observed. The RPE contained phagosomes that also showed rod-opsin labeling (Fig. 11)
.
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Although synaptic structures, such as groups of ribbons and spherules surrounded by vesicles of electrolucent content were occasionally observed in places as distant as the IS of the PCs (Fig. 15) , most of these structures were seen in subnuclear locations, either separated or close to the synaptic active sites (Fig. 16) . Myeloid bodies, such as were described in the IS, also were common features of the subnuclear cytoplasm (Fig. 14) .
The OPL, which mainly represents photoreceptor endings and cellular processes of the INL, was rather well developed. Numerous synaptic contacts were observed, and photoreceptor cell pedicles occasionally showed a complex organization, with several synaptic active sites (Fig. 17) . Rod-opsin–like immunolabeling extended to the plasma membrane of most of the pedicles (Fig. 18) , although with lower density than in the OS. Indeed, with all three antibodies used against rod-opsin, labeling over the entire ONL was found in LM sections, confirming the EM observations.
Some cells showing wide cytoplasmic areas without organellae, corresponding to the metachromatic cells observed in semithin sections, were seen dispersed through both the ONL and INL (Fig. 19) and occasionally in the ganglion cell layer (GCL). Their nuclei, which were round and heterochromatic, resembled those of PCs. Furthermore, synaptic structures (Figs. 19 19a) and single cilia (Fig. 20) were also present in these cells.
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The anti-calretinin antibody labeled several cellular somata present in both the inner and the outer margins of the INL, corresponding to a subpopulation of horizontal and amacrine cells, respectively, and also in the GCL (Fig. 23) . The OPL and IPL were labeled as well, but no sublayers were seen in the IPL (Fig. 23) , such as those observed in other rodents.
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At the EM level, the INL presented differentiated cell types (Fig. 24) . Müller cells displayed round or oval dense nuclei; remarkably, some of these nuclei were displaced toward the ONL. We attempted to classify the remainder of the nuclei mainly by their position in the INL. Nuclei located in the horizontal cell sublayer, close to the OPL, were round and less electrodense than those of Müller cells (Fig. 24) . The amacrine cell sublayer was occupied by cells with large, round electrolucent nuclei, with prominent nucleoli, showing arborization of the cytoplasm toward the IPL (Figs. 24 25) . In the middle region of the INL, nuclei with characteristics similar to the amacrine nuclei were observed that probably correspond to bipolar cells (Fig. 24) . Processes of bipolar cells could be seen penetrating the IPL and exhibiting diverse types of synapses, some of them with ribbonlike, dense structures (Figs. 25 25a) . These bipolar processes also received input, the so-called reciprocal synapses, from amacrine cells (Fig. 25a) . Some axosomatic synapses were seen on amacrine cells (Fig. 26) . In the IPL, which was rather well developed, other types of synapses were observed. Of the two main types of synaptic vesicles distinguished, most had electrolucent contents, but a minority displayed electrodense contents (Fig. 26) .
In the GCL, two main types of cells were observed (Fig. 27) . Type I ganglion cells displayed rounded nuclei of large size, approximately 8 µm in diameter, and reduced heterochromatic component. The nucleus of type II cells, approximately 5 µm in diameter, presented invaginations of the nuclear envelope and more heterochromatin than that of type I. Nucleoli were prominent in both types. In the type II cells, the nucleus and the entire soma were smaller than in type I cells. Many rER cisterns, a well-developed Golgi apparatus, and numerous mitochondria were observed in type I cells (Fig. 28) . Some synapses were detected in the ganglion cell somata (Figs. 29 29a) . Occasionally, ganglion cells did not form a single stratified row, but appeared to be organized in a column invading the IPL (Fig. 30) .
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The retina of the mole rat showed a quite normal organization. All the layers and cell types of the mammalian retina were detectable, with the exception of cone cells, whose presence, despite some circumstantial evidence, could not yet be directly demonstrated.
The observed morphology of the PCs did not correspond to the classic rod or cone cells, because the OS did not display the normal stacked organization, but were rudimentary and composed of whorls of membranes. These are quite similar to those reported in the mole retina26 and, particularly remarkable, in the OS of the avian pinealocytes.27 28 29 Although in most avian species, pinealocytes are not connected to second-order neurons, some exceptions occur, such as passerine birds, in which these neurons are abundant.27
In sighted rodents, such as the mouse, rod and cone nuclei show some differential ultrastructural features, which allow them to be distinguished.30 In Spalax, the nuclei observed in the ONL all had a similar appearance and the rod-opsin immunolabeling indicated that in our samples the majority, if not all, of the cells in this layer were rods. In fact, we did not detect immunolabeling in Spalax with CERN-906, an antibody that is a good marker for the green cone cells in other rodents, such as mouse.31 Our antibodies have been shown before to be monospecific for these Spalax phototransduction-related proteins as well.14 32 This does not necessarily imply, of course, that cone cells are entirely absent in the mole rat retina. In fact, David-Gray et al.13 32 have provided evidence that the mole rat retina expresses a green-cone–like opsin, which is one of the most "red-shifted" photopigments found in rodents. However, the location of this opsin has not been established yet, and it need not originate in cone cells.
The PC pedicles in the mole rat retina did not exhibit the degree of organization reported in most sighted mammals. The presence of ribbon fields, sometimes in regions distant from the synaptic site, the so called "active zone",33 suggests a storage of part of the synaptic machinery. Similar synaptic ribbon plasticity has also been observed in the retina of hibernating squirrels,34 and, in fact, in the pineal of most mammals, as well.35 The mammalian rod cell normally uses a single active zone at its pedicle.36 It might thus be suspected that some photoreceptor endings in the mole rat retina exhibiting multiple synaptic sites correspond to cone cells. Actually, images of other mammalian cone terminals reported in the literature37 show a much more complex organization. Some investigators38 39 have reported that cone pedicles have approximately 20 active zones, many more than the examples observed in our samples. However, the presence of calretinin-positive horizontal cells in the Spalax retina (see later discussion) may also reflect the presence of cone-cells, because the three types of horizontal cells described in sighted mammals all interconnect cone pedicles, either with other cone cells or with rod cell spherules.40 Hence, the question of whether cone cells persist in Spalax is still open and requires more specific cone-directed assays.
Even though that some displaced cells were found, the Spalax retina displayed a stratified organization similar to that reported for sighted vertebrates. Although calretinin-positive horizontal cells have also been found in other species, including a rodent such as the rat,41 the calretinin-positive distribution found in Spalax strongly resembles the one identified in the mouse with anti-calbindin antibodies.20
The lamination of the IPL reported in other mammals20 was absent in Spalax, and this indicates that the inner layers of its retina have a lower degree of organization than that observed in sighted animals. Nevertheless, as indicated by morphology and the immunolabeling at the LM and the EM levels, amacrine, bipolar, and ganglion cells were well represented, and synaptic contacts among them were well established. Calretinin-immunopositive amacrine cells, which are the so called "AII" or "rod amacrine cells" in other mammals,42 43 seemed to represent most of the amacrine population in Spalax. The pattern of synaptic contacts in the IPL was diverse and not easy to interpret in ultrastructural images without the use of any type of labeling, which is outside the context of the present study. It is widely accepted, however, that ribbonlike densifications in the IPL belong exclusively to bipolar cells. To distinguish cone and rod bipolar cells by their ultrastructure or the position of their terminals in the IPL is problematic, because these aspects may vary, depending on the species. Taking this into account, the images we present could correspond to cone as well as rod bipolar cells. To differentiate at the EM level between AII amacrine cells and other types at the EM level is not easy, either. In other mammals, AII cells receive input from rod bipolar cells, but they usually make chemical output synapses with cone bipolar cells and ganglion cells. Considering that the population of cone bipolar cells, if present, would be a small minority, we present the most likely explanation, in our opinion, for the synaptic contacts observed between bipolar and amacrine cells in Figures 25 and 26 .
The cell type showing organellae-free cytoplasmic areas described in our results undoubtedly had the characteristics of the rod cells described in the ONL, with the exception that no OS was present. It is not clear presently why they occupy such a displaced position in the retina. Rod opsin IR and also the presence of established synapses suggest that these cells may have retained some kind of photoreceptor function.
Taking into account all these considerations, we conclude that the retina of Spalax presents the morphology of a functional organ, with no evidence of a severe differentiation-related degenerative process. In addition, in view of the absence of apoptotic cells in our samples, which were taken from adult individuals, there is no evidence for a slow aging-connected degenerative process, as is demonstrated, for instance, in the rds mouse, which also displays rudimentary OS.44
Strong evidence for a photosensory function of the Spalax retina was provided by the presence of intact synaptic organization throughout the retinal layers. This was further supported by the abundant presence of PC pedicles and strong rod-opsin and transducin immunolabeling. In addition, as Janssen et al.14 have demonstrated, the Spalax rod visual pigment is fully functional, and light stimulation triggers c-fos expression in the SCN.9 Also, rod-opsin–positive phagosomes are detectable in the RPE, demonstrating that the OS undergo their typical turnover process.
Although the strongest rod-opsin labeling was observed in the rudimentary OS, the entire cell membrane exhibited a positive reaction. This could indicate that the entire PC has become photoactive, complying with the loss of pressure to maintain proper image-detection. In fact, most of the light reaching the eye of the mole rat probably enters laterally through an unpigmented area of the hairy skin12
and not frontally, because of the strong filtering effect of the pigmentary cap. In addition, the folds observed in the Spalax retina, quite similar to those found in the avian pineal29
and in the retina of megachiroptean bats,45
would no longer interfere with image resolution. We confirm the LM observation of Janssen et al.14
that -transducin IR is restricted to the rudimentary OS at the TEM level. This may restrict a photosensory function to the OS, but we cannot exclude that the putatively lower levels of transducin near the plasma membrane may yet have escaped immunodetection. The release of the severe spatial constraints, imposed on rhodopsin in the highly organized disc membranes of the OS in sighted animals, may also explain why Spalax rhodopsin complies with much larger carbohydrate antennae.14
All the mentioned morphologic features indicate that the Spalax retina has adopted features of the pineal of nonmammalian vertebrates and hence suggest strongly, in line with earlier suggestions,12 13 14 30 that it has evolved toward a nonvisual photosensory function. The arguments for this affirmation presented in this manuscript can be summarized as follows.
The subcutaneous location of the Spalax eye and its diffuse light input are similar to the situation faced by the avian pinealocytes. The abnormal OS organization, a likely evolutionary acquisition, is also similar to avian pinealocytes, wherein the function in photoperiodic physiology does not require neatly stacked visual pigment. The synaptic ribbon fields and their plasticity have also been reported in the pinealocytes of mammalian and nonmammalian vertebrates.35 Finally, the retinal folds observed in our samples resemble those found in the avian pineal gland29 and in the retina of some nocturnal mammals.45 In fact, the histologic structure of the pineal organ of some higher vertebrates, such as avians, has been described as a "folded retina."46
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Submitted for publication December 10, 2001; revised March 14, 2002; accepted March 22, 2002.
Commercial relationships policy: N.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked "advertisement" in accordance with 18 U.S.C. 
1734 solely to indicate this fact.
Corresponding author: Rafael Cernuda-Cernuda, Departamento de Morfología y Biología Celular (8a planta), Facultad de Medicina, Universidad de Oviedo, 33071 Oviedo, Spain; rcernuda{at}correo.uniovi.es.
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-B-crystallin of the blind subterranean mole-rat: high homology with sighted mammals Gene 264,45-49[Medline][Order article via Infotrieve]
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) was noted in a frontal view of the eye. However, pigment was absent in the areas surrounding the frontal cap (arrows). Bar, 0.4 mm.
