Mitochondrial function after associating liver partition and portal vein ligation for staged hepatectomy in an experimental model.

BACKGROUND: Associating liver partition and portal vein ligation for staged hepatectomy (ALPPS) is a two-stage strategy to induce rapid regeneration of the remnant liver. The technique has been associated with high mortality and morbidity rates. This study aimed to evaluate mitochondrial function, biogenesis and morphology during ALPPS-induced liver regeneration. METHODS: Male Wistar rats (n = 100) underwent portal vein ligation (PVL) or ALPPS. The animals were killed at 0 h (without operation), and 24, 48, 72 or 168 h after intervention. Regeneration rate and proliferation index were assessed. Mitochondrial oxygen consumption and adenosine 5'-triphosphate (ATP) production were measured. Mitochondrial biogenesis was evaluated by protein level measurements of peroxisome proliferator-activated receptor γ co-activator (PGC) 1-α, nuclear respiratory factor (NRF) 1 and 2, and mitochondrial transcription factor α. Mitochondrial morphology was evaluated by electron microscopy. RESULTS: Regeneration rate and Ki-67 index were significantly raised in the ALPPS group compared with the PVL group (regeneration rate at 168 h: mean(s.d.) 291·2(21·4) versus 245·1(13·8) per cent, P < 0·001; Ki-67 index at 24 h: 86·9(4·6) versus 66·2(4·9) per cent, P < 0·001). In the ALPPS group, mitochondrial function was impaired 48 h after the intervention compared with that in the PVL group (induced ATP production); (complex I: 361·9(72·3) versus 629·7(165·8) nmol per min per mg, P = 0·038; complex II: 517·5(48·8) versus 794·8(170·4) nmol per min per mg, P = 0·044). Markers of mitochondrial biogenesis were significantly lower 48 and 72 h after ALPPS compared with PVL (PGC1-α at 48 h: 0·61-fold decrease, P = 0·045; NRF1 at 48 h: 0·48-fold decrease, P = 0·028). Mitochondrial size decreased significantly after ALPPS (0·26(0·05) versus 0·40(0·07) µm2 ; P = 0·034). CONCLUSION: Impaired mitochondrial function and biogenesis, along with the rapid energy-demanding cell proliferation, may cause hepatocyte dysfunction after ALPPS. Surgical relevance Associating liver partition and portal vein ligation for staged hepatectomy (ALPPS) is a well known surgical strategy that combines liver partition and portal vein ligation. This method induces immense regeneration in the future liver remnant. The rapid volume increase is of benefit for resectability, but the mortality and morbidity rates of ALPPS are strikingly high. Moreover, lagging functional recovery of the remnant liver has been reported recently. In this translational study, ALPPS caused an overwhelming inflammatory response that interfered with the peroxisome proliferator-activated receptor γ co-activator 1-α-coordinated, stress-induced, mitochondrial biogenesis pathway. This resulted in the accumulation of immature and malfunctioning mitochondria in hepatocytes during the early phase of liver regeneration (bioenergetic destabilization). These findings might explain some of the high morbidity if confirmed in patients.

Photopigment coexpression in mammals: comparative and developmental aspects.

In mammals, each cone had been thought to contain only one single type of photopigment. It was not until the early 1990s that photopigment coexpression was reported. In the house mouse, the distribution of color cones shows a characteristic division. Whereas in the upper retinal field the ratio of short wave to middle-to-long wave cones falls in the usual range (1:10), in the ventral retinal field M/L-pigment expression is completely missing. In the transitional zone, numerous dual cones are detectable (spatial coexpression). In other species without retinal division, dual cones appear during development, suggesting that M/L-cones develop from S-cones. Dual elements represent a transitory stage in M/L-cone differentiation that disappear with maturation (transitory coexpression). These two phenomena seem to be mutually exclusive in the species studied so far. In the comparative part of this report the retinal cone distribution of eight rodent species is reported. In two species dual cones appear in adult specimens without retinal division, and dual elements either occupy the dorsal peripheral retina, or make up the entire cone population. This is the first observation proving that all cones of a retina are of dual nature. These species are good models for the study of molecular control of opsin expression and renders them suitable sources of dual cones for investigations on the role and neural connections of this peculiar cone type. In the developmental part, the retinal maturation of other species is examined to test the hypothesis of transitory coexpression. In these species S-pigment expression precedes that of the M/L-pigment, but dual cones are either identified in a small number or they are completely missing from the developing retina. These results exclude a common mechanism for M/L-cone maturation: they either transdifferentiate from S-cones or develop independently.

Nonvisual photoreceptors of the deep brain, pineal organs and retina.

The role of the nonvisual photoreception is to synchronise periodic functions of living organisms to the environmental light periods in order to help survival of various species in different biotopes. In vertebrates, the so-called deep brain (septal and hypothalamic) photoreceptors, the pineal organs (pineal- and parapineal organs, frontal- and parietal eye) and the retina (of the "lateral" eye) are involved in the light-based entrain of endogenous circadian clocks present in various organs. In humans, photoperiodicity was studied in connection with sleep disturbances in shift work, seasonal depression, and in jet-lag of transmeridional travellers. In the present review, experimental and molecular aspects are discussed, focusing on the histological and histochemical basis of the function of nonvisual photoreceptors. We also offer a view about functional changes of these photoreceptors during pre- and postnatal development as well as about its possible evolution. Our scope in some points is different from the generally accepted views on the nonvisual photoreceptive systems. The deep brain photoreceptors are hypothalamic and septal nuclei of the periventricular cerebrospinal fluid (CSF)-contacting neuronal system. Already present in the lancelet and representing the most ancient type of vertebrate nerve cells ("protoneurons"), CSF-contacting neurons are sensory-type cells sitting in the wall of the brain ventricles that send a ciliated dendritic process into the CSF. Various opsins and other members of the phototransduction cascade have been demonstrated in telencephalic and hypothalamic groups of these neurons. In all species examined so far, deep brain photoreceptors play a role in the circadian and circannual regulation of periodic functions. Mainly called pineal "glands" in the last decades, the pineal organs actually represent a differentiated form of encephalic photoreceptors. Supposed to be intra- and extracranially outgrown groups of deep brain photoreceptors, pineal organs also contain neurons and glial elements. Extracranial pineal organs of submammalians are cone-dominated photoreceptors sensitive to different wavelengths of light, while intracranial pineal organs predominantly contain rod-like photoreceptor cells and thus scotopic light receptors. Vitamin B-based light-sensitive cryptochromes localized immunocytochemically in some pineal cells may take part in both the photoreception and the pacemaker function of the pineal organ. In spite of expressing phototransduction cascade molecules and forming outer segment-like cilia in some species, the mammalian pineal is considered by most of the authors as a light-insensitive organ. Expression of phototransduction cascade molecules, predominantly in young animals, is a photoreceptor-like characteristic of pinealocytes in higher vertebrates that may contribute to a light-percepting task in the perinatal entrainment of rhythmic functions. In adult mammals, adrenergic nerves--mediating daily fluctuation of sympathetic activity rather than retinal light information as generally supposed--may sustain circadian periodicity already entrained by light perinatally. Altogether three phases were supposed to exist in pineal entrainment of internal pacemakers: an embryological synchronization by light and in viviparous vertebrates by maternal effects (1); a light-based, postnatal entrainment (2); and in adults, a maintenance of periodicity by daily sympathetic rhythm of the hypothalamus. In addition to its visual function, the lateral eye retina performs a nonvisual task. Nonvisual retinal light perception primarily entrains genetically-determined periodicity, such as rod-cone dominance, EEG rhythms or retinomotor movements. It also influences the suprachiasmatic nucleus, the primary pacemaker of the brain. As neither rods nor cones seem to represent the nonvisual retinal photoreceptors, the presence of additional photoreceptors has been supposed. Cryptochrome 1, a photosensitive molecule identified in retinal nerve cells and in a subpopulation of retinal photoreceptors, is a good candidate for the nonvisual photoreceptor molecule as well as for a member of pacemaker molecules in the retina. When comparing various visual and nonvisual photoreceptors, transitory, "semi visual" (directional) light-perceptive cells can be detected among them, such as those in the parietal eye of reptiles. Measuring diffuse light intensity of the environment, semivisual photoreceptors also possess some directional light perceptive capacity aided by complementary lens-like structures, and screening pigment cells. Semivisual photoreception in aquatic animals may serve for identifying environmental areas of suitable illumination, or in poikilotermic terrestrial species for measuring direct solar irradiation for thermoregulation. As directional photoreceptors were identified among nonvisual light perceptive cells in the lancelet, but eyes are lacking, an early appearance of semivisual function, prior to a visual one (nonvisual --> semivisual --> visual?) in the vertebrate evolution was supposed.

Comparative ultrastructure and cytochemistry of the avian pineal organ.

The breeding of birds is expected to solve problems of nourishment for the growing human population. The function of the pineal organ synchronizing sexual activity and environmental light periods is important for successful reproduction. Comparative morphology of the avian pineal completes data furnished by experiments on some frequently used laboratory animals about the functional organization of the organ. According to comparative histological data, the pineal of vertebrates is originally a double organ (the "third" and the "fourth eye"). One of them often lies extracranially, perceiving direct solar radiation, and the other, located intracranially, is supposed to measure diffuse brightness of the environment. Birds have only a single pineal, presumably originating from the intracranial pineal of lower vertebrates. Developing from the epithalamus, the avian pineal organ histologically seems not to be a simple gland ("pineal gland") but a complex part of the brain composed of various pinealocytes and neurons that are embedded in an ependymal/glial network. In contrast to organs of "directional view" that develop large photoreceptor outer segments (retina, parietal pineal eye of reptiles) in order to decode two-dimensional images of the environment, the "densitometer"-like pineal organ seems to increase their photoreceptor membrane content by multiplying the number of photoreceptor perikarya and developing follicle-like foldings of its wall during evolution ("folded retina"). Photoreceptor membranes of avian pinealocytes can be stained by antibodies against various photoreceptor-specific compounds, among others, opsins, including pineal opsins. Photoreceptors immunoreacting with antibodies to chicken pinopsin were also found in the reptilian pineal organ. Similar to cones and rods representing the first neurons of the retina in the lateral eye, pinealocytes of birds possess an axonal effector process which terminates on the vascular surface of the organ as a neurohormonal ending, or forms ribbon-containing synapses on pineal neurons. Serotonin is detectable immunocytochemically on the granular vesicles accumulated in neurohormonal terminals. Pinealocytic perikarya and axon terminals also bind immunocytochemically recognizable excitatory amino acids. Peripheral autonomic fibers entering the pineal organ through its meningeal cover terminate near blood vessels. Being vasomotor fibers, they presumably regulate the blood supply of the pineal tissue according to the different levels of light-dependent pineal cell activity.

Immunocytochemical demonstration of visual pigments in the degenerate retinal and pineal photoreceptors of the blind cave salamander (Proteus anguinus).

Visual pigments in the regressed eye and pineal of the depigmented neotenic urodele, the blind cave salamander (Proteus anguinus anguinus), were studied by immunocytochemistry with anti-opsin antibodies. The study included light- and electron-microscopic investigations of both the eye and the pineal organ. A comparison was made with the black pigmented subspecies Proteus anguinus parkelj (black proteus), which has a normal eye structure. In the retina of the black proteus, we found principal rods, red-sensitive cones and a third photoreceptor type, which might represent a blue- or UV-sensitive cone. Photoreceptors in the regressed eye of the blind cave salamanders from the Planina cave contained degenerate outer segments, consisting of a few whorled discs and irregular clumps of membranes. The great majority of these outer segments showed immunolabelling for the red-sensitive cone opsin and only a few of them were found to be positive for rhodopsin. An even more pronounced degeneration was observed in the photoreceptors of the animals derived from the Otovec doline, which are completely devoid of an outer segment, most of them not even possessing an inner segment. Even in some of these highly degenerate cells, the presence of rhodopsin could be detected in the plasma membrane; however, immunoreactions with antibodies recognizing cone visual pigment were negative. In the pineals of all studied animals, the degenerate photoreceptor outer segments were recognized exclusively by the antibody against the red-sensitive cone opsin. The presence of immunopositive visual pigments indicates the possibility of a retained light sensitivity in the blind cave salamander photoreceptors.

Cone differentiation with no photopigment coexpression.

PURPOSE: To decide whether the transitory coexpression of cone visual pigments described in the developing rat and gerbil retina is a universal feature of dichromatic mammalian species. METHODS: The rabbit, a species widely used in eye research, was selected for the study and a search made for the presence of cones that bound more than one cone antibody during the first postnatal week. To plot the densities of individual cone types and to colocalize the two visual pigments, immunocytochemistry on retinal wholemounts and consecutive tangential sections, respectively, were used. RESULTS: The sequence in which the visual pigments began to be expressed was the same as that observed in other mammals: first, rhodopsin; second, blue pigment; and last, green pigment. The striking increase in blue cone density numbers observed in the rat, however, did not occur in the rabbit. Instead, some days after the first blue cones appeared, the green cones also started to express their visual pigment, and this cone type soon outnumbered the blue cones. Within the limits of the immunocytochemical method, it was established that unlike the developing rat, the presence of double-labeled cones was not a character of the rabbit retina. CONCLUSIONS: Visual pigment coexpression is an interesting phenomenon of retinal development, however, it is not the exclusive scenario of photoreceptor differentiation. Each species must be carefully studied before deciding whether its retinal cones synthesize both pigments during retinal development.

Photoreceptor cells in the Xenopus retina.

This review summarizes our present state of knowledge about spectrally different photoreceptor cell types in the Xenopus retina. The classification of the photoreceptors was based on morphology, combined with immunolabelling with various anti-visual pigment antibodies and other molecular probes on semithin sections and retinal wholemounts. The majority of photoreceptors is represented by rods. Altogether 97-98% of the total rod population consists of the principal ("red") rods that are selectively labeled by N-terminal specific anti-bovine rhodopsin monoclonal antibodies (mAbs) and are maximally sensitive to green light. The other, rare, blue-sensitive rod type ("green rod") is thinner, not stained by these antibodies but binds C-terminal specific anti-rhodopsin mAbs. The major representatives of the cones are red-sensitive and consist of a morphologically heterogeneous group comprising both (principal and accessory) members of double cones, as well as large single cones. Outer segments in this group are selectively labeled by mAb COS-1, specific to the L/M group of cone visual pigments. Another, relatively rare cone type is similar in size, but slightly smaller than the large single cone and is not labeled by mAb COS-1. This cone type is assumed to have a blue-sensitive cone visual pigment. The third, least abundant, and immunocytochemically distinct cone type is a small single (miniature) cone, which binds mAb OS-2 relatively strongly, and anti-rhodopsin mAbs 4B4 and 1D4 weakly. By exclusion, this small single cone may be identical with the UV-sensitive cone. Further studies are needed, however, to identify the color sensitivity of the latter two cone types.

Degeneration of cone photoreceptors induced by expression of the Mas1 protooncogene.

Although transgenic expression of oncogenes typically leads to tumorigenesis, oncogene expression directed to the rod photoreceptors leads to cell death without tumor formation. To evaluate the cellular and functional changes induced in cone photoreceptors by an oncogene, the Mas1 protooncogene was targeted to the cones of transgenic mice by the human red/green opsin promoter. Mas1 was chosen because of its exclusive expression in the nervous system and its homology to opsin. The overall histologic appearance of the transgenic retina was normal and retinal tumors were never observed. While rod-mediated electroretinograms were normal in all respects, cone-mediated responses were diminished in direct relationship to the level of transgene expression as determined by Northern blot analysis. Responses of UV- and green-sensitive cones were reduced equivalently, and Northern analysis and immunocytochemistry indicated that cone photoreceptor densities were markedly diminished throughout transgenic retinas. These results indicate that oncogene expression in cones induces cell death without tumor formation and support the possibility that aberrant oncogene expression may underlie some forms of hereditary retinal diseases. The Mas1 transgenic mice may be useful in understanding the cone photoreceptor degeneration that occurs in cone dystrophies and age-related macular degeneration and in evaluating potential therapies for these disorders.

Photoreceptor distribution in the retinas of subprimate mammals.

Relevant data on the distribution of color cones are summarized, with special emphasis on the marked dorsoventral asymmetries observed in a number of mammalian species. In addition, an overview is given of studies that demonstrate the coexistence of two visual pigments within the same cone cell. The biological significance of these phenomena is discussed in conjunction with comparative immunocytochemical analyses of subprimate retinas. Based on various cone distribution patterns and temporal and spatial visual pigment coexpression, two models of cone photoreceptor differentiation are suggested.

The pineal organ as a folded retina: immunocytochemical localization of opsins.

The most simple pineal complex (the pineal and parapineal organs of lampreys), consists of saccular evaginations of the diencephalic roof, and has a retina-like structure containing photoreceptor cells and secondary neurons. In more differentiated vertebrates, the successive folding of the pineal wall multiplies the cells and reduces the lumen of the organ, but the pattern of the histological organization remains similar to that of lampreys; therefore, we consider the histological structure of the pineal organ of higher vertebrates as a 'folded retina'. The cell membrane of several pineal photoreceptor outer-segments of vertebrates immunoreact with anti-retinal opsin antibodies supporting the view of retina-like organization of the pineal. Some other pineal outer segments do not react with retinal anti-opsin antibodies, a result suggesting the presence of special pineal photopigments in different types of pinealocytes that obviously developed during evolution. The chicken pinopsin, detected in the last years, may represent one of these unknown photopigments. Using antibodies against chicken pinopsin, we compared the immunoreactivity of different photoreceptors of the pineal organs from cyclostomes to birds at the light and electron microscopic levels. We found pinopsin immunoreaction on all pinealocytes of birds and on the rhodopsin-negative large reptilian pinealocytes. As the pinopsin has an absorption maximum at 470 nm, these avian and reptilian immunoreactive pinealocytes can be regarded as green-blue light-sensitive photoreceptors. Only a weak immunoreaction was observed on the frog and fish pinealocytes and no reaction was seen in cyclostomes and in the frontal organ of reptiles. Some photoreceptors of the retina of various species also reacted the pinopsin antibodies, therefore, pinopsin must have certain sequential similarity to individual retinal opsins of some vertebrates.

The photoreceptors and visual pigments of the garter snake (Thamnophis sirtalis): a microspectrophotometric, scanning electron microscopic and immunocytochemical study.

Scanning electron microscopy, immunocytochemistry, and single cell microspectrophotometry were employed to characterize the photoreceptors and visual pigments in the retina of the garter snake, Thamnophis sirtalis. The photoreceptor population was found to be comprised entirely of cones, of which four distinct types were identified. About 45.5% of the photoreceptors are double cones consisting of a large principal member joined near the outer segment with a much smaller accessory member. About 40% of the photoreceptors are large single cones, and about 14.5% are small single cones forming two subtypes. The outer segments of the large single cones and both the principal and accessory members of the doubles contain the same visual pigment, one with peak absorbance near 554 nm. The small single cones contain either a visual pigment with peak absorbance near 482 nm or one with peak absorbance near 360 nm. Two classes of small single cones could be distinguished also by immunocytochemistry and scanning electron microscopy. The small single cones with the 360-nm pigment provide the garter snake with selective sensitivity to light in the near ultraviolet region of the spectrum. This ultraviolet sensitivity might be important in localization of pheromone trails.

A 221-bp fragment of the mouse opsin promoter directs expression specifically to the rod photoreceptors of transgenic mice.

Mutations in the human rod opsin gene have been shown to segregate with autosomal dominant retinitis pigmentosa (ADRP) and photoreceptor degeneration in transgenic mice. While these degenerations are characterized by the primary degeneration of rods, cones eventually die as well. To determine whether this subsequent cone degeneration is the result of expression of mutant rod opsin in the cones, the retinal cell-type specificity of a 221-bp fragment of the mouse rod opsin promoter was evaluated. Two transgenic mouse lines generated by injecting a fusion gene comprised of a 221-bp fragment of the mouse rod opsin promoter and the simian virus 40 large tumor antigen gene (Tag) were examined. The expression of Tag causes photoreceptor cell degeneration in members of both transgenic lines. However, the two lines differed with respect to the level of Tag expression and the rate and extent of photoreceptor cell degeneration. Immunocytochemical localization of opsin and Tag in surviving photoreceptor cells was determined and the results were confirmed by reverse transcriptase polymerase chain reaction (RT-PCR). Rod- and cone-mediated function was evaluated by electroretinography (ERG). In the higher Tag-expressing transgenic line only one row of nuclei remained in the outer nuclear layer at postnatal day (P) 150. While these nuclei showed no antigenicity for rod opsin or Tag, they did stain with an antibody that reacts with both rod and cone S-antigens (arrestins), indicating that these cells were surviving photoreceptor nuclei. Positive staining with peanut agglutinin, which uniquely decorates matrix domains surrounding cones in the normal retina, confirmed that the surviving photoreceptor nuclei were of cone origin. RT-PCR substantiated the results from immunostaining; amplification product was obtained using blue cone opsin transcripts but not from either Tag or rod opsin transcripts. The second transgenic mouse line exhibited a much slower photoreceptor cell death that was associated with low levels of Tag transgene transcript. At P120, approximately 50% of photoreceptors remained and an approximately 45% reduction in the rod ERG a-wave was observed. Cone-mediated ERGs, however, were normal. The results demonstrate the rod-specific expression of Tag as directed by the 221-bp fragment of the mouse rod opsin promoter and suggest that the cone degeneration in ADRP or transgenic mice associated with mutations in the rod opsin gene is a secondary effect of rod degeneration.

Immunoreactive pinopsin in pineal and retinal photoreceptors of various vertebrates.

Pinopsin is a pineal specific opsin newly identified in the pineal of birds which has an absorption maximum at 470 nm. As the opsin content of photoreceptors in the pineal complex of several species is not yet known, in the present work, we studied their pinopsin immunoreactivity in various vertebrates from cyclostomes to mammals. We also compared the immunoreactivity of pineal photoreceptors to that of retinal cones and rods of each animal. For the immunocytochemistry, we raised antibodies in rabbits against a 14 amino acids containing part of the chicken pinopsin molecule. The immunoreaction was performed at the electron microscopic level. The pineal organs show a great diversity in vertebrates: there is a pineal organ present from cyclostomes to mammals, in addition, there is a parapineal organ in cyclostomes and fishes, a frontal organ in frogs and a parietal eye in several reptiles. We detected a strong pinopsin immunoreaction on most of the pinealocytes of birds and on the large photoreceptor-type of the pineal of reptiles. Rod-type photoreceptors of the avian retina and a cone of the reptile retina was immunoreactive as well. According to the known absorption maximum of pinopsin, the immunoreactivity may indicate a green-blue light-sensitivity for these photoreceptors. The immunoreactivity was less pronounced or absent in mammals as well as in less differentiated species. The pineal organ of snakes and the parietal eye of reptiles equally failed to exhibit pinopsin immunoreactive photoreceptors, presumably, due to the absence of green-blue light-sensitive photoreceptors of pinopsin-type in these species.

Distribution of cone photoreceptors in the mammalian retina.

The retina of mammals contains various amounts of cone photoreceptors that are relatively evenly distributed and display a radially or horizontally oriented area of peak density. In most mammalian species two spectrally different classes of cone can be distinguished with various histochemical and physiological methods. These cone classes occur in a relatively constant ratio, middle-to-longwave sensitive cones being predominant over short-wave cones. Recent observations do not support the idea that each cone subpopulation is uniformly distributed across the retina. With appropriate type-specific markers, unexpected patterns of colour cone topography have been revealed in certain species. In the mouse and the rabbit, the "standard" uniform pattern was found to be confined exclusively to the dorsal retina. In a ventral zone of variable width all cones express short-wave pigment, a phenomenon whose biological significance is not known yet. Dorso-ventral asymmetries have been described in lower vertebrates, matching the spectral distribution of light reaching the retina from various sectors of the visual field. It is not clear, however, whether the retinal cone fields in mammals carry out a function similar to that of their counterparts in fish and amphibians. Since in a number of mammalian species short-wave cones are the first to differentiate, and the expression of the short-wave pigment seems to be the default pathway of cone differentiation, we suggest that the short-wave sensitive cone fields are rudimentary areas conserving an ancestral stage of the photopigment evolution.

The opossum photoreceptors--a model for evolutionary trends in early mammalian retina.

The topography and spectral characteristics of mammalian photoreceptors correlate with both, the present ecological demands and the evolutionary history. The South American Opossum is a marsupial mammal with unspecialized habitus and crepuscular lifestyle. A sparse population of cones (max. = 3000/mm2) can be differentiated into four subtypes by morphological, topographical and immunocytochemical criteria. In spite of this unusual diversity the cone types can be split into two functional groups: The population of single cones labeled by antibody OS-2 for short wavelength sensitive pigments was ubiquitous but at very low densities (200/mm2). The single cones labeled by antibody (COS-1) against long wavelength sensitive pigments constitute the dominant population in the area centralis (2300/mm2). These two single cone types correlate with the pair typically present in placental mammals. Discrimination of spatial and color contrast may be provided by this "modern" set. The COS-1 labeled double and single cones bearing an oil droplet, display a different pattern by being restricted to the inferior (non-tapetal) half of the retina (max = 800/mm2). This additional set of cones with oil droplets and long wavelength pigments is a conservative feature of the opossum retina and other marsupials. As an accessory cone system it is possibly providing enhanced sensitivity at mesopic conditions. During the early evolution of nocturnal mammals with its prominent expansion of rod vision these cone types were conserved but then were lost in placental mammals. Thus the unique features of mammalian retinas are the result of two evolutionary steps: first a reduction of cone based vision, followed by a secondary differentiation of photopic vision and behaviour relying on the remaining set of cones.

Microspectrophotometric and immunocytochemical identification of ultraviolet photoreceptors in geckos.

Retinas of the nocturnal geckos, Hemidactylus turcicus, Hemidactylus garnotii, and Teratoscincus scincus, were studied with microspectrophotometry and immunocytochemistry against various visual pigment epitopes to reveal UV-sensitive photoreceptors. From 6-20% of the thinner members of type C double photoreceptors, earlier believed to be blue-sensitive, were found to contain a UV-absorbing visual pigment with lambda max at 363-366 nm. The pigment had bleaching and dichroic properties typical of other photoreceptor cell types of the retina. Presumptive UV-sensitive cells in retinal sections were "negatively" labeled as they did not react with either the cone-specific monoclonal antibody COS-1 or with the anti-rhodopsin polyclonal serum AO, which together labeled all of the remaining photoreceptor types (green-sensitive A singles, B doubles, and thicker members of C doubles, as well as the blue-sensitive majority of thinner members of C doubles). UV cells were moderately stained with the mAb K42-41 produced against the 5-6 loop of bovine rhodopsin, which also moderately labeled blue-sensitive cells. mAb OS-2 strongly stained all outer segments, including the UV-sensitive ones. Similarities between gecko UV visual pigments, and UV visual pigments of other vertebrates, as well as possible functional significance of these cells are discussed.

Photoreceptors in a primitive mammal, the South American opossum, Didelphis marsupialis aurita: characterization with anti-opsin immunolabeling.

The retinas of placental mammals appear to lack the large number and morphological diversity of cone subtypes found in diurnal reptiles. We have now studied the photoreceptor layer of a South American marsupial (Didelphis marsupialis aurita) by peanut agglutinin labeling of the cone sheath and by labeling of cone outer segments with monoclonal anti-visual pigment antibodies that have been proven to consistently label middle-to-long wavelength (COS-1) and short-wavelength (OS-2) cone subpopulations in placental mammals. Besides a dominant rod population (max. = 400,000/mm2) four subtypes of cones (max. = 3000/mm2) were identified. The outer segments of three cone subtypes were labeled by COS-1: a double cone with a principal cone containing a colorless oil droplet, a single cone with oil droplet, and another single cone. A second group of single cones lacking oil droplets was labeled by OS-2 antibody. The topography of these cone subtypes showed striking anisotropies. The COS-1 labeled single cones without oil droplets were found all over the retina and constituted the dominant population in the area centralis located in the temporal quadrant of the upper, tapetal hemisphere. The population of OS-2 labeled cones was also ubiquitous although slightly higher in the upper hemisphere (200/mm2). The COS-1 labeled cones bearing an oil droplet, including the principal member of double cones, were concentrated (800/mm2) in the inferior, non-tapetal half of the retina. The two spectral types of single cones resemble those of dichromatic photopic systems in most placental mammals. The additional set of COS-1 labeled cones is a distinct marsupial feature. The presence of oil droplets in this cone subpopulation, its absence in the area centralis, and the correlation with the non-tapetal inferior hemisphere suggest a functional specialization, possibly for mesopic conditions. Thus, sauropsid features have been retained but probably with a modified function.

Two different visual pigments in one retinal cone cell.

The retina of the mouse, rabbit, and guinea pig is divided into a superior area dominated by green-sensitive (M) cones and an inferior area in which cones possess practically only short wavelength-sensitive (S) photopigments. The present study shows that the transitional zone between these retinal areas is populated by cones labeled by both the M and S cone photopigment-specific antibodies COS-1 and OS-2. It is concluded that the overwhelming majority of the transitional cones express both visual pigments. A small population of the transitional cones was strongly labeled exclusively by OS-2 (genuine S cones). The results indicate that, in contrast to the generally accepted idea of one visual pigment per one cone cell, cones of certain mammalian species can express different opsins simultaneously under natural conditions. We speculate that the coexpression may be due to the overlapping of regulatory factors determining the M and S fields.