Search

Background: Diabetic men are less likely to suffer prostate cancer, and insulin signalling through insulin receptors has been long considered. However, the role of insulin-dependent glucose transporters has yet to be elucidated. The unique metabolic properties of prostate cancer are attributed to the central role of androgens. Androgen-sensitive tumour cells have higher mitochondrial activity, while castration-resistant cells exhibit aerobic glycolysis. In addition, to glycolysis, one of the hallmarks of cancer metabolism is increased glucose uptake. However, the prostate's oncogenic value of glucose transporters (GLUTs) needs to be better characterized. This research aims to discover the relevance of insulin-dependent glucose transporters to cancer progression and their importance in the protective role of diabetes in prostate cancer. Methods: Androgen-sensitive LNCaP and androgen-insensitive PC-3 cells were used in vitro. Castration-resistant LNCaP-R cells and cells overexpressing GLUT1 or GLUT4 were established from LNCaP cell line. In addition, TRAMP (Transgenic Adenocarcinoma of Mouse Prostate) mice and prostatic samples from patients were employed. Results: We found that androgens stimulate insulin-independent glucose transporters, while androgen independence is associated with GLUT4 overexpression. The ectopic overexpression of GLUT4 promotes the characteristics of a castration-resistant phenotype. Metabolomics confirmed that hormone-resistant prostate cancer cells show an oxidative metabolism with a clear enrichment in amino acid metabolism. Diabetic TRAMP mice showed total tumour regression, while insulin administration restored proliferation and recovered GLUT4 levels. The levels of GLUT4 increase along with tumour progression in TRAMP mice, and it is reduced by castration and streptozotocin-induced diabetes. Finally, the levels of GLUT4 accumulation in tumour tissues compared to normal epithelial in patients' samples showed a clear co-location with nuclear AR. Conclusion: Here it is confirmed the relevance of insulin-mediated glucose uptake through GLUT4 with prostate cancer progression and its relation to the reduced occurrence of prostate cancer in diabetic men.

Neuroindole melatonin, a hormone synthesized during the night mainly&mdash, but not exclusively&mdash, by the pineal gland of all vertebrates, functions as an adapting signal to the light-dark cycle. Its antioxidant, neuroprotective, anti-inflammatory, and antitumor properties are all well-known and widely reported. Melanoma is one of the most common carcinomas among developed countries and a type of tumor particularly difficult to fight back in medium/advanced stages. In contrast to other types of cancer, influence of melatonin on melanoma has been scarcely investigated. Thus, we have chosen the murine melanoma model B16-F10 cell line to study antiproliferative and antitumoral actions of melatonin. For this purpose, we combined both, cell culture and in vivo models. Melatonin reduced either, growth rate or migration of B16-F10 cells. Furthermore, melanin synthesis was altered by melatonin, promoting its synthesis. Melatonin also induced a G2/M cell cycle arrest and altered the cytoskeletal organization. To corroborate these results, we tested the effect of melatonin in the in vivo model of B16-F10 cell injection in the tail vein, which causes numerous lung metastases. Two different strategies of melatonin administration were used, namely, in drinking water, or daily intraperitoneal injection. However, contrary to what occurred in cell culture, no differences were observed between control and melatonin treated groups. Results obtained led us to conclude that melatonin exerts an antiproliferative and anti-migrating effect on this melanoma model by interfering with the cytoskeleton organization, but this pharmacological effect cannot be translated in vivo as the indole did not prevent metastasis in the murine model, suggesting that further insights into the effects of the indole in melanoma cells should be approached to understand this apparent paradox.

Melatonin is a well-known, nighttime-produced indole found in bacteria, eukaryotic unicellulars, animals or vascular plants. In vertebrates, melatonin is the major product of the pineal gland, which accounts for its increase in serum during the dark phase, but it is also produced by many other organs and cell types. Such a wide distribution is consistent with its multiple and well-described functions which include from the circadian regulation and adaptation to seasonal variations to immunomodulatory and oncostatic actions in different types of tumors. The discovery of its antioxidant properties in the early 1990s opened a new field of potential protective functions in multiple tissues. A special mention should be made regarding the nervous system, where the indole is considered a major neuroprotector. Furthermore, mitochondria appear as one of the most important targets for the indole's protective actions. Melatonin's mechanisms of action vary from the direct molecular interaction with free radicals (free radical scavenger) to the binding to membrane (MLT1A and MLT1B) or nuclear receptors (RZR/RORα). Receptor binding has been associated with some, but not all of the indole functions reported to date. Recently, two new mechanisms of cellular uptake involving the facilitative glucose transporters GLUT/SLC2A and the proton-driven oligopeptide transporter PEPT1/2 have been reported. Here we discuss the potential importance that these newly discovered transport systems could have in determining the actions of melatonin, particularly in the mitochondria. We also argue the relative importance of passive diffusion vs active transport in different parts of the cell.

Biennial Meeting for the Society for Free Radical Research International (SFRRI 2021) (20th. 2021. Virtual)

In the present study, we characterize the antinociceptive effects produced by the chemokine CCL4 in mice. The intraplantar administration of very low doses of CCL4 (0.1-3 pg) produced bilateral antinociception assessed by the unilateral hot-plate test (UHP) without evoking chemotactic responses at the injection site. Moreover, the subcutaneous administration of CCL4 (3-100 pg/kg) also yielded bilateral antinociception in the UHP and the paw pressure test and reduced the number of spinal neurons that express Fos protein in response to noxious stimulation. The implication of peripheral CCR5 but not CCR1 in CCL4-evoked antinociception was deduced from the inhibition produced by systemic but not intrathecal, administration of the CCR5 antagonist DAPTA, and the inefficacy of the CCR1 antagonist J113863. Besides, the inhibition observed after subcutaneous but not intrathecal administration of naloxone demonstrated the involvement of peripheral opioids and the efficacy of naltrindole but not cyprodime or nor-binaltorphimine supported the participation of δ-opioid receptors. In accordance, plasma levels of met-enkephalin, but not β-endorphin, were augmented in response to CCL4. Likewise, CCL4-evoked antinociception was blocked by the administration of an anti-met-enk antibody. Leukocyte depletion experiments performed with cyclophosphamide, anti-Ly6G, or anti-CD3 antibodies indicated that the antinociceptive effect evoked by CCL4 depends on circulating T lymphocytes. Double immunofluorescence experiments showed a four times more frequent expression of met-enk in CD4

Melatonin, N-acetyl-5-methoxytryptamine, is an indole mainly synthesized from tryptophan in the pineal gland and secreted exclusively during the night in all the animals reported to date. While the pineal gland is the major source responsible for this night rise, it is not at all the exclusive production site and many other tissues and organs produce melatonin as well. Likewise, melatonin is not restricted to vertebrates, as its presence has been reported in almost all the phyla from protozoa to mammals. Melatonin displays a large set of functions including adaptation to light: dark cycles, free radical scavenging ability, antioxidant enzyme modulation, immunomodulatory actions or differentiation–proliferation regulatory effects, among others. However, in addition to those important functions, this evolutionary ‘ancient’ molecule still hides further tools with important cellular implications. The major goal of the present review is to discuss the data and experiments that have addressed the relationship between the indole and glucose. Classically, the pineal gland and a pinealectomy were associated with glucose homeostasis even before melatonin was chemically isolated. Numerous reports have provided the molecular components underlying the regulatory actions of melatonin on insulin secretion in pancreatic beta-cells, mainly involving membrane receptors MTNR1A/B, which would be partially responsible for the circadian rhythmicity of insulin in the organism. More recently, a new line of evidence has shown that glucose transporters GLUT/SLC2A are linked to melatonin uptake and its cellular internalization. Beside its binding to membrane receptors, melatonin transportation into the cytoplasm, required for its free radical scavenging abilities, still generates a great deal of debate. Thus, GLUT transporters might constitute at least one of the keys to explain the relationship between glucose and melatonin. These and other potential mechanisms responsible for such interaction are also discussed here.

We show here that the intraplantar administration of CCL5 in mice produces hyperalgesia at low doses but activates compensatory antinociceptive mechanisms at doses slightly higher. Thus, the injection of 3–10 ng of CCL5 evoked thermal hyperalgesia through the activation of CCR1 and CCR5 receptors, as demonstrated by the inhibitory effect exerted by the selective antagonists J113863 (0.01–0.1 μg) and DAPTA (0.3–3 μg), respectively. The prevention of this hyperalgesia by diclofenac (1–10 μg), the inhibitors of COX-1 SC-560 (0.1–1 μg) or COX-2 celecoxib (1–5 μg), the TRPV1 antagonist capsazepine (0.03–0.3 μg) or the TRPA1 antagonist HC030031 (10–50 μg) demonstrates the involvement of prostaglandin synthesis and TRP sensitization in CCL5-evoked hyperalgesia. Doses of CCL5 higher than 17 μg did not evoke hyperalgesia. However, this effect was restored by the administration of naloxone-methiodide (5 μg), nor-binaltorphimine (10 mg/kg) or an anti-dynorphin A antibody (0.62–2.5 ng). The administration of 30 ng of CCL5 also induced hyperalgesia in mice with reduced number of circulating white blood cells in response to cyclophosphamide or with selective neutrophil depletion induced by an anti-Ly6G antibody. In fact, the number of neutrophils present in paws treated with 30 ng of CCL5 was greater than in paws receiving the administration of the hyperalgesic dose of 10 ng. Finally, the expression of the endogenous opioid peptide dynorphin A was demonstrated by double immunofluorescence assays in these neutrophils attracted by CCL5. These results support previous data describing the hyperalgesic properties of CCL5 and constitute the first indication that a chemokine of the CC group can activate endogenous analgesic mechanisms.

The mammalian Period1 gene is rhythmically expressed and its proteins are found within the nucleus of the cells of the suprachiasmatic nuclei (SCN), the central circadian pacemaker in mammals; however, whether the target of the PER1 proteins is also the nucleus in the retinal peripheral clock cells is yet to be determined. Using an anti-PER1 protein antibody in Western blot analyses, we found three isoforms (75, 110 and 140 kDa) in extracts of the SCN, as well as in other different parts of the brain, whereas just two isoforms (75 and 110 kDa) were detected in the retinal extracts. We have observed that PER1 immunolabelling has a cytoplasmic location in many cells of the ganglion cell layer and in a few cells in the inner nuclear layer of the mouse retina. This cellular location was seen in any of the tissue samples taken at 4 h intervals, either in the day/night cycle or in constant darkness, of both wild type and rd mice. Unlike this situation, PER1 isoforms were nuclear proteins in the SCN cells as well as in other parts of the brain of the same animals. No circadian changes were found for these clock proteins in the neural retina. These findings suggest that PER1 proteins play roles in the retina different from those established in the SCN.

Extraretinal photoreceptors located within the medio-basal hypothalamus regulate the photoperiodic control of seasonal reproduction in birds. An action spectrum for this response describes an opsin photopigment with a λmax of ∼492nm. Beyond this however, the specific identity of the photopigment remains unresolved. Several candidates have emerged including rod-opsin; melanopsin (OPN4); neuropsin (OPN5); and vertebrate ancient (VA) opsin. These contenders are evaluated against key criteria used routinely in photobiology to link orphan photopigments to specific biological responses. To date, only VA opsin can easily satisfy all criteria and we propose that this photopigment represents the prime candidate for encoding daylength and driving seasonal breeding in birds. We also show that VA opsin is co-expressed with both gonadotropin-releasing hormone (GnRH) and arginine-vasotocin (AVT) neurons. These new data suggest that GnRH and AVT neurosecretory pathways are endogenously photosensitive and that our current understanding of how these systems are regulated will require substantial revision.

Endogenous rhythms of mammals are controlled by the clock located in the suprachiasmatic nucleus (SCN). The molecular mechanism of a clock involves transcription/translation-based feedback loops in which the expression of the so called “clock genes” is suppressed periodically by their protein products. Previous studies reported influence of the eye itself on the circadian oscillation of the SCN, apart from the well-known photic readjustment of the central clock. With this in mind, we decided to analyze the mPer1 clock gene expression in the retinally degenerate (rd) mouse SCN by means of immunohistochemical techniques. Our objective was to detect possible alterations of the daily endogenous oscillation of PER1 protein in the SCN of these rd mice, as well as to make clear whether or not this protein was involved in the resetting of the central clock in a manner similar to wild-type animals. We found that the endogenous levels of PER1 protein were reduced in the SCN of rd mice throughout the 24-h cycle, which suggests that loss of classic photoreceptors influences somehow the main mechanism of the SCN clock. Light stimulation induced a parallel increase of Per1 expression at the subjective night, but not at the subjective day, in both rd and wild-type mice. Therefore, SCN readjustment by light in the rd mice occurs with a pattern similar to wild-type controls, despite the reduced PER1 protein levels detected. The effect of retinal degeneration on the circadian system and the possible interactions between the retinal and the SCN clocks are discussed.

Pneumolysin (PLY) is an important virulence factor of Streptococcus pneumoniae . We examined the ability of three murine monoclonal antibodies (MAbs) to PLY (PLY-4, PLY-5, and PLY-7) to affect the course of pneumococcal pneumonia in mice. The intravenous administration of antibodies PLY-4 and PLY-7 protected the mice from the lethal effect of the purified toxin. Mice treated with PLY-4 before intranasal inoculation of S. pneumoniae type 2 survived longer (median survival time, 100 h) than did untreated animals (median survival time, 60 h) ( P < 0.0001). The median survival time for mice treated with a combination of PLY-4 and PLY-7 was 130 h, significantly longer than that for mice given isotype-matched indifferent MAbs ( P = 0.0288) or nontreated mice ( P = 0.0002). The median survival time for mice treated with a combination of three MAbs was significantly longer (>480 h) than that for mice treated with PLY-5 (48 h; P < 0.0001), PLY-7 (78 h; P = 0.0007), or PLY-4 (100 h; P = 0.0443) alone. Similarly, the survival rate for mice treated with three MAbs (10 of 20 mice) was significantly higher than the survival rate obtained with PLY-5 (1 of 20; P = 0.0033), PLY-4 (2 of 20; P = 0.0138), or PLY-7 (3 of 20; P = 0.0407) alone. These results suggest that anti-PLY MAbs act with a synergistic effect. Furthermore, MAb administration was associated with a significant decrease in bacterial lung colonization and lower frequencies of bacteremia and tissue injury with respect to the results for the control groups.

The vertebrate retina is known to mediate both visual and non-image-forming photic responses. With the use of statistical analyses of sections immunohistochemically labelled with a polyclonal antiserum against the activated form of protein CREB (p-CREB), a transcription factor which participates in some neural responses to stimuli, we have observed that the piriform cortex of both wild-type and retinally degenerate (rd) mice respond to light stimulation independently of the circadian time in which the stimulus was given. Responses in visually blind (rd/rd) mice corroborate the hypothesis that there must be neural connections between the retina and cortical brain areas other than those involved in image processing, and strongly support the idea that since these mice lack rods and cones, the melanopsin retinal ganglion cells could mediate this non-visual light input.

Item does not contain fulltext The purpose of the present investigation was to provide a detailed description of the encephalic photoreceptors of Xenopus laevis at the light microscopic level and to determine their relationship with the neurosecretory cells of the hypothalamus in order to further our understanding of photoperiodic regulation of seasonal rhythms. Numerous opsin-positive neurons were found in the hypothalamic magnocellular preoptic nucleus and their axonal processes were seen to run laterally towards the basal regions of the brain, reaching the neural lobe of the hypophysis. Analysis of labelling with different antisera in adjacent sections, as well as double-immunolabelling carried out on the same section, revealed that mesotocin immunoreactivity was present in most of the opsin-positive neurons; however, no evidence for opsin and vasotocin coexpression was found in any of the sections analysed. The close localization of LHRH and opsin/mesotocin fibers in some regions of the brain, such as the median eminence, suggests that some interaction between these two systems might exist. In conclusion, in this study we provide the first strong evidence that the hypothalamic mesotocinergic neurons, which have been proved to be connected to the GnRH system in other species, are directly involved in photoreception in Xenopus laevis. These findings represent a novel contribution to our understanding of how light influences the seasonal reproductive cycles in lower vertebrates.

In an attempt to clarify its possible physiological role, we studied the eye of the Zambian mole rat Cryptomys anselli by light, electron and confocal microscopy using conventional staining as well as immunolabelling with rod and cone cell markers. The small eyes of Cryptomys are located superficially and display all features typical of sighted animals: iris, pupil and well-developed lens, separating the anterior chamber and the vitreous. The retina shows a well stratified organization and the folds described in blind subterranean or nocturnal mammals were not observed. The major population of the photoreceptor cells in the Cryptomys retina consists of rod cells, again with a morphology quite similar to that found in sighted animals. The relatively short outer segments contain numerous well-stacked disks and show a strong rod-opsin as well as transducin immunoreaction. Synapses were evident in the spherules, the round basal processes of the rod cell, but they lacked the precise organization reported for sighted mammals. Cone cells were present as well, as indicated by peanut lectin staining, but no immunolabelling with polyclonal M/L-opsin antisera was detectable. The presence of cone cells was also suggested by some basal processes at the outer plexiform layer which displayed several synaptic active sites and irregular contours. While the other retinal layers also showed an organization typical of sighted mammals, there were signs of less tightly preserved morphology as well. Displaced rods and amacrine and/or ganglion cells were observed, and some sparse rod spherules penetrated into the inner nuclear layer. A major reduction was observed in the number of ganglion cells, estimated from the number of axons in the optic nerve, that was very low (approximately 1000 per retina on average) relative to sighted mammals. The data we have suggest a slow, ongoing loss of cells with ageing. Apoptotic nuclei, mainly corresponding to photoreceptor cells and ganglion cells, were detected in young individuals, and an overall reduction in the thickness of the retina was observed in older animals. The morphological data presented here allow some first speculations on the physiological role of the Cryptomys eye and will hopefully trigger detailed studies on the chronobiology and the anatomy of the retinal projections and of the visual cortex of this remarkable species.

Lateral line organ, a superficial sensory system in amphibia and fish which provides the animal with information about its surrounding environment, is divided classically into two main different types, ordinary and specialized, whose functions are mechanoreceptive and electroreceptive, respectively. Although it has great diversity, the basic sensory unit, which is usually called "neuromast," is composed of sensory cells embedded in accessory cells. The functions of the latter are to support the sensory cells and to secrete the material that covers the organs, forming a cupular structure or filling a canal which enables the organ to communicate with the exterior. Sensory cells of mechanoreceptive neuromasts have a tuft of processes included in the cupular material; these are a kinocilium and a group of stereocilia with a typical staircase arrangement. The displacement of the stereocilia towards or away from the kinocilium produces different stimuli. The electroreceptive organs are more diverse. They include ampullary and tuberous organs. The latter can be subdivided into different types: knollenorgans, mormyromasts, gymnarchomasts, etc. All of these present a great diversity among species, but their morphology is less reported than that of the mechanoreceptive organs. This paper summarizes the structural features of the main different types of lateral line organs, as well as their taxonomic distribution and different patterns of distribution along the surface of the animal.

The melanopsin-expressing retinal ganglion cells are specialized in measuring irradiance for several functions, including daily photoentrainment and regulation of pupil size. In the present study, these cells were analyzed in mice during their perinatal period, from embryonic day (E) 15 to postnatal day (P) 1. Melanopsin expression was detected at E15 in cells that did not co-express the transcription factor Brn3a. Under light/dark (LD) cycles, the number of melanopsin-expressing cells did not change between E16 and E19, while a very significant increase was observed during the short interval around birth, between E19 (the day before birth) and P0 (the day of birth). As these samples were collected after lights on, to determine whether such increase in melanopsin expression was driven by light, we also analyzed samples collected 0-4 hours after birth (during the night period), which revealed that the cell number increase was already present and, therefore, was not induced by the early post-birth light exposure. To clarify the role of ambient light conditions during this period, P1 retinas from pups under constant light or darkness conditions were also analyzed and compared to those of mice under LD cycles. No variation in the number of immunostained cells was detected among the groups studied, indicating that ambient conditions did not provoke the increase in melanopsin expression detected. Rather, it might have been induced by either a maternal or a developmental signal and is likely related to the first connections between the retina and the suprachiasmatic nucleus reported by other authors.

Melanopsin, an opsin protein expressed in mammalian retinal ganglion cells (RGCs), makes them responsive to light. Such photosensitive RGCs form the retinohypothalamic tract (RHT) that provides signals to the suprachiasmatic nucleus (SCN), the master regulator of circadian rhythms. The SCN is adjusted daily to the environmental day/night cycle by signal inputs incoming from the RHT. In the present work we have studied, using immunohistochemistry techniques, the types and number of cells which expressed melanopsin during the postnatal development of pigmented C3H/He mice maintained in a standard daily cycle (12-h light/12-h dark). Our results clearly show for the first time that the retina maintains a rather constant number of melanopsin-expressing RGCs from the first postnatal day and, thus, demonstrate that no loss of these photosensitive cells occurs during postnatal development. This supports the general idea that the non-image-forming system, in which these cells are involved, is functional at the very early postnatal stage.

In addition to some other functions, melanopsin-expressing retinal ganglion cells (RGCs) constitute the principal mediators of the circadian photoentrainment, a process by which the suprachiasmatic nucleus (the central clock of mammals), adjusts daily to the external day/night cycle. In the present study these RGCs were immunohistochemically labelled using a specific polyclonal antiserum raised against mouse melanopsin. A daily oscillation in the number of immunostained cells was detected in mice kept under a light / dark (LD) cycle. One hour before the lights were on (i.e., the end of the night period) the highest number of immunopositive cells was detected while the lowest was seen four hours later (i.e., within the first hours of the light period). This finding suggests that some of the melanopsin-expressing RGCs “turn on” and “off” during the day/night cycle. We have also detected that these daily variations already occur in the early postnatal development, when the rod/cone photoreceptor system is not yet functional. Two main melanopsin-expressing cell subpopulations could be found within the retina: M1 cells showed robust dendritic arborization within the OFF sublamina of the inner plexiform layer (IPL), whilst M2 cells had fine dendritic processes within the ON sublamina of the IPL. These two cell subpopulations also showed different daily oscillations throughout the LD cycle. In order to find out whether or not the melanopsin rhythm was endogenous, other mice were maintained in constant darkness for six days. Under these conditions, no defined rhythm was detected, which suggests that the daily oscillation detected either is light-dependent or is gradually lost under constant conditions. This is the first study to analyze immunohistochemically the daily oscillation of the number of melanopsin-expressing cells in the mouse retina.

The purpose of this study was to characterize the morphological abnormalities in the retinas of chicks (Gallus gallus) suffering from the autosomal recessive disease, retinopathy, globe enlarged (rge/rge).rge/rge affected and age matched control retinas were examined from hatch up to 730 days of age. Thickness of retinal layers at six retinal regions was measured from plastic embedded sections. Morphological features were examined on semi-thin sections by light microscopy and on ultra-thin sections by transmission electron microscopy. Immunohistochemistry was performed using a panel of several different antibodies. Additionally, comparative counting of rod outer segments, rows of cells in the inner nuclear layer, and ganglion cells per unit length was performed.The earliest changes observed in rge/rge retinas were disorganization of the outer plexiform layer and abnormal location of the endoplasmic reticulum of the photoreceptors. In rge/rge retinas, cone pedicles were larger, irregular in shape, and usually contained multivesicular bodies. In addition, synaptic ribbons of the cone pedicles and rod spherules in rge/rge retinas were less numerous compared to controls. Large glycogen deposits progressively accumulated in the perinuclear cytoplasm associated with the abnormally located endoplasmic reticuli in accessory cones and rods. Total retinal thickness progressively decreased with age in rge/rge birds. This was accompanied by a decrease in the number of cells in the inner nuclear layer and a decrease in the number of rod outer segments (OSs). Several changes were detected in the rge/rge retinas using immunohistochemistry, including mislocalized opsin immunoreactivity of rod photoreceptors, a decrease in number and disorganization of opsin positive rod OSs (especially in the peripheral regions), a decrease in number of tyrosine hydroxylase positive neurites in the distal inner plexiform layer, and activation of macroglial and microglial cells.As we previously reported, the rge/rge chick has vision loss that is not the result of photoreceptor loss and is unusual in that electroretinographic responses, although abnormal, are maintained until well after vision loss has developed. The phenotype is associated with a developmental disruption of both rod and cone photoreceptor synaptic terminals that progresses with age. It is possible that these changes may be indicative of abnormal circuitry within the outer plexiform layer, and that they underlie the progressive loss of vision in rge/rge birds. Other early changes suggesting photoreceptor abnormality are dilation of photoreceptor cell bodies, abnormal positioning of endoplasmic reticulum in the perinuclear region that is associated with abnormal glycogen deposition, and mislocalization of opsin immunoreactivity in rods. The rge/rge birds develop globe enlargement after the morphological and electroretinographic abnormalities. Globe enlargement in chicks can be induced by a number of different environmental factors. It is possible that abnormal signaling of photoreceptors to inner retinal cells could induce excessive ocular growth in the rge/rge birds. Many of the morphological changes such as retinal thinning seen in older rge/rge birds may be partly the result of the considerable globe enlargement that occurs later in the disease process. Molecular genetic studies to identify the causal gene mutation should help explain the morphological features of the rge/rge phenotype and clarify their association with vision loss and electroretinographic abnormalities.

The effect of cone- and rod-cell loss on the activation of transcription factor CREB (by phosphorilation at Ser133) was examined in the pacemaker of mammals, the suprachiasmatic nucleus (SCN). For this purpose, brain sections of rd/rd and wild-type C3H mice were immunolabelled with a polyclonal antibody that recognises p-CREB, i.e., the activated form of the protein. Both rd/rd and wild-type mice maintained in constant darkness showed a circadian variation of p-CREB nuclear staining: the number of immunopositive nuclear pixels at the subjective night was higher than the one observed at the subjective day. However, some differences were detected between both groups: (1) p-CREB immunolabelling in the SCN of rd/rd mice was significantly reduced throughout the 24-h cycle; (2) the time in which the activation of CREB begins to increase at the subjective night in these mice is delayed with regard to wild-type mice. When a light stimulus was given at the subjective night p-CREB inmunostaining significantly increased in the SCN of both rd/rd and wild-type mice when compared to basal levels, while no significant effect was found when the stimulus was given at the subjective day. Taken together, our results suggest that despite lower levels of p-CREB, indicating that something is altered in the SCN of rd/rd mice, the main mechanisms of the clock (e.g., circadian oscillation, readjustment by light) are still fully functional in these mice. The present study supports the idea that the CREB/CRE pathway is a component of the circadian clock molecular mechanism.

In an attempt to clarify its possible physiological role, we studied the eye of the Zambian mole rat Cryptomys anselli by light, electron and confocal microscopy using conventional staining as well as immunolabelling with rod and cone cell markers. The small eyes of Cryptomys are located superficially and display all features typical of sighted animals: iris, pupil and well-developed lens, separating the anterior chamber and the vitreous. The retina shows a well stratified organization and the folds described in blind subterranean or nocturnal mammals were not observed. The major population of the photoreceptor cells in the Cryptomys retina consists of rod cells, again with a morphology quite similar to that found in sighted animals. The relatively short outer segments contain numerous well-stacked disks and show a strong rod-opsin as well as transducin immunoreaction. Synapses were evident in the spherules, the round basal processes of the rod cell, but they lacked the precise organization reported for sighted mammals. Cone cells were present as well, as indicated by peanut lectin staining, but no immunolabelling with polyclonal M/L-opsin antisera was detectable. The presence of cone cells was also suggested by some basal processes at the outer plexiform layer which displayed several synaptic active sites and irregular contours. While the other retinal layers also showed an organization typical of sighted mammals, there were signs of less tightly preserved morphology as well. Displaced rods and amacrine and/or ganglion cells were observed, and some sparse rod spherules penetrated into the inner nuclear layer. A major reduction was observed in the number of ganglion cells, estimated from the number of axons in the optic nerve, that was very low (approximately 1000 per retina on average) relative to sighted mammals. The data we have suggest a slow, ongoing loss of cells with ageing. Apoptotic nuclei, mainly corresponding to photoreceptor cells and ganglion cells, were detected in young individuals, and an overall reduction in the thickness of the retina was observed in older animals. The morphological data presented here allow some first speculations on the physiological role of the Cryptomys eye and will hopefully trigger detailed studies on the chronobiology and the anatomy of the retinal projections and of the visual cortex of this remarkable species.

The retina of the blind mole rat Spalax ehrenbergi was compared with other vertebrate photosensitive organs in an attempt to correlate its histologic organization with a presumptive nonvisual photoreceptor role.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.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.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.

The ontogeny of photic responsiveness in the retina and the suprachiasmatic nucleus (SCN) of C57BL/6J mouse was studied using the enhanced expression of the immediate early gene c-fos as a marker of neuronal activation. c-fos expression was assessed by immunocytochemical localisation of its protein product. Light induction of Fos-like protein in the retina and SCN occurred first at postnatal day four (PD 4). At this stage of development, some cells in the inner part of the neuroblastic layer and in the ganglion cell layer showed positive immunoreaction; the number of Fos-like positive cells increased with age until it reached adult levels by PD 15. Induction of Fos-like expression at PD 4 in the SCN mainly occurred in the ventrolateral region, the region that receives the greatest density of retinal innervation. These results indicate that retinal input can activate cells in the SCN even before eyelids open, and the SCN can be stimulated by photic inputs as early as day 4 after birth.

Rd mutant mice are visually blind but they maintain the ability of synchronising their circadian rhythms to the external light-dark cycles. We used immunocytochemical procedures to detect light-induced Fos expression in the rd mice retina. We found that Fos is expressed in the rd retina in an unattenuated pattern through the entire life of the animal. Furthermore, we have found that cells expressing Fos are distributed throughout the whole retina, while opsin expression takes place only in the dorsal half of the retina in the 1-year old rd mice. Finally, we found that light induces Fos expression in the rd retina at the same levels during the subjective day as during the subjective night, whereas in the suprachiasmatic nucleus (SCN), Fos is stimulated by light only during the subjective night. Our results support the hypothesis that new, undiscovered photoreceptors are implicated in light perception for the circadian system.

An anti-Fos protein antiserum was used to elucidate the diurnal expression of Fos protein in the normal and degenerate rd/rd mice retina. We have found that Fos expression is stimulated in cells of both inner nuclear layer (INL) and ganglion cell layer (GCL) at the onset of light period and reaches its maximum after 2 h. After which, the number of stained nuclei decreases along the light/dark cycle until almost no reaction is observed at the end of dark period. This expression pattern was similar in both normal and rd/rd mice although degenerate retinas showed a much lower number of stained nuclei. Aged rd animals also show Fos expression in GCL and INL in response to light stimuli suggesting that severely degenerate retinas are still able to transduce light stimulus.

Neurotrophins and their receptors (p75 and Trk family of receptors) play an important role in the survival of different populations of neurons in the central and peripheral nervous system. Expression of p75, TrkA, TrkB and TrkC was examined in mouse retinas by means immunohistochemistry in the postnatal development of normal and rd/rd mice (C57BL/6J). The rd/rd mice suffer a degeneration that causes a massive lost of photoreceptor cells. Results showed immunoreactivity to all three Trk proteins in both normal and rd/rd mice during the first 21 postnatal days, but some variations in intensity and localization were found. p75 immunoreaction was only present in rd/rd mice at the end of the degeneration process. These results could indicate a role of neurotrophins and their receptors in both the postnatal development of mouse retina and the degeneration process of rd/rd mice.

The vertebrate eye mediates both image-forming and non–image-forming photoreception. Image-forming photoreception (vision) enables the animal to detect and track objects in the environment, whereas non–image-forming photoreception is responsible for the measurement of ambient irradiance, so that, for example, the internal circadian biological clock can be synchronized with the astronomical day, a process called photoentrainment.1,2 The hypothalamic suprachiasmatic nucleus (SCN), which is considered the central circadian pacemaker of mammals, is adjusted on a daily basis to the environmental light/dark cycle1 by the detection of light by melanopsin-expressing, intrinsically photosensitive retinal ganglion cells (ipRGCs).3–6 Such ipRGCs transmit this light information to the SCN by way of the retinohypothalamic tract.7,8 These cells also project to other brain areas involved in pupil constriction, promotion of sleep, gaze control, image-forming vision, and other activities.9 Moreover, ipRGCs constitute the principal conduits for rod-cone input involved in non–image-forming responses, including circadian photoentrainment.10 In fact, the destruction of these cells altered the effects of light on circadian rhythms.10–12 Therefore, the rod and cone photoreceptors and the ipRGCs are complementary in providing signals for nonvisual photoreceptive functions. In mice, at least 70% of the RGCs generated during retinal development die through programed cell death during the postnatal period13; however, as we previously demonstrated in pigmented mice, no diminution in the number of melanopsin-expressing cells occurs during postnatal development.14 ipRGCs are responsive to light from birth.15,16 Moreover, the SCN begins to function as a circadian pacemaker during late fetal development.17 Depending on the intensity of the stimulus, light was able to induce expression of the immediate early gene c-fos in the SCN at postnatal day (P) 0 to P118 or at P4.19 Taken together, these data indicate that the melanopsin-based system is functional as early as the day of birth. Previous studies have demonstrated that melanopsin expression shows daily oscillation.20–22 Such rhythm was also demonstrated in neonatal albino rats and neonatal pigmented mice,22,23 when rods and cones are not yet fully developed. Hannibal et al.21 and Mathes et al.,24 using albino rats, also reported differential regulation of melanopsin expression in response to continuous darkness (DD) or continuous light (LL). Such changes in melanopsin expression were also detected in albino rat pups.23 This suggests that ipRGCs can adapt their responsiveness to the external illumination conditions by regulating their melanopsin content even in the absence of functional rod-cone photoreceptors. Among the ipRGCs, two main morphologic types have been previously identified: M1 cells, with their dendritic arborization in the OFF sublayer of the inner plexiform layer (IPL), and M2 cells, with their dendrites forming a plexus in the ON sublayer of the IPL. Recently, two isoforms of melanopsin, Opn4S and Opn4L, have been identified. M1 cells express both melanopsin isoforms, whereas M2 cells express only the Opn4L isoform.25 Different electrophysiological responses,26 as well as different brain projections,27 were reported for these two cell subpopulations. In a previous study,22 we detected a different daily oscillation for M1 and M2 cells that was already present in the early postnatal period. Albino animals are often used as models in numerous studies concerning the retina, despite the fact that most mutations causing albinism provoke anomalous retinal development, including lower numbers of rods, incomplete development of the central retina, and chiasmatic abnormalities.28 Therefore, it should be taken into account that results obtained in albino models are not fully comparable with those of pigmented animals. To better understand the development of the ipRGCs, the present study analyzed for the first time in albino mice these cells and their main subpopulations within the postnatal period under standard 12-hour light/12-hour dark cycles. Furthermore, the effects of exposure to constant light or constant darkness on the postnatal development of these cells were also studied.