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Parkinson’s disease (PD) evolves over an extended and variable period in humans; several years prior to the onset of classical motor symptoms, cognitive deficits as well as sleep and biological rhythm disorders develop and worsen with disease progression, significantly impacting the quality of life of patients. The gold standard MPTP macaque model of PD recapitulates the progression of motor and non-motor symptoms over contracted periods of time.Here, this multidisciplinary and multiparametric study follows, in five animals, the steady progression of motor and non-motor symptoms and describes their reversal following bilateral grafts of neural precursors in diverse functional domains of the basal ganglia.Results show unprecedented recovery from cognitive symptoms in addition to a strong clinical motor recuperation. Both motor and cognitive recovery and partial circadian rhythm recovery correlate with the degree of graft integration into the host environment as well as with in-vivo levels of striatal dopaminergic innervation and function.Given inter-individuality of disease progression and recovery the present study underlines the importance of longitudinal multidisciplinary assessments in view of clinical translation and provides empirical evidence that integration of neural precursors following transplantation efficiently restores function at multiple levels in parkinsonian non-human primates.One Sentence SummaryEmpirical evidence that cell therapy efficiently reverts cognitive and clinical motor symptoms in the non-human primate model of Parkinson’s disease.

Cognitive deficits as well as disorders of sleep and biological rhythms constitute non-motor symptoms that significantly impact quality of life in Parkinson’s disease patients. Few studies have evaluated the impact of cell replacement therapy on such non-motor symptoms. Here we used a multidisciplinary approach to assess the therapeutic potential of bilateral grafts of neural stem cells in a macaque model of Parkinson’s disease on both motor and non-motor markers of functional recovery. Grafts led to varying degrees of functional recovery while sham experiments did not. We show unprecedented recovery from cognitive symptoms in addition to a clear clinical motor recuperation. Motor and cognitive recovery but not circadian rhythm recovery correlated with the degree of graft integration into the host environment and with in-vivo levels of striatal dopaminergic transporters and function. This study provides empirical evidence that neural stem cells transplantation efficiently restore function at multiple levels in Parkinsonian non-human primates. We demonstrate the promising potential of multiple-sites neural stem cells grafts for Parkinson’s disease but furthermore underline the crucial importance of such multidisciplinary approaches for an effective clinical translation.

Successful antidepressant (AD) treatments are still difficult to achieve. Recently, bright light stimulation (BLS) was shown effective in non-seasonal depression but its mode of action remains elusive. We demonstrate here, using a new mouse model of depression resistant to ADs including ketamine, that chemogenetic activation of lateral habenula (LHb) astroglia prevented the potentiating effect of BLS on the AD response. Additionally, the beneficial action of BLS was associated with upregulation of a specific part of the prefrontal cortex opioid system. These results show that improved behavioral outcome produced by BLS requires habenular astroglia and endogenous opioids as crucial buffer systems.

INTRODUCTION The interaction among cell-autonomous circadian oscillators—daily cycles of activity–rest and feeding–fasting—produces diurnal rhythms in gene expression in almost all animal tissues. These rhythms control the timing of a wide range of functions across different organs and brain regions, affording optimal fitness. Chronic disruption of these rhythms predisposes to and are hallmarks of numerous diseases and affective disorders. RATIONALE Time-series gene expression studies in a limited number of tissues from rodents have shown that 10 to 40% of the genome exhibits a ~24-hour rhythm in expression in a tissue-specific manner. However, rhythmic expression data from diverse tissues and brain regions from humans or our closest primate relatives is rare. Such multitissue diurnal gene expression data are necessary for gaining mechanistic understanding of how spatiotemporal orchestration of gene expression maintains normal physiology and behavior. We used a RNA sequencing technique to assess gene expression in major tissues and brain regions from baboons (a primate closely related to humans) housed under a defined 24-hour light–dark and feeding–fasting schedule. RESULTS We assessed gene expression in 64 different tissues and brain regions of male baboons, collected every 2 hours over the 24-hour day. Tissue-specific transcriptomes in baboon were comparable with that from humans (Human GTEx data set). We detected >25,000 expressed transcripts, including protein-coding and -noncoding RNAs. Nearly 11,000 genes were commonly expressed in all tissues. These universally expressed genes (UEGs) encoded for basic cellular functions such as transcription, RNA processing, DNA repair, protein homeostasis, and cellular metabolism. The remainders were expressed in distinct sets of tissues, with ~1500 genes expressed exclusively in a single tissue. Rhythmic transcripts were found in all tissues, but the number of cycling transcripts varied from ~200 to >3000 in a given tissue, with only limited overlap in the repertoire of rhythmic transcripts between tissues. Of the 11,000 UEGs, the vast majority (96.6%) showed 24-hour rhythmicity in at least one tissue. A majority (>80%) of the 18,000 protein-coding genes detected also exhibited 24-hour rhythms in expression. The most enriched rhythmic transcripts across tissues were core clock components and their immediate output targets. However, their relative abundance and robustness of daily rhythms varied across tissues. Considered at the organismal level, global rhythmic transcription in 64 tissues organized into bursts of peak transcription, during early morning and late afternoon (when 11,000 transcripts reach their peak level). By contrast, during a relative “quiescent phase” in early evening that coincides with the onset of sleep and no food intake, only 700 rhythmic transcripts reach their peak expression level. CONCLUSION The daily expression rhythms in >80% of protein-coding genes, encoding diverse biochemical and cellular functions, constitutes by far the largest regulatory mechanism that integrates diverse biochemical functions within and across cell types. From a translational point of view, rhythmicity may have a major impact in health because 82.2% of genes coding for proteins that are identified as druggable targets by the U.S. Food and Drug Administration show cyclic changes in transcription.

Although a wide range of physiological functions regulated by dopamine (DA) display circadian variations, the role of DA in the generation and/or modulation of these rhythms is unknown. In Parkinson's disease (PD) patients, in addition to the classical motor symptoms, disturbances of the pattern of daily rest/wake cycles are common non-motor symptoms. We investigated daily and circadian rhythms of rest/activity behaviors in a transgenic MitoPark mouse model with selective inactivation of mitochondrial transcription factor A (Tfam) resulting in a slow and progressive degeneration of DA neurons in midbrain structures. Correlated with this, MitoPark mice show a gradual reduction in locomotor activity beginning at about 20 weeks of age. In a light–dark cycle, MitoPark mice exhibit a daily pattern of rest/activity rhythms that shows an age-dependent decline in both the amplitude and the stability of the rhythm, coupled with an increased fragmentation of day/night activities. When the circadian system is challenged by exposure to constant darkness or constant light conditions, control littermates retain a robust free-running circadian locomotor rhythm, whereas in MitoPark mice, locomotor rhythms are severely disturbed or completely abolished. Re-exposure to a light/dark cycle completely restores daily locomotor rhythms. MitoPark mice and control littermates express similar masking behaviors under a 1 h light/1 h dark regime, suggesting that the maintenance of a daily pattern of rest/activity in arrhythmic MitoPark mice can be attributed to the acute inhibitory and stimulatory effects of light and darkness. These results imply that, in addition to the classical motor abnormalities observed in PD, the loss of the midbrain DA neurons leads to impairments of the circadian control of rest/activity rhythms.

Purpose of the study Major Depression (MD) is the most frequent psychiatric disorders. Despite the large range of available antidepressants, MD treatments remain unsatisfactory due to delayed onset of action and partial therapeutic responsiveness. Hence, more effective antidepressant treatments are urgently required. Recently, an increased awareness of light on mood has been highlighted. Light was shown to synchronize the circadian clock and modulate brain structures involved in sleep regulation, mood and cognition, via light sensitive melanopsin retinal ganglion cells (ipRGCs). Aberrant light cycles and/or disrupted circadian rhythmicity produce depressive states. Inversely, luminotherapy has been proven to be effective in seasonal depression but also in unipolar depression [1]. However, the underlying mechanisms of light are still unknown. Several studies reveal that i) ipRGCs play an important role in mood regulation induced by light [2] and ii) circadian and 5-HT systems are two important interactive regulatory brain networks that modulate mood. The purpose of this study was to decipher the neurobiological mechanisms by which light potentiates the antidepressant response. Methods An original model of treatment resistant depression [3] was used, based on a modified version of the forced swimming test. Briefly, mice were put in a tank filled with water (25°C) during 10 minutes 5 consecutive days (4 hours after lights off). Then, one group of mice were exposed to standard light (100 lux) and another group received add-on one hour of bright light (1000 lux, one hour before lights off) during 9 weeks. Weekly, during 2 months (2 hours after lights off), the immobility time was analyzed during the 4 first minutes of the swimming time. In parallel, the locomotor activity of the mice was assessed in their home-cage using the Circadian Activity Monitoring System. Drugs were injected 30 minutes prior to FST on week 8 and 9. Results In both groups of mice, the immobility time, observed in day 2 to 5, was significantly increased (+40%) and remained stable for at least 8 weeks while a single dose of the NMDA receptor antagonist ketamine (3 mg/kg, i.p) failed to reverse this pseudo-depressive behavior. Interestingly, only the combination of ketamine (3 mg/kg) and the muscarinic antagonist scopolamine (0.1 mg/kg) associated with bright light stimulation was able to decrease the immobility time. No difference was observed in locomotor activity, suggesting that the immobility evaluation corresponds to a behavioral despair. Moreover, a reduced sucrose preference was observed in stressed mice. Conclusion In this novel, validated model of depression [3], we showed that the immobility observed after 5 days of swim stress could remain stable for more than 5 weeks and this mainly because the repeated stress was performed during the dark phase [4]. Repeated bright light exposure, one hour per day during 8 weeks, potentiates the antidepressant effect of a sub-effective combination of ketamine and scopolamine [5]. Together, these results suggest, for the first time in treatment resistant mice, that light, in combination of a pharmacological treatment, can unveil an antidepressant response. Studies are currently ongoing to determine the role of ipRGCs in this surprising response.

Light is a potent stimulus for regulating circadian, hormonal, and behavioral systems. In addition, light therapy is effective for certain affective disorders, sleep problems, and circadian rhythm disruption. These biological and behavioral effects of light are influenced by a distinct photoreceptor in the eye, melanopsin containing intrinsically photosensitive retinal ganglion cells (ipRGCs), in addition to the conventional rods and cones. We summarize the neurophysiology of this newly described sensory pathway and consider implications for the measurement, production and application of light. A new light-measurement strategy taking account of the complex photoreceptive input to these non-visual responses is proposed for use by researchers, and simple suggestions for artificial/architectural lighting are provided for regulatory authorities, lighting manufacturers, designers and engineers.

The retinal circadian clock is crucial for optimal regulation of retinal physiology and function, yet its cellular location in mammals is still controversial. We used laser microdissection to investigate the circadian profiles and phase relations of clock gene expression and Period gene induction by light in the isolated outer (rods/cones) and inner (inner nuclear and ganglion cell layers) regions in wild-type and melanopsin-knockout (Opn 4 (-/-) ) mouse retinas. In the wild-type mouse, all clock genes are rhythmically expressed in the photoreceptor layer but not in the inner retina. For clock genes that are rhythmic in both retinal compartments, the circadian profiles are out of phase. These results are consistent with the view that photoreceptors are a potential site of circadian rhythm generation. In mice lacking melanopsin, we found an unexpected loss of clock gene rhythms and of the photic induction of Per1-Per2 mRNAs only in the outer retina. Since melanopsin ganglion cells are known to provide a feed-back signalling pathway for photic information to dopaminergic cells, we further examined dopamine (DA) synthesis in Opn 4 (-/-) mice. The lack of melanopsin prevented the light-dependent increase of tyrosine hydroxylase (TH) mRNA and of DA and, in constant darkness, led to comparatively high levels of both components. These results suggest that melanopsin is required for molecular clock function and DA regulation in the retina, and that Period gene induction by light is mediated by a melanopsin-dependent, DA-driven signal acting on retinal photoreceptors.

In mammals, non-visual responses to light involve intrinsically photosensitive melanopsin-expressing retinal ganglion cells (ipRGCs) that receive synaptic inputs from rod and cone photoreceptors. Several studies have shown that cones also play a role in light entrainment, photic responses of the suprachiasmatic nucleus (SCN), pupil constriction, and sleep induction. These studies suggest that cones are mainly involved in the initial response to light, whereas melanopsin provides a sustained input for nonvisual responses during continued light exposure. Based on this idea, we explored the effects of the absence of middle-wavelength (MW)-cones on the temporal responses of circadian behavior and clock gene expression in light. In mice lacking MW-cones, our results show a reduction in behavioral phase shifts in response to light stimulations of short duration at 480 and 530 nm, but no alteration for short-wavelength (360-nm) light exposures. Similarly, induction of the period gene mPer1 and mPer2 mRNAs in the SCN are attenuated in response to light exposures of mid to long wavelengths. Modeling of the photoresponses shows that mice lacking MW-cones have an overall reduction in sensitivity that increases with longer wavelengths. The differences in photic responsiveness are consistent with the idea that cones provide a strong initial phasic input to the circadian system at light-onset and may confer a priming effect on ipRGC responses to sub-threshold light exposures. In summary, the contribution of MW-cones is essential for the normal expression of phase shifts and clock gene induction by light in mammals. (Author correspondence: ouria.benyahya@inserm.fr)

Mammalian retinal photoreceptors form an irradiance detection system that drives many nonvisual responses to light such as pupil reflex and resetting of the circadian clock. To understand the role of pupil size in circadian light responses, pupil diameter was pharmacologically manipulated and the effect on behavioral phase shifts at different irradiance levels was studied in the Syrian hamster. Dose-response curves for steady-state pupil size and for behavioral phase shifts were constructed for 3 pupil conditions (dilated, constricted, and control). Retinal irradiance was calculated from corneal irradiance, pupil size, retinal surface area, and absorption of ocular media. The sensitivity of photic responses to retinal irradiance is approximately 1.5 log units higher than to corneal irradiance. When plotted against corneal irradiance, pharmacological pupil constriction reduces the light sensitivity of the circadian system, but pupil dilation has no effect. As expected, when plotted against retinal irradiance all dose-response curves superimposed, confirming that the circadian system responds to photon flux on the retina. Pupil dilation does not increase the circadian response to increasing irradiance, since the response of the circadian system attains saturation at irradiance levels lower than those required to induce pupil constriction. The main finding shows that due to the different response sensitivities, the effect of pupil constriction on the light sensitivity of the circadian system in the hamster under natural conditions is virtually negligible. We further suggest the existence of distinct modulating mechanisms for the differential retinal irradiance sensitivity of the pupil system and the circadian system, which enables the different responses to be tuned to their specific tasks while using similar photoreceptive input.

Objectif Les cellules ganglionnaires a melanopsine (ipRGC), avec les cones et bâtonnets, forment un reseau retinien de photoreception sous-tendant l’effet non visuel (NV) de la lumiere. L’objectif de notre travail est de determiner la contribution individuelle de ces substrats neuronaux dans les reponses du cerveau NV. Methodes Notre strategie experimentale repose sur les proprietes spectrales, spatiales differentes des cones et ipRGCs. Vingt-huit sujets (within-subject design) ont ete exposes consecutivement a 4 stimuli lumineux de 50 min chacun, de 19–23 h. Le stimulus fut compose d’une lumiere blanche centrale (20°, 7000 lux, LBC) focalisee sur la fovea (cones), et d’une lumiere en peripherie (20–220°, 300 lux) enrichie en bleu (BE) afin d’activer les ipRGCs, ou rouge (RE) afin de limiter cette activation. L’EEG, le reflexe pupillaire (PLR), les performances cognitives et visuelles ont ete mesurees pendant l’exposition. Resultats Nos resultats preliminaires ne montrent pas de difference entre RE vs BE dans la dynamique de l’activite beta (13,5–32 Hz) et ondes lentes (1–7 Hz). Les performances cognitives sont similaires. L’allumage differee de la LBC provoque une constriction supplementaire de la pupille (+10 %, p Conclusion Nos resultats suggerent que la dynamique/sensibilite a la lumiere varie pour chaque reponse NV. Le PLR recoit un message photoreceptif impliquant les ipRGC et cones. De maniere surprenante, un stimulus lumineux presente dans le champ visuel peripherique, et de faible intensite (300 lux), peut recruter les ipRGC et activer les fonctions NV.

Le glaucome est une neuropathie optique chronique aboutissant a la degenerescence des cellules ganglionnaires retiniennes. Des etudes recentes montrent que l’atteinte glaucomateuse s’etend des cellules ganglionnaires jusqu’aux centres visuels centraux. La degenerescence cellulaire est observee dans les couches magno, parvo et koniocellulaires du noyau genicule lateral, et ces modifications sont associees a l’augmentation de la pression intraoculaire et a l’atteinte du nerf optique. Il existe egalement, parallelement a la perte des cellules ganglionnaires, des modifications tissulaires au sein meme du cortex visuel. Dans un modele de rat glaucomateux, nous avons montre recemment que la perte des cellules ganglionnaires induit une reduction des projections retiniennes non seulement sur les structures centrales visuelles, mais egalement sur le systeme circadien. Des etudes chez l’homme suggerent que la degenerescence des cellules ganglionnaires retiniennes au cours du glaucome pourrait aboutir a une alteration de la voie retino-hypoathalamique permettant la synchronisation des rythmes circadiens.

Calcium-binding proteins are involved in numerous functional roles in the retina and are widely distributed in almost all retinal neurons. The present study aimed to characterize the distribution of the calcium-binding proteins calbindin, calretinin, parvalbumin and recoverin in relation to retinal cell types in a strepsirhine primate (mouse lemur, Microcebus) in comparison with primate species of the three main haplorhine lineages (marmoset, macaque and human), as well as a rodent (gerbil, Taterillus). The main findings show that whereas the recoverin antibody labels both rod and cone photoreceptors in all species, calbindin consistently labels cones, but not rods, in the haplorhine primates marmoset, macaque and human, but none of the photoreceptors in the mouse lemur. Marmoset and macaque also show a distinct label of cone outer segments with calretinin. Depending on the species, bipolar cells express calbindin and/or recoverin, while amacrine, horizontal and ganglion cells are labeled to varying degrees with calbindin, calretinin and parvalbumin. Haplorhine and strepsirhine primates clearly differ in the expression of calcium-binding protein expression in horizontal cells. In all haplorhine species, horizontal cells are densely labeled with parvalbumin whereas in mouse lemur horizontal cells express calbindin but not parvalbumin. Several characteristics of the calcium-binding immunostaining in the retina of the mouse lemur are similar to those observed in the rodent, and distinguish this species from the diurnal haphorhine primates. These differences may be related to adaptations of retinal structure and function to the nocturnal niche, since nocturnal strepsirhine and haphorhine (Tarsius and Aotus) primates share some features of calcium-binding expression.

Illumination of different areas of the human retina elicits differences in acute light-induced suppression of melatonin. The aim of this study was to compare changes in plasma melatonin levels when light exposures of equal illuminance and equal photon dose were administered to superior, inferior, and full retinal fields. Nine healthy subjects participated in the study. Plexiglass eye shields were modified to permit selective exposure of the superior and inferior halves of the retinas of each subject. The Humphrey Visual Field Analyzer was used both to confirm intact full visual fields and to quantify exposure of upper and lower visual fields. On study nights, eyes were dilated, and subjects were exposed to patternless white light for 90 min between 0200 and 0330 under five conditions: (1) full retinal exposure at 200 lux, (2) full retinal exposure at 100 lux, (3) inferior retinal exposure at 200 lux, (4) superior retinal exposure at 200 lux, and (5) a dark-exposed control. Plasma melatonin levels were determined by radioimmunoassay. ANOVA demonstrated a significant effect of exposure condition ( F = 5.91, p < 0.005). Post hoc Fisher PLSD tests showed significant ( p [#003C] 0.05) melatonin suppression of both full retinal exposures as well as the inferior retinal exposure; however, superior retinal exposure was significantly less effective in suppressing melatonin. Furthermore, suppression with superior retinal exposure was not significantly different from that of the dark control condition. The results indicate that the inferior retina contributes more to the light-induced suppression of melatonin than the superior retina at the photon dosages tested in this study. Findings suggest a greater sensitivity or denser distribution of photoreceptors in the inferior retina are involved in light detection for the retinohypothalamic tract of humans.

The induction of the proto-oncogene c-fos, and its phosphoprotein product Fos, has been extensively used to study the effects of light on the circadian pacemaker in the suprachiasmatic nucleus (SCN). Experimental approaches to the quantification of Fos induction have mainly been based on immunohistochemistry and subsequent measure of Fos immunoreactivity (IR) in sections of the SCN. In this study, the authors compare several methods of quantification using optical density image analysis or counts of Fos-IR labeled cells. To assess whether optical density measures using image analysis reflect the amount of Fos in brain tissue, the authors developed standards of known concentrations of Fos protein in an agar matrix. The agar standards were sectioned and treated simultaneously with sections of the SCN from animals exposed to different levels of irradiance. Optical density was found to be proportional to the quantity of Fos in the sections, indicating that this measure accurately reflects relative levels of Fos protein induction. Quantification by optical density analysis allows an objective measure in which the various parameters, conditions of illumination, and threshold can be maintained constant throughout the analysis. Counting cells by visual observation is more subjective because threshold values cannot be precisely defined and can vary according to the observer, illumination, degree of label, and other factors. In addition, cell counts involving direct visual observation, automated cell counts, or stereological methods do not take into account the difference in the density of label between cells, thus giving equal weight to lightly or densely stained cells. These measures are more or less weakly correlated with measures of optical density and thus do not accurately reflect the amount of bound Fos protein in the tissue sections. In contrast, labeled surface area as measured by image analysis shows a linear relationship with optical density. The main outcome of this study is that computer-assisted image analysis provides an accurate and rapid method to determine the relative amount of Fos protein in the SCN and the effects of light on intracellular signaling mechanisms involved in the circadian clock.

The delayed appearance of motor symptoms in PD poses a crucial challenge for early detection of the disease. We measured the binding potential of the selective dopamine active transporter (DAT) radiotracer [(11)C]PE2I in MPTP-treated macaque monkeys, thus establishing a detailed profile of the nigrostriatal DA status following MPTP intoxication and its relation to induced motor and non-motor symptoms. Clinical score and cognitive performance were followed throughout the study. We measured longitudinally in vivo the non-displaceable binding potential to DAT in premotor, motor-recovered (i.e. both non-symptomatic) and symptomatic MPTP-treated monkeys. Results show an unexpected and pronounced dissociation between clinical scores and [(11)C]PE2I-BP(ND) during the premotor phase i.e. DAT binding in the striatum of premotor animals was increased around 20%. Importantly, this broad increase of DAT binding in the caudate, ventral striatum and anterior putamen was accompanied by i) deteriorated cognitive performance, showing a likely causal role of the observed hyperdopaminergic state (Cools, 2011; Cools and D'Esposito, 2011) and ii) an asymmetric decrease of DAT binding at a focal point of the posterior putamen, suggesting that increased DAT is one of the earliest, intrinsic compensatory mechanisms. Following spontaneous recovery from motor deficits, DAT binding was greatly reduced as recently shown in-vivo with other radiotracers (Blesa et al., 2010, 2012). Finally, high clinical scores were correlated to considerably low levels of DAT only after the induction of a stable parkinsonian state. We additionally show that the only striatal region which was significantly correlated to the degree of motor impairments is the ventral striatum. Further research on this period should allow better understanding of DA compensation at premature stages of PD and potentially identify new diagnosis and therapeutic index.

Light is a powerful stimulant for human alertness and cognition, presumably acting through a photoreception system that heavily relies on the photopigment melanopsin. In humans, evidence for melanopsin involvement in light-driven cognitive stimulation remains indirect, due to the difficulty to selectively isolate its contribution. Therefore, a role for melanopsin in human cognitive regulation remains to be established. Here, sixteen participants underwent consecutive and identical functional MRI recordings, during which they performed a simple auditory detection task and a more difficult auditory working memory task, while continuously exposed to the same test light (515 nm). We show that the impact of test light on executive brain responses depends on the wavelength of the light to which individuals were exposed prior to each recording. Test-light impact on executive responses in widespread prefrontal areas and in the pulvinar increased when the participants had been exposed to longer (589 nm), but not shorter (461 nm), wavelength light, more than 1 h before. This wavelength-dependent impact of prior light exposure is consistent with recent theories of the light-driven melanopsin dual states. Our results emphasize the critical role of light for cognitive brain responses and are, to date, the strongest evidence in favor of a cognitive role for melanopsin, which may confer a form of “photic memory” to human cognitive brain function.

In the blind subterranean mole rat Spalax ehrenbergi superspecies complete ablation of the visual image-forming capability has been accompanied by an expansion of the bilateral projection from the retina to the suprachiasmatic nucleus. We have cloned the open reading frame of a visual pigment from Spalax that shows >90% homology with mammalian rod pigments. Baculovirus expression yields a membrane protein with all functional characteristics of a rod visual pigment (λmax = 497 ± 2 nm; pK a of meta I/meta II equilibrium = 6.5; rapid activation of transducin in the light). We not only provide evidence that this Spalax rod pigment is fully functional in vitro but also show that all requirements for a functional pigment are present in vivo. The physiological consequences of this unexpected finding are discussed. One attractive option is that during adaptation to a subterranean lifestyle, the visual system of this mammal has undergone mosaic reorganization, and the visual pigments have adapted to a function in circadian photoreception.

The mole rat is a solitary, subterranean and photoperiodic rodent. We investigated its rest activity behavior under several lighting conditions, complemented our observations with light-induced c-fos expression, and compared the activity behavior of two chromosomal forms (2n = 58 and 60). The 26 mole rats had a clear overall preference for activity in the light or dark period, but prolonged recordings in five individuals showed that the initial preference was not stable in the nocturnal animals, they became diurnal. A 6-h advance of the light-dark (LD) cycle induced a shift of activity and the previous LD preference was reestablished. The large daily variability of activity onset did not allow this study to determine whether the animals were entrained to the LD cycle upon release into constant darkness (DD) or whether activity had been masked by light. The period of the motor activity rhythm in DD free ran in more than 50% of the animals. No differences in activity were observed between the two karyotypes. Immunohistochemistry for c-fos expression in the nucleus suprachiasmaticus at different circadian times showed that c-fos was induced only in animals exposed to a 1-h light pulse during the subjective night, but not during the subjective day or in control animals in the absence of a light pulse. The large intra- and inter-individual variability in daily motor activity both in LD and in DD suggest only a weak photic entrainment of the circadian clock to light of approximately 100 lux, and possibly a weak regulation of behavior by the circadian clock.

The availability of naturally occurring and transgenic retinal mutants has made the mouse an attractive experimental model to address questions regarding photoentrainment of circadian rhythms. However, very little is known about the retinal cells and the retinal projections to the nuclei of the murine circadian timing system. Furthermore, the effect of inherited retinal degeneration on these projections is not understood. In this report, we have used pseudorabies virus as a neuroanatomical tract tracer in mice to address a series of questions: Which retinal cells mediate circadian responses to light? What is the nature of the retinohypothalamic projection? What is the impact of the inherited retinal disorder, retinal degenerate (rd/rd), on the structures of the photoentrainment pathway? Our results show that a class ofretinal ganglion cell, morphologically similar to the type III ganglion cells of the rat, appears to project to central circadian structures of the mouse. They are few in number and sparsely distributed throughout the retina. The low number and broad distribution of these specialized retinal ganglion cells may be an adaptive mechanism to integrate environmental irradiance without compromising the spatial resolution required for vision. In addition, viral infection of conelike and rodlike photoreceptors and amacrinelike cells suggest that these cells may mediate or contribute to circadian responses to light. Inherited retinal degeneration has no obvious effect on the anatomy of the retinal cells or their projections to the circadian axis. These anatomical findings are consistent with our previous findings showing that aged rd/rd mice are capable of regulating their circadian rhythms by light with unattenuated sensitivity.

Disturbances of the daily sleep/wake cycle are common non-motor symptoms of Parkinson's disease (PD). However, the impact of dopamine (DA) depletion on circadian rhythms in PD patients or non-human primate (NHP) models of the disorder have not been investigated. We evaluated alterations of circadian rhythms in NHP following MPTP lesion of the dopaminergic nigro-striatal system. DA degeneration was assessed by in vivo PET ([(11)C]-PE2I) and post-mortem TH and DAT quantification. In a light∶dark cycle, control and MPTP-treated NHP both exhibit rest-wake locomotor rhythms, although DA-depleted NHP show reduced amplitude, decreased stability and increased fragmentation. In all animals, 6-sulphatoxymelatonin peaks at night and cortisol in early morning. When the circadian system is challenged by exposure to constant light, controls retain locomotor rest-wake and hormonal rhythms that free-run with stable phase relationships whereas in the DA-depleted NHP, locomotor rhythms are severely disturbed or completely abolished. The amplitude and phase relations of hormonal rhythms nevertheless remain unaltered. Use of a light-dark masking paradigm shows that expression of daily rest-wake activity in MPTP monkeys requires the stimulatory and inhibitory effects of light and darkness. These results suggest that following DA lesion, the central clock in the SCN remains intact but, in the absence of environmental timing cues, is unable to drive downstream rhythmic processes of striatal clock gene and dopaminergic functions that control locomotor output. These findings suggest that the circadian component of the sleep-wake disturbances in PD is more profoundly affected than previously assumed.

The deterioration of sleep in the older population is a prevalent feature that contributes to a decrease in quality of life. Inappropriate entrainment of the circadian clock by light is considered to contribute to the alteration of sleep structure and circadian rhythms in the elderly. The present study investigates the effects of aging on non-visual spectral sensitivity to light and tests the hypothesis that circadian disturbances are related to a decreased light transmittance. In a within-subject design, eight aged and five young subjects were exposed at night to 60 minute monochromatic light stimulations at 9 different wavelengths (420-620 nm). Individual sensitivity spectra were derived from measures of melatonin suppression. Lens density was assessed using a validated psychophysical technique. Although lens transmittance was decreased for short wavelength light in the older participants, melatonin suppression was not reduced. Peak of non-visual sensitivity was, however, shifted to longer wavelengths in the aged participants (494 nm) compared to young (484 nm). Our results indicate that increased lens filtering does not necessarily lead to a decreased non-visual sensitivity to light. The lack of age-related decrease in non-visual sensitivity to light may involve as yet undefined adaptive mechanisms.

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There is increasing evidence that melanopsin-expressing ganglion cells (ipRGCs) are altered in retinal pathologies. Using a streptozotocin-induced (STZ) model of diabetes, we investigated the impact of diabetic retinopathy on non-visual functions by analyzing ipRGCs morphology and light-induced c-Fos and Period 1–2 clock genes in the central clock (SCN). The ability of STZ-diabetic mice to entrain to light was challenged by exposure animals to 1) successive light/dark (LD) cycle of decreasing or increasing light intensities during the light phase and 2) 6-h advance of the LD cycle. Our results show that diabetes induces morphological changes of ipRGCs, including soma swelling and dendritic varicosities, with no reduction in their total number, associated with decreased c-Fos and clock genes induction by light in the SCN at 12 weeks post-onset of diabetes. In addition, STZ-diabetic mice exhibited a reduction of overall locomotor activity, a decrease of circadian sensitivity to light at low intensities, and a delay in the time to re-entrain after a phase advance of the LD cycle. These novel findings demonstrate that diabetes alters clock genes and behavioral responses of the circadian timing system to light and suggest that diabetic patients may show an increased propensity for circadian disturbances, in particular when they are exposed to chronobiological challenges.

In addition to the hallmark motor disorders in Parkinson's disease (PD) patients, nonmotor symptoms have attracted increasing attention. Among the nonmotor symptoms, sleep disturbances and cognitive deficits are frequently reported and contribute to a decrease in the quality of life. The pathophysiology of cognitive and sleep-wake abnormalities in PD is poorly understood partially due to the lack of appropriate animal models that fully replicate the entire pathological and behavioral spectrum of the disease. In this study, we undertook a long-term evaluation of circadian, locomotor and cognitive abilities in both acute and chronic MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine)-treated mouse models. Activity rhythms and locomotor activity were assayed under light-dark cycles, constant darkness, or constant light, re-entrainment to shifts of the light-dark cycle, and a behavioral masking paradigm. Cognitive abilities were assessed using a radial water maze task. Although both acute and chronic treatment regimes induced 70% degeneration of dopaminergic neurons in the substantia nigra, neither circadian nor cognitive alterations were observed even after nearly 1 yr. During aging, there was a significant decrease of locomotor activity and of several circadian parameters without any exacerbation in MPTP-treated animals. These results emphasize the limitations of the MPTP-treated mouse as an animal model of nonmotor symptoms of PD in addition to the already well-documented inadequacy to replicate cardinal motor features of the disease.

Studies of simian and prosimian primates have demonstrated a remarkable interspecific constancy in the pattern of innervation of the magnocellular and parvocellular layers of the dorsal lateral geniculate nucleus (dLGN) by retinal afferents. The characteristic organization of this nucleus in primates, as well as its apparent phylogenetic stability, have led to the proposal that the distinct laminar arrangement of the dLGN is one of the diagnostic characters that define the Order Primates. Here, we describe the distribution of retinal afferents to the dLGN of Tarsius, the single contemporary member of an ancient lineage of primates. In this genus, the more superficial layer of the dLGN receives projections from the ipsilateral eye, a unique situation among the members of the Order Primates. This observation adds support to the proposal that Tarsius does not share a more recent common ancestry with simians as compared with lemuriform and lorisiform primates.

Purpose Although several methods have been proposed to evaluate lens transmittance, to date there is no consensual in vivo approach in clinical practice. The aim of this study was to compare ocular lens density and transmittance measurements obtained by an improved psychophysical scotopic heterochromatic flicker photometry (sHFP) technique to the results obtained by three other measures: a psychophysical threshold technique, a Scheimpflug imaging technique, and a clinical assessment using a validated subjective scale. Methods Forty-three subjects (18 young, 9 middle aged, and 16 older) were included in the study. Individual lens densities were measured and transmittance curves were derived from sHFP indexes. Ocular lens densities were compared across methods by using linear regression analysis. Results The four approaches showed a quadratic increase in lens opacification with age. The sHFP technique revealed that transmittance decreased with age over the entire visual spectrum. This decrease was particularly pronounced between young and older participants in the short (53.03% decrease in the 400-500 nm range) wavelength regions of the light spectrum. Lens density derived from sHFP highly correlated with the values obtained with the other approaches. Compared to other objective measures, sHFP also showed the lowest variability and the best fit with a quadratic trend (r2 = 0.71) of lens density increase as a function of age. Conclusions The sHFP technique offers a practical, reliable, and accurate method to measure lens density in vivo and predict lens transmittance over the visible spectrum. An accurate quantification of lens transmittance should be obtained in clinical practice, but also in research in visual and nonvisual photoreception.

Many physiological and pathological conditions are associated with a change in the crystalline lens transmittance. Estimates of lens opacification, however, generally rely on subjective rather than objective measures in clinical practice. The goal of our study was to develop an improved psychophysical heterochromatic flicker photometry technique combined with existing mathematical models to evaluate the spectral transmittance of the human ocular media noninvasively. Our results show that it is possible to accurately estimate ocular media density in vivo in humans. Potential applications of our approach include basic research and clinical settings on visual and nonimage-forming visual systems.

Light emitting diodes (LEDs) are being used increasingly as light sources in life sciences applications such as in vision research, fluorescence microscopy and in brain-computer interfacing. Here we present an inexpensive but effective visual stimulator based on light emitting diodes (LEDs) and open-source Arduino microcontroller prototyping platform. The main design goal of our system was to use off-the-shelf and open-source components as much as possible, and to reduce design complexity allowing use of the system to end-users without advanced electronics skills. The main core of the system is a USB-connected Arduino microcontroller platform designed initially with a specific emphasis on the ease-of-use creating interactive physical computing environments. The pulse-width modulation (PWM) signal of Arduino was used to drive LEDs allowing linear light intensity control. The visual stimulator was demonstrated in applications such as murine pupillometry, rodent models for cognitive research, and heterochromatic flicker photometry in human psychophysics. These examples illustrate some of the possible applications that can be easily implemented and that are advantageous for students, educational purposes and universities with limited resources. The LED stimulator system was developed as an open-source project. Software interface was developed using Python with simplified examples provided for Matlab and LabVIEW. Source code and hardware information are distributed under the GNU General Public Licence (GPL, version 3).

Many nonvisual functions are regulated by light through a photoreceptive system involving melanopsin-expressing retinal ganglion cells that are maximally sensitive to blue light. Several studies have suggested that the ability of light to modulate circadian entrainment and to induce acute effects on melatonin secretion, subjective alertness, and gene expression decreases during aging, particularly for blue light. This could contribute to the documented changes in sleep and circadian regulatory processes with aging. However, age-related modification in the impact of light on steady-state pupil constriction, which regulates the amount of light reaching the retina, is not demonstrated. We measured pupil size in 16 young (22.8 ± 4 years) and 14 older (61 ± 4.4 years) healthy subjects during 45-second exposures to blue (480 nm) and green (550 nm) monochromatic lights at low (7 × 1012 photons/cm2/s), medium (3 × 1013 photons/cm2/s), and high (1014 photons/cm2/s) irradiance levels. Results showed that young subjects had consistently larger pupils than older subjects for dark adaptation and during all light exposures. Steady-state pupil constriction was greater under blue than green light exposure in both age groups and increased with increasing irradiance. Surprisingly, when expressed in relation to baseline pupil size, no significant age-related differences were observed in pupil constriction. The observed reduction in pupil size in older individuals, both in darkness and during light exposure, may reduce retinal illumination and consequently affect nonvisual responses to light. The absence of a significant difference between age groups for relative steady-state pupil constriction suggests that other factors such as tonic, sympathetic control of pupil dilation, rather than light sensitivity per se, account for the observed age difference in pupil size regulation. Compared to other nonvisual functions, the light sensitivity of steady-state pupil constriction appears to remain relatively intact and is not profoundly altered by age.

The retinal innervation, cytoarchitectural, and immunohistochemical organization of the suprachiasmatic nucleus (SCN) was studied in the domestic sheep. The SCN is a large elongated nucleus extending rostrocaudally for roughly 3 mm in the hypothalamus. The morphology is unusual in that the rostral part of the nucleus extends out of the main mass of the hypothalamus onto the dorsal aspect of the optic chiasm. Following intraocular injection of wheat-germ agglutininhorseradish peroxidase or tritiated amino acids, anterograde label is distributed throughout the SCN. Retinal innervation of the SCN is bilaterally symmetric or predominantly ipsilateral. Quantitative image analysis demonstrates that, although the amount of autoradiographic label is greatest in the ventral and central parts of the nucleus, density varies progressively between different regions. In addition to the SCN, retinal fibers are also seen in the medial preoptic area, the anterior and lateral hypothalamic areas, the dorsomedial hypothalamus, the retrochiasmatic area, and the basal telencephalon. Whereas the SCN can be identified using several techniques, complete delineation of the nucleus requires combined tract tracing, cytoarchitectural, and histochemical criteria. Compared with the surrounding hypothalamic regions, the SCN contains smaller, more densely packed neurons, and is largely devoid of myelinated fibers. Cell soma sizes are smaller in the ventral SCN than in the dorsal or lateral parts, but an obvious regional transition is lacking. Using Nissl, myelin, acetylcholinesterase, and cytochrome oxidase staining, the SCN can be clearly distinguished in the rostral and medial regions, but is less differentiated toward the caudal pole. Immunohistochemical demonstration of several neuropeptides shows that the neurochemical organization of the sheep SCN is heterogeneous, but that it lacks a distinct compartmental organization. Populations of different neuropeptide-containing cells are found throughout the nucleus, although perikarya positive for vasoactive intestinal polypeptide and fibers labeled for methionine-enkephalin are predominant ventrally; neurophysine-immunoreactive cells are more prominent in the dorsal region and toward the caudal pole. The results suggest that the intrinsic organization of the sheep SCN is characterized by gradual regional transitions between different zones.

BACKGROUND: It is increasingly recognized that non-motor symptoms are a prominent feature of Parkinson's disease and in the case of cognitive deficits can precede onset of the characteristic motor symptoms. Here, we examine in 4 monkeys chronically treated with low doses of the neurotoxin MPTP the early and long-term alterations of rest-activity rhythms in relationship to the appearance of motor and cognitive symptoms. METHODOLOGY/PRINCIPAL FINDINGS: Behavioral activity recordings as well as motor and cognitive assessments were carried out continuously and in parallel before, during and for several months following MPTP-treatment (12-56 weeks). Cognitive abilities were assessed using a task that is dependent on the functional integrity of the fronto-striatal axis. Rest-activity cycles were monitored continuously using infrared movement detectors of locomotor activity. Motor impairment was evaluated using standardized scales for primates. Results show that MPTP treatment led to an immediate alteration (within one week) of rest-activity cycles and cognitive deficits. Parkinsonian motor deficits only became apparent 3 to 5 weeks after initiating chronic MPTP administration. In three of the four animals studied, clinical scores returned to control levels 5-7 weeks following cessation of MPTP treatment. In contrast, both cognitive deficits and chronobiological alterations persisted for many months. Levodopa treatment led to an improvement of cognitive performance but did not affect rest-activity rhythms in the two cases tested. CONCLUSIONS/SIGNIFICANCE: Present results show that i) changes in the rest activity cycles constituted early detectable consequences of MPTP treatment and, along with cognitive alterations, characterize the presymptomatic stage; ii) following motor recovery there is a long-term persistence of non-motor symptoms that could reflect differential underlying compensatory mechanisms in these domains; iii) the progressive MPTP-monkey model of presymptomatic ongoing parkinsonism offers possibilities for in-depth studies of early non-motor symptoms including sleep alterations and cognitive deficits.

This study investigates the possibility of an endogenous circadian rhythm in retinal cone function in humans. A full-field cone electroretinogram (ERG) was performed every 2 h for 24 h under continuous rod-saturating ambient white light (53 ± 30 lux; pupils dilated) in nine healthy subjects. Distinct circadian variations were superimposed upon a gradual decrease in cone responsiveness to light, demonstrated most reliably in the implicit times of b-wave and oscillatory potentials, and to a lesser extent in amplitude and a-wave implicit times. After mathematical correction of the linear trend, the cone response was found to be greatest around 20:00 h and least around 06:00 h. The phase of the ERG circadian rhythm was not synchronized with the phase of the salivary melatonin rhythm measured the previous evening. Melatonin levels measured under constant light on the day of ERG assessments were suppressed by 53% on average compared to melatonin profiles obtained previously under near-total darkness in seven participants. The progressive decline in cone responsiveness to light over the 24 h may reflect an adaptation of the cone-driven retinal system to constant light, although the mechanism is unclear. The endogenous rhythm of cone responsiveness to light may be used as an additional index of central or retinal circadian clock time.

Retinal projections and visual thalamo-cortical connections were studied in the subterranean mole rat, belonging to the superspecies Spalax ehrenbergi, by anterograde and retrograde tracing techniques. Quantitative image analysis was used to estimate the relative density and distribution of retinal input to different primary visual nuclei. The visual system of Spalax presents a mosaic of both regressive and progressive morphological features. Following intraocular injections of horseradish peroxidase conjugates, the retina was found to project bilaterally to all visual structures described as receiving retinal afferents in non-fossorial rodents. Structures involved in form analysis and visually guided behaviors are reduced in size by more than 90%, receive a sparse retinal innervation, and are cytoarchitecturally poorly differentiated. The dorsal lateral geniculate nucleus, as defined by cyto- and myelo-architecture, cytochrome oxidase, and acetylcholinesterase distribution as well as by afferent and efferent connections, consists of a narrow sheet 3-5 neurons thick, in the dorsal thalamus. Connections with visual cortex are topographically organized but multiple cortical injections result in widespread and overlapping distributions of geniculate neurons, thus indicating that the cortical map of visual space is imprecise. The superficial layers of the superior colliculus are collapsed to a single layer, and the diffuse ipsilateral distribution of retinal afferents also suggests a lack of precise retinotopic relations. In the pretectum, both the olivary pretectal nucleus and the nucleus of the optic tract could be identified as receiving ipsilateral and contralateral retinal projections. The ventral lateral geniculate nucleus is also bilaterally innervated, but distinct subdivisions of this nucleus or the intergeniculate leaflet could not be distinguished. The retina sends a sparse projection to the dorsal and lateral terminal nuclei of the accessory optic system. The medial terminal nucleus is not present. In contrast to the above, structures of the "non-image forming" visual pathway involved in photoperiodic perception are well developed in Spalax. The suprachiasmatic nucleus receives a bilateral projection from the retina and the absolute size, cytoarchitecture, density, and distribution of retinal afferents in Spalax are comparable with those of other rodents. A relatively hypertrophied retinal projection is observed in the bed nucleus of the stria terminalis. Other regions which receive sparse visual input include the lateral and anterior hypothalamic areas, the retrochiasmatic region, the sub-paraventricular zone, the paraventricular hypothalamic nucleus, the anteroventral and anterodorsal nuclei, the lateral habenula, the mediodorsal nucleus, and the basal telencephalon.(ABSTRACT TRUNCATED AT 400 WORDS)

The retinal projection to the basal telencephalon was studied in eight species of primates from the suborders Strepsirhini and Haplorhini, including one anthropoid primate, the gibbon. Animals received an intraocular injection of tritiated amino acids and the distribution of retinal fibers and terminals was demonstrated by autoradiographic techniques in horizontal and coronal sections. In all species a discrete group of labeled retinal fibers is observed to branch off from the dorsolateral aspect of the optic tract at the level of the suprachiasmatic nucleus. These fibers, destined to the basal telencephalon, are topographically distinct from the retinal fibers which innervate the suprachiasmatic nucleus and medial hypothalamic regions. The fibers of the retinotelencephalic tract course dorsally above the supraoptic nucleus through the lateral hypothalamic area and then proceed further rostrally and laterally below the diagonal band of Broca towards the olfactory tubercle. Within the olfactory tubercle, terminal distribution of label is observed in the mediocaudal region along the granular cell layer II. In the macaque this cellular layer shows a characteristic thickening in the region of retinal terminals which is evident in both coronal and horizontal section. In some species this labeled region is seen within the superficial bulge of the tubercle on the ventral aspect of basal telencephalon. In all primates the retinal projection to olfactory tubercle is bilateral. In prosimians label is predominantly contralateral to the injected eye, in New World monkeys label is equally distributed on both sides of the brain and in Old World monkeys label is mainly found ipsilaterally. Retinal fibers were also seen in the periamygdaloid region but never extended as far as piriform cortex. These results, in addition to previous studies in other mammalian orders, confirm that the basal telencephalon, and in particular the olfactory tubercle, constitutes a region of visual and olfactory convergence. This sensory integration may be related to photic and chemosensory modulation of reproductive physiology and behavior.

African mole-rats (family Bathyergidae) are strictly subterranean rodent species that are rarely exposed to environmental light. Morphological and physiological adaptations to the underground environment include a severely reduced eye size and regressed visual system. Responses of the circadian system to light, however, appear to be intact, since mole-rats are able to entrain their circadian activity rhythms to the light-dark cycle and light induces Fos expression in the suprachiasmatic nucleus (SCN). Social organization varies from solitary species to highly elaborated eusocial structures, characterized by a distinct division of labor and in which one reproductive female regulates the behavior and reproductive physiology of other individuals in the colony. The authors studied light-induced Fos expression in the SCN to increasing light intensities in four mole-rat species, ranging from strictly solitary to highly social. In the solitary Cape mole-rat, light induces significant Fos expression in the SCN, and the number of Fos-immunopositive cells increases with increasing light intensity. In contrast, Fos induction in the SCN of social species was slightly greater than, but not statistically different from, the dark-control animals as is typical of most rodents. One species showed a trend for an increase in expression with increased light, whereas a second species showed no trend in expression. In the naked mole-rat, Fos expression appeared higher in the dark-controls than in the animals exposed to light, although the differences in Fos expression were not significant. These results suggest a gradient in the sensitivity of the circadian system to light in mole-rats, with a higher percentage of individuals that are unresponsive to light in correlation with the degree of sociality. In highly social species, such as the naked mole-rat that live in a relatively stable subterranean milieu in terms of food availability, temperature, constant darkness, and devoid of 24-h cyclic environmental cues, the temporal coordination of rest-wake activities may be dependent on social interactions and social status rather than on photic regulation of the circadian timing system.

The phase and period responses to short light pulses were studied in the jerboa, a seasonal, hibernating, nocturnal rodent from the Atlas region in Morocco. The jerboa, which is a saltatory species, showed precise activity onsets and offsets under a light-dark (LD) cycle using infrared captors to record locomotor activity. When released into constant darkness (DD), the majority of animals showed a circadian period (tau)24 h (mean tau = 23.89 +/- 0.13 h) and a lengthening of the activity span, alpha. Animals were subsequently exposed to up to eight 15-min light pulses, each separated by at least 2 wks, for up to 160 days in DD. During this span, most individuals maintained robust circadian rhythmicity, with clearly defined activity onsets and offsets, similar levels of total activity, duration of alpha, and percent activity occurring during the subjective night. The phase response curve (PRC) is typical of other nocturnal rodents, with light eliciting delays during late subjective day and early subjective night (CT8-CT19) and advances during late subjective night to early subjective day (CT19-CT2). A dead zone, when light had no effect on phase, is observed during mid-subjective day (CT3-CT8). A few individuals showed large (9 h) Type 0 phase resetting near the singularity region (CT19) that resulted in a complete phase reversal, but otherwise displayed normal phase-shifting responses at other CT times. The tau response curve showed a decrease in period from early to late subjective night with increases at other times, but these changes were small (maximum9 min) and highly variable. There was a distinct tendency for animals that had an initial short tau in DD to conserve a short tau during the series of light pulses and, inversely, for animals with long tau to conserve a long tau. This suggests possible constraints on the plasticity of variation of tau in relation to the endogenous period of the animal.

In addition to rods and cones, the human retina contains light-sensitive ganglion cells that express melanopsin, a photopigment with signal transduction mechanisms similar to that of invertebrate rhabdomeric photopigments (IRP). Like fly rhodopsins, melanopsin acts as a dual-state photosensitive flip-flop in which light drives both phototransduction responses and chromophore photoregeneration that bestows independence from the retinoid cycle required by rods and cones to regenerate photoresponsiveness following bleaching by light. To explore the hypothesis that melanopsin in humans expresses the properties of a bistable photopigment in vivo we used the pupillary light reflex (PLR) as a tool but with methods designed to study invertebrate photoreceptors. We show that the pupil only attains a fully stabilized state of constriction after several minutes of light exposure, a feature that is consistent with typical IRP photoequilibrium spectra. We further demonstrate that previous exposure to long wavelength light increases, while short wavelength light decreases the amplitude of pupil constriction, a fundamental property of IRP difference spectra. Modelling these responses to invertebrate photopigment templates yields two putative spectra for the underlying R and M photopigment states with peaks at 481 nm and 587 nm respectively. Furthermore, this bistable mechanism may confer a novel form of "photic memory" since information of prior light conditions is retained and shapes subsequent responses to light. These results suggest that the human retina exploits fly-like photoreceptive mechanisms that are potentially important for the modulation of non-visual responses to light and highlights the ubiquitous nature of photoswitchable photosensors across living organisms.

At the optic chiasm retinal fibers either cross the midline, or remain uncrossed. Here we trace hemispheric pathways through the marmoset chiasm and show that fibers from the lateral optic nerve pass directly toward the ipsilateral optic tract without any significant change in fiber order and without approaching the midline, while those from medial regions of the nerve decussate directly. Anterograde labeling from one eye shows that the two hemispheric pathways remain segregated through the proximal nerve and chiasm with the uncrossed confined laterally. Retrograde labeling from the optic tract confirms this. This clearly demonstrates that hemispheric pathways are segregated through the primate chiasm. Previous chiasmatic studies have been undertaken mainly on rodents and ferrets. In these species there is a major change in fiber order pre-chiasmatically, where crossed and uncrossed fibers mix, reflecting their embryological history when all fibers approach the midline prior to their commitment to innervate either hemisphere. This pattern was thought to be common to placental mammals. In marsupials there is no change in fiber order and uncrossed fibers remain confined laterally through nerve and chiasm, again, reflecting their developmental history when all uncrossed fibers avoid the midline. Recently it has been shown that this distinction is not a true dichotomy between placental mammals and marsupials, as fiber order in tree shrews and humans mirrors the marsupial pattern. Architectural differences in the mature chiasm probably reflect different developmental mechanisms regulating pathway choice. Our results therefore suggest that both the organization and development of the primate optic chiasm differ markedly from that revealed in rodents and carnivores.

Glaucoma is a widespread ocular disease and major cause of blindness characterized by progressive, irreversible damage of the optic nerve. Although the degenerative loss of retinal ganglion cells (RGC) and visual deficits associated with glaucoma have been extensively studied, we hypothesize that glaucoma will also lead to alteration of the circadian timing system. Circadian and non-visual responses to light are mediated by a specialized subset of melanopsin expressing RGCs that provide photic input to mammalian endogenous clock in the suprachiasmatic nucleus (SCN). In order to explore the molecular, anatomical and functional consequences of glaucoma we used a rodent model of chronic ocular hypertension, a primary causal factor of the pathology. Quantitative analysis of retinal projections using sensitive anterograde tracing demonstrates a significant reduction (approximately 50-70%) of RGC axon terminals in all visual and non-visual structures and notably in the SCN. The capacity of glaucomatous rats to entrain to light was challenged by exposure to successive shifts of the light dark (LD) cycle associated with step-wise decreases in light intensity. Although glaucomatous rats are able to entrain their locomotor activity to the LD cycle at all light levels, they require more time to re-adjust to a shifted LD cycle and show significantly greater variability in activity onsets in comparison with normal rats. Quantitative PCR reveals the novel finding that melanopsin as well as rod and cone opsin mRNAs are significantly reduced in glaucomatous retinas. Our findings demonstrate that glaucoma impacts on all these aspects of the circadian timing system. In light of these results, the classical view of glaucoma as pathology unique to the visual system should be extended to include anatomical and functional alterations of the circadian timing system.

Connections of the accessory optic system (AOS) with the pretectum are described in the macaque monkey. Injections of tritiated amino acids in the pretectum demonstrate a major contralateral projection to the dorsal (DTN), lateral (LTN), and medial (MTN) terminal nuclei of the AOS and a sparser projection to the ipsilateral LTN. Injections of retrograde tracers, Fast Blue (FB), or wheat germ agglutinin horseradish peroxidase (WGA-HRP) plus nonconjugated horseradish peroxidase (HRP) in the LTN show that the pretectal-LTN projection originates from two nuclei. The main source of pretectal efferents to the LTN is from the pretectal olivary nucleus (OPN) and is entirely contralateral. This projection, which appears unique to primates, originates from the large multipolar cells of the OPN. In addition to this projection, the nucleus of the optic tract (NOT) projects to the ipsilateral LTN, as in nonprimates. Injection of WGA-HRP in the pretectum shows a reciprocal predominantely ipsilateral projection from the LTN to the pretectum. Retinas were observed after injection of FB in the LTN. The retinal ganglion cells projecting to the AOS are mainly distributed near the fovea and in the nasal region of the contralateral eye, suggesting a nasotemporal pattern of decussation. The demonstration of a directmore » connection between LTN and OPN forces to a reconsideration of the functional role of the AOS. Previous descriptions of luminance responsive cells in the LTN support a possible participation of this nucleus in the control of the pupillary light reflex.« less

In mammals, nonvisual responses to light have been shown to involve intrinsically photosensitive retinal ganglion cells (ipRGC) that express melanopsin and that are modulated by input from both rods and cones. Recent in vitro evidence suggests that melanopsin possesses dual photosensory and photoisomerase functions, previously thought to be a unique feature of invertebrate rhabdomeric photopigments. In cultured cells that normally do not respond to light, heterologous expression of mammalian melanopsin confers light sensitivity that can be restored by prior stimulation with appropriate wavelengths. Using three different physiological and behavioral assays, we show that this in vitro property translates to in vivo, melanopsin-dependent nonvisual responses. We find that prestimulation with long-wavelength light not only restores but enhances single-unit responses of SCN neurons to 480-nm light, whereas the long-wavelength stimulus alone fails to elicit any response. Recordings in Opn4-/- mice confirm that melanopsin provides the main photosensory input to the SCN, and furthermore, demonstrate that melanopsin is required for response enhancement, because this capacity is abolished in the knockout mouse. The efficiency of the light-enhancement effect depends on wavelength, irradiance, and duration. Prior long-wavelength light exposure also enhances short-wavelength—induced phase shifts of locomotor activity and pupillary constriction, consistent with the expression of a photoisomerase-like function in nonvisual responses to light.

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