Responses of synaptic ribbons in pineal photoreceptors under normal and experimental lighting conditions

The size of synaptic ribbons (SR) in photoreceptor cells of the goldfish pineal organ was quantified over 24-h light:dark cycles of long (16:8) and short (10:14) photoperiods during summer and winter months, respectively. The amplitude of both rhythms was similar with peak values occurring toward the latter part of the photophase or early dark. When fish were entrained to the long photoperiod and exposed to continual light, SR size continued to increase during the expected dark time. The effect of extending the photoperiod into the expected dark time was diminished when fish were entrained to a short photoperiod and presented with 6 h of darkness at the end of the 24-h period. The size increase in response to environmental lighting is believed to reflect a greater demand for either vesicle attachment sites or neurotransmitter storage sites since vesicles (neurotransmitter) have been hypothesized to accumulate in the synaptic pedicles during inhibition by light. From a comparative standpoint it is noteworthy that synaptic ribbons (vesicle-crowned rods) in mammals react in a similar manner to both normal and experimental lighting conditions.     

Apoptotic death of photoreceptors in the streptozotocin-induced diabetic rat retina

Aims/hypothesis Neurodegenerative changes in the diabetic retina occurring before diabetic retinopathy could be inevitable by the altered energy (glucose) metabolism, in the sense that dynamic image-processing activity of the retinal neurons is exclusively dependent on glucose. We therefore investigated the morphological changes in the neural retina, including neuronal cell death, of a streptozotocin-induced model of diabetes.Methods Streptozotocin was intravenously injected. Rats were maintained hyperglycaemic without insulin treatment for 1 week and 4, 8, 12, and 24 weeks, respectively. Diabetic retinas were processed for histology, electron microscopy, and immunohistochemistry using the TUNEL method.Results A slight reduction in the thickness of the inner retina was observed throughout the diabetic retinas and a remarkable reduction was seen in the outer nuclear layer 24 weeks after the onset of diabetes. The post-synaptic processes of horizontal cells in the deep invaginations of the photoreceptors showed degeneration changes from 1 week onwards. A few necrotic ganglion cells were observed after 4 weeks. At 12 weeks, some amacrine cells and a few horizontal cells showed necrotic features. Three to seven cellular layers in the outer nuclear layer and nerve terminals, rolled by the fine processes of the Müller cells near the somata of the degenerated ganglion cells, were apparent at 24 weeks. Apoptosis appeared in a few photoreceptor cells at 4 weeks, and the number of apoptotic photoreceptors increased thereafter.Conclusion/interpretation These findings suggest that the visual loss associated with diabetic retinopathy could be attributed to an early phase of substantial photoreceptor loss, in addition to later microangiopathy.Abbreviations TUNEL Terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling - GCL ganglion cell layer - IPL inner plexiform layer - INL inner nuclear layer - OPL outer plexiform layer - ONL outer nuclear layer     

Adhesion GPCR Latrophilin 3 regulates synaptic function of cone photoreceptors in a trans-synaptic manner

Cone photoreceptors mediate daylight vision in vertebrates. Changes in neurotransmitter release at cone synapses encode visual information and is subject to precise control by negative feedback from enigmatic horizontal cells. However, the mechanisms that orchestrate this modulation are poorly understood due to a virtually unknown landscape of molecular players. Here, we report a molecular player operating selectively at cone synapses that modulates effects of horizontal cells on synaptic release. Using an unbiased proteomic screen, we identified an adhesion GPCR Latrophilin3 (LPHN3) in horizontal cell dendrites that engages in transsynaptic control of cones. We detected and characterized a prominent splice isoform of LPHN3 that excludes a element with inhibitory influence on transsynaptic interactions. A gain-of-function mouse model specifically routing LPHN3 splicing to this isoform but not knockout of LPHN3 diminished Ca_V1.4 calcium channel activity profoundly disrupted synaptic release by cones and resulted in synaptic transmission deficits. These findings offer molecular insight into horizontal cell modulation on cone synaptic function and more broadly demonstrate the importance of alternative splicing in adhesion GPCRs for their physiological function.

Vision is a key sensory modality essential for the survival of most living organisms. In mammals, it is enabled by the retina: a neural structure composed of more than 60 distinct neurons each uniquely wired into the circuitry and with particular roles in image processing (1, 2). Vision begins with the detection of light by rod and cone photoreceptors. Rod photoreceptor cells are exquisitely sensitive to light and mediate vision at low light levels (3, 4). However, most vertebrates including humans rely on cone cells for daytime vision (5). Accordingly, cones have an extremely broad range of light sensitivity spanning 6 to 7 orders of magnitude (6), quickly adapting to changes in luminance and providing high spatial and temporal visual acuity (5, 7, 8). The molecular, cellular, and circuit mechanisms that allow cones to perform their tasks has been a subject of intense interest, providing groundbreaking discoveries that illuminate fundamental organizational principles that govern signal processing by neural circuits in general.The capture and processing of photons by the phototransduction cascade of cones generates graded changes in membrane potential: hyperpolarizing to light and depolarizing with darkness (7). These voltage signals alter the ongoing rate of neurotransmitter glutamate release at the cone synapse to relay information about light and dark to the retinal circuitry (9). The molecular entity that mediates this transformation is the L-type voltage-gated Ca²⁺ channel, Ca_V1.4 (10–12). It is located at specialized active zones containing synaptic ribbons and couples light-driven changes in voltage to changes in local Ca²⁺ levels thereby regulating the vesicular fusion machinery (13, 14). The Ca_V1.4 channel forms a macromolecular complex with a number of synaptic molecules and thus plays a pivotal role in both the structural and functional organization of the presynaptic active zone of photoreceptors (15). Accordingly, changes in Ca_V1.4 function imposed by binding partners or environment have a tremendous impact on the synaptic communication of cone photoreceptors and vision (16–18).Cones form synaptic contacts with three types of neurons. They synapse with postsynaptic ON- and OFF-type bipolar cells (BC) to relay visual information to the downstream neuronal circuitry (19, 20). Cones also contact lateral inhibitory neurons known as horizontal cells (HCs) that connect adjacent to BC dendrites, forming a tripartite synaptic triad (20). This elaborate synaptic arrangement of cones is a site of major influence on how visual information is processed contributing to unique cone physiology and adaptive capacity for daylight detection (21, 22).The function of HCs and their physiological mechanisms are particularly intriguing. HCs powerfully modulate synaptic transmission at cone synapses (23). Light-evoked hyperpolarization of HCs counteracts light-induced suppression of glutamate release from cone terminals, thereby providing strong negative feedback (23, 24). Because each HC contacts multiple cones, this negative influence on surrounding cones is a major mechanism for producing lateral inhibition, a classical feature of signal processing in the retina that enhances contrast and spatial resolution of vision (25). In addition, feedback from individual HC dendrites to specific cone terminals and ribbons can also act locally, fine-tuning synaptic output to local illumination gradients (26–29).While the role of HCs from a circuit perspective is well understood, the mechanisms that they use to provide negative feedback are subject to debate and controversy. At least three different explanations have been provided: direct ephaptic effects (30, 31), changes in synaptic pH (28, 32, 33), and modulation by GABA released from HCs (34, 35). These models are not necessarily mutually exclusive and unifying theories have been proposed (35, 36). Importantly, one of the central effects invariably observed in response to HC feedback is modulation of the Ca_V1.4 function at the active zones of cone terminals (32, 37). However, there is a significant void in our understanding of molecular mechanisms by which HCs modulate transmission of cone signals, mostly due to a paucity of players known to operate at this synapse. Identification and functional characterization of molecular elements involved in coordinating HC influence in cone synapses can transform our understanding of this enigmatic area of visual neuroscience.Here, we performed an unbiased proteomic profiling of proteins selectively enriched in cone synapses. This led to identification of an adhesion G protein–coupled receptor (aGPCR), latrophilin3 (LPHN3), whose role in retina physiology, photoreceptor synaptic development, and function was previously unexplored. We show that alternative splicing of LPHN3 in the retina generates unique isoforms with distinct properties. Using mouse models, we demonstrate that changes in LPHN3 splicing regulate cone synaptic transmission transsynaptically by affecting Ca_V1.4 function. These findings reveal a molecular player with a pivotal role in regulating synaptic function of cone photoreceptors.     

Daily Profiles of N-Acetyltransferase Measured at a Single Time in Rat Pineal Glands, Retinas, and Harderian Glands

Serotonin N-acetyltransferase activity (NAT) exhibited a daily cycle in light:dark (LD) 14:10 when it was measured in pineal glands taken from rats killed at a sequence of time points. The ratio of peak subjective night NAT to minimum subjective day NAT was 10.9/0.3 nmol per pineal gland per hour. When the rats were placed in constant dark the rhythm persisted (8.2/0.02). When the rats were placed in constant light the rhythm persisted with markedly attenuated amplitude (0.6/0.02).
We also measured NAT profiles in rat pineal glands, Harderian glands, and retinas with alternative methods. We kept rats on six LD 14:10 light-dark cycles with lights-out beginning at midnight, 2 AM, 4 AM, 6 AM, 8 AM, or 10 AM and killing all the rats at one time point, 10 AM. We examined the NAT time profiles 4, 8, and 11 days following placement of the rats in the phase-shifted cycles. In addition, we measured the NAT profile in LD 2:22 and LD 22:2 by keeping the rats on twelve cycles for 11 days and killing all the rats at one time.
Pineal NAT exhibited a rhythm in all the cycles: peak-dark/nadir-light values (nmol product per gland per hour) were 15.6/0.1 in LD 14:10, controls killed at successive time points. The ratios for the profiles obtained using the one time point procedure were 16.7/0.1 in LD 14:10 8.5/0.2 in LD 22.2, and 12.9/0.2 in LD 2.22. Increasing the photoperiod reduced the time to the NAT peak.
In LD 14:10, Harderian NAT was 31–39 nmol per gland per hour but the peak/ nadir radio was only 1.2; retinal NAT was low (0.2–0.7 nmol per retina per hour) and had only a 3.5-fold peak/nadir ratio.     

Melatonin as an antioxidant in retinal photoreceptors

Dark-adapted, single photoreceptors isolated from the frog retina produce reactive oxygen species (ROS) after about 1 min of illumination with saturating light that we verified by their oxidation of preloaded dihydrorhodamine 123 (DHR) into the fluorescent rhodamine 123 (RHO). In this preparation we tested the antioxidant effects of vitamin E and of melatonin. Melatonin at picomolar and low nanomolar concentrations was determined to be 100 times more potent in inhibiting the light-induced oxidative processes than was vitamin E. On the contrary, both compounds exerted potent prooxidant effects at micromolar concentrations that is above the physiological levels of melatonin. This provides evidence that physiological concentrations of melatonin in a living cell may exert protective actions against a natural oxidant stimulus (light). This helps to define the functional role of endogenous melatonin in photoreceptors, which by their physiological characteristics, are among the marked producers of ROS in the organism.     

Bright daytime light enhances circadian amplitude in a diurnal mammal

Mammalian circadian rhythms are orchestrated by a master pacemaker in the hypothalamic suprachiasmatic nuclei (SCN), which receives information about the 24 h light–dark cycle from the retina. The accepted function of this light signal is to reset circadian phase in order to ensure appropriate synchronization with the celestial day. Here, we ask whether light also impacts another key property of the circadian oscillation, its amplitude. To this end, we measured circadian rhythms in behavioral activity, body temperature, and SCN electrophysiological activity in the diurnal murid rodent Rhabdomys pumilio following stable entrainment to 12:12 light–dark cycles at four different daytime intensities (ranging from 18 to 1,900 lx melanopic equivalent daylight illuminance). R. pumilio showed strongly diurnal activity and body temperature rhythms in all conditions, but measures of rhythm robustness were positively correlated with daytime irradiance under both entrainment and subsequent free run. Whole-cell and extracellular recordings of electrophysiological activity in ex vivo SCN revealed substantial differences in electrophysiological activity between dim and bright light conditions. At lower daytime irradiance, daytime peaks in SCN spontaneous firing rate and membrane depolarization were substantially depressed, leading to an overall marked reduction in the amplitude of circadian rhythms in spontaneous activity. Our data reveal a previously unappreciated impact of daytime light intensity on SCN physiology and the amplitude of circadian rhythms and highlight the potential importance of daytime light exposure for circadian health.

In mammals, near-24-h (circadian) rhythms in physiology and behavior are orchestrated by a master clock located in the hypothalamic suprachiasmatic nuclei (SCN) (1, 2). The SCN clock generates a circadian rhythm in electrical activity, with neurons significantly more excited during the day (up state) than at night (down state) (3, 4). This endogenous rhythm is synchronized (entrained) to the external 24-h light–dark (LD) cycle via input from the retina (5). Thus, light exposure in the circadian night induces adjustments in circadian phase to ensure that internal time faithfully reflects external (celestial) time. Conceptual and mathematical models of light’s impact on the clock address this ability to reset circadian phase (the basis of entrainment) (6–9). However, there is evidence that another fundamental property of circadian rhythms, their amplitude, may also be influenced by light.It is well established that the amplitude and reliability of 24-h rhythms in some aspects of physiology and behavior can be enhanced by increasing daytime light exposure (10–20). Such effects may be explained by the ability of light to directly engage some of the systems under circadian control (e.g., increasing alertness and body temperature, Tb) (21–23) and thus enhance rhythm amplitude de facto, without impacting the circadian clock itself. However, reports that enhanced daytime light can also lead to higher production of melatonin on the subsequent night, many hours after light exposure has ceased (12, 24–26), pose a challenge to that explanation. The long-lasting nature of that effect raises the possibility that daytime light may have a more fundamental impact on circadian amplitude. We set out here to address this possibility by using laboratory rodents to ask whether increasing daytime irradiance produces persistent improvements in circadian amplitude and whether this can be traced back to changes in the physiological activity of the SCN circadian oscillator itself.A challenge to studying the impact of daytime light exposure in common laboratory models (mice, hamsters, and rats) is that they are nocturnal and employ strategies to avoid light in the day (such as curling up asleep). We therefore used a diurnal rodent, Rhabdomys pumilio (the four-striped mouse) (27–29) which is active through the day in both the laboratory and wild, ensuring good exposure to modulations in daytime light intensity. We find that increasing irradiance across a range equivalent to that from dim indoor lighting to natural daylight enhances the reproducibility and robustness of behavioral and physiological rhythms at the whole-animal level that persist into subsequent free run in constant darkness. This effect is associated with profound differences in the electrophysiological activity of the SCN, with bright daytime light producing persistent increases in SCN excitability and enhancing the amplitude of the circadian variation in spontaneous neuronal activity.     

Evolutionary transformation of rod photoreceptors in the all-cone retina of a diurnal garter snake

Vertebrate retinas are generally composed of rod (dim-light) and cone (bright-light) photoreceptors with distinct morphologies that evolved as adaptations to nocturnal/crepuscular and diurnal light environments. Over 70 years ago, the “transmutation” theory was proposed to explain some of the rare exceptions in which a photoreceptor type is missing, suggesting that photoreceptors could evolutionarily transition between cell types. Although studies have shown support for this theory in nocturnal geckos, the origins of all-cone retinas, such as those found in diurnal colubrid snakes, remain a mystery. Here we investigate the evolutionary fate of the rods in a diurnal garter snake and test two competing hypotheses: (i) that the rods, and their corresponding molecular machinery, were lost or (ii) that the rods were evolutionarily modified to resemble, and function, as cones. Using multiple approaches, we find evidence for a functional and unusually blue-shifted rhodopsin that is expressed in small single “cones.” Moreover, these cones express rod transducin and have rod ultrastructural features, providing strong support for the hypothesis that they are not true cones, as previously thought, but rather are modified rods. Several intriguing features of garter snake rhodopsin are suggestive of a more cone-like function. We propose that these cone-like rods may have evolved to regain spectral sensitivity and chromatic discrimination as a result of ancestral losses of middle-wavelength cone opsins in early snake evolution. This study illustrates how sensory evolution can be shaped not only by environmental constraints but also by historical contingency in forming new cell types with convergent functionality.How complex structures can arise has long fascinated evolutionary biologists, and the evolution of the eye, as noted by Charles Darwin (1), is perhaps the most famous example. Within the vertebrate eye, the light-sensing photoreceptors are complex, highly specialized cellular structures that can be divided into two general types based on their distinct morphologies and functions: cones, which are active during the day and contain cone opsin pigments, and rods, which mediate dim-light vision and contain rhodopsin (RH1) (2–4). The visual pigments contained in cone photoreceptors are classified into four different subtypes that mediate vision across the visible spectrum from the UV to the red (2). Although most vertebrate retinas are duplex, containing both cones and rods, squamate reptiles (lizards and snakes) are unusual, not only in having highly variable photoreceptor morphologies, but also for several instances of the absence of an entire class of photoreceptors, resulting in simplex retinas composed of only cones or rods (4).In a seminal book published in 1942, Walls (4) hypothesized that, during evolution, vertebrate photoreceptors could transform from one type to another, a process that he termed photoreceptor “transmutation.” As key examples of his theory, Walls (4) highlighted anatomical changes in the photoreceptors of snakes and geckos, two groups within which there have been significant shifts in diurnal and nocturnal activity patterns. Although several subsequent studies have investigated this hypothesis in geckos (5–9), whether the evolutionary transmutation of photoreceptors can happen in snakes remains an open question (10). Walls also noted a number of peculiar morphological adaptations in snake eyes, which he proposed were due to a subterranean phase early in snake evolution that led to degeneration of the ophidian visual system, resulting in loss of features common to other terrestrial vertebrates (4).Colubrid snakes are an ideal group to study Walls’s hypothesis of transmutation, due to their highly variable photoreceptor morphologies that range from all-cone in, at least some, diurnal species, such as Thamnophis (garter snakes), to all-rod in some nocturnal species, as well as species with the presumed ancestral condition of duplex retinas (4, 11). Previous studies in the diurnal colubrid Thamnophis have demonstrated an all-cone retina (4, 11–14), consisting of double cones and large single cones that express a long-wavelength pigment [presumably long wavelength-sensitive opsin (LWS)], and two classes of small single cone, one with a short-wavelength pigment [presumably short wavelength-sensitive 1 opsin (SWS1)] and the other with a middle-wavelength pigment, the identity of which is unclear (14). However, the ancestral condition for colubrids is likely to have been a duplex retina containing both rods and cones, similar to snakes such as pythons and boas, which have rods that express RH1, large single cones that express LWS, and small single cones that express SWS1 (Fig. 1) (4, 10, 11, 15, 16). The SWS2 and RH2 opsins, present ancestrally in vertebrates, appear to have been lost early in the evolution of snakes, perhaps as a result of their proposed fossorial origins (10, 17, 18).Open in a separate windowFig. 1.Illustration of evolutionary pathways for two alternative hypotheses for the evolution of an all-cone retina from a duplex ancestor in diurnal colubrids. In hypothesis 1 the rod photoreceptors, along with RH1, are lost, and an additional cone type is derived from duplication of an existing cone or retained from an ancestral condition that was lost in other snakes. In hypothesis 2 the rod photoreceptor is evolutionarily modified into a cone photoreceptor, maintaining expression of RH1 and other rod-specific phototransduction machinery.Based on these findings, we can formulate two main hypotheses for the evolution of the all-cone retina of diurnal colubrids from the duplex ancestral condition (Fig. 1). The first is that the rods were lost, and RH1 and other components of the visual transduction cascade unique to rod photoreceptors were either lost or targeted to cones. The second hypothesis is that the rods were evolutionarily modified to resemble the appearance, and presumably the function, of cones. If the rods were modified to resemble cones, we might expect a subset of cones to possess molecular components, such as RH1, and morphological features consistent with a rod ancestry. To test these hypotheses, we examined the photoreceptors and visual pigments of a diurnal garter snake (Thamnophis proximus) by combining multiple methodologies including sequencing and molecular evolutionary analyses of opsin genes, microspectrophotometry (MSP) of intact photoreceptor cells, in vitro expression of visual pigments, and scanning and transmission electron microscopy (SEM and TEM) and immunohistochemistry of T. proximus retinas. The combined results of these experiments provide strong evidence that RH1 and other components of the rod visual transduction machinery are expressed in a subset of cone-like photoreceptors with rod ultrastructural features, and that the RH1-expressing “cones” are not true cones, as previously thought, but rather are modified (i.e., “transmuted”), cone-like rods. Our results shed new light on the evolutionary origins of the all-cone retinas of diurnal colubrid snakes, demonstrating how ancestral losses can be compensated by evolutionary modification of existing cellular structures.     

The spectral composition of evening light and individual differences in the suppression of melatonin and delay of sleep in humans

The effect of light on circadian rhythms and sleep is mediated by a multi-component photoreceptive system of rods, cones and melanopsin-expressing intrinsically photosensitive retinal ganglion cells. The intensity and spectral sensitivity characteristics of this system are to be fully determined. Whether the intensity and spectral composition of light exposure at home in the evening is such that it delays circadian rhythms and sleep also remains to be established. We monitored light exposure at home during 6-8wk and assessed light effects on sleep and circadian rhythms in the laboratory. Twenty-two women and men (23.1±4.7yr) participated in a six-way, cross-over design using polychromatic light conditions relevant to the light exposure at home, but with reduced, intermediate or enhanced efficacy with respect to the photopic and melanopsin systems. The evening rise of melatonin, sleepiness and EEG-assessed sleep onset varied significantly (P<0.01) across the light conditions, and these effects appeared to be largely mediated by the melanopsin, rather than the photopic system. Moreover, there were individual differences in the sensitivity to the disruptive effect of light on melatonin, which were robust against experimental manipulations (intra-class correlation=0.44). The data show that light at home in the evening affects circadian physiology and imply that the spectral composition of artificial light can be modified to minimize this disruptive effect on sleep and circadian rhythms. These findings have implications for our understanding of the contribution of artificial light exposure to sleep and circadian rhythm disorders such as delayed sleep phase disorder.     

Hydroxyindole-O-methyltransferase mRNA expression in a subpopulation of photoreceptors in the chicken retina

Abstract: Hydroxyindole O -methyltransferase (HIOMT, EC 2.1.1.4) catalyzes the final step in the synthesis of melatonin in the pineal gland and retina. HIOMT mRNA was localized by in situ hybridization in the chicken retina to some, but clearly not all, photoreceptors, while in the pineal gland, most pinealocytes displayed a positive hybridization signal. The in situ hybridization localization was confirmed by immunocytochemistry, using an antibody directed against a synthetic chicken HIOMT peptide. Western blot analysis demonstrated an immunoreactive protein of about 40 kilodaltons in the pineal, but the HIOMT protein was below detectable levels in the retina. However, the HIOMT-peptide antibody did identify a modestly immunoreactive subpopulation of retinal photoreceptors. These observations suggest that, in the chicken, melatonin biosynthetic activity is located mainly in a subpopulation of retinal photoreceptors and in most pinealocytes.     

Human cone elongation responses can be explained by photoactivated cone opsin and membrane swelling and osmotic response to phosphate produced by RGS9-catalyzed GTPase

Human cone outer segment (COS) length changes in response to stimuli bleaching up to 99% of L- and M-cone opsins were measured with high resolution, phase-resolved optical coherence tomography (OCT). Responses comprised a fast phase (∼5 ms), during which COSs shrink, and two slower phases (1.5 s), during which COSs elongate. The slower components saturated in amplitude (∼425 nm) and initial rate (∼3 nm ms⁻¹) and are well described over the 200-fold bleaching range as the sum of two exponentially rising functions with time constants of 80 to 90 ms (component 1) and 1,000 to 1,250 ms (component 2). Measurements with adaptive optics reflection densitometry revealed component 2 to be linearly related to cone pigment bleaching, and the hypothesis is proposed that it arises from cone opsin and disk membrane swelling triggered by isomerization and rate-limited by chromophore hydrolysis and its reduction to membrane-localized all-trans retinol. The light sensitivity and kinetics of component 1 suggested that the underlying mechanism is an osmotic response to an amplified soluble by-product of phototransduction. The hypotheses that component 1 corresponds to G-protein subunits dissociating from the membrane, metabolites of cyclic guanosine monophosphate (cGMP) hydrolysis, or by-products of activated guanylate cyclase are rejected, while the hypothesis that it corresponds to phosphate produced by regulator of G-protein signaling 9 (RGS9)-catalyzed hydrolysis of guanosine triphosphate (GTP) in G protein–phosphodiesterase complexes was found to be consistent with the results. These results provide a basis for the assessment with optoretinography of phototransduction in individual cone photoreceptors in health and during disease progression and therapeutic interventions.

Cone photoreceptors initiate the high-acuity daytime vision of healthy humans. The loss of central retinal cone function, as occurs in age-related macular degeneration, is personally devastating and medically extremely costly (1, 2). Over the past 25 y, thanks to the development of optical coherence tomography (OCT) (3, 4) and the application of adaptive optics (AO) to retinoscopy (5–7), there have been enormous advances in imaging the cone mosaic at single-cell resolution in the living eye. Very recently, nanometer-scale measurements of the optical path length (OPL) of rod and cone outer segments (COSs) have made it possible to “optoretinographically” quantify light-driven length changes of individual human COSs (8–14), affording a cell by cell assessment of normal cone function and of dysfunction in disease states (15), with great potential value for evaluating therapeutic interventions. Other than the dependence of rod outer segment (ROS) elongation on the G protein transducin (16), no specific molecular mechanisms of light-driven outer segment elongation, however, have been identified.In this investigation, hypotheses for the molecular mechanisms of human COS elongation are proposed and tested. The kinetics and bleaching sensitivity of the COS responses were measured in response to brief light stimuli ranging 200-fold in intensity, up to and including intensities that bleach the entire complement of cone opsin. The bleach-level dependence and kinetics strongly constrain hypotheses that could underlie COS elongation, rejecting G-protein subunits dissociated from the membrane, by-products of phosphodiesterase-catalyzed cyclic guanosine monophosphate (cGMP) hydrolysis or of guanylate cyclase synthesis of cGMP, and changes in ionic concentrations. Application of a model of cone phototransduction appropriate for the extreme bleaching levels involved supports the hypotheses that COS elongation is partly driven by the osmotic response to free phosphate (P_i) produced by the regulator of G-protein signaling 9 (RGS9)-catalyzed hydrolysis of guanosine triphosphate (GTP) in the G_tcα–phosphodiesterase (PDE) complex and partly driven by bleaching-induced swelling of cone opsin (17) and disk membrane expansion attendant chromophore hydrolysis and its reduction to all-trans retinol (at-ROL) (18).     

Phase Shift of Daily Profiles of N-Acetyltransferase in the Rat Pineal Gland

Pineal N-acetyltransferase activity (NAT) has a circadian rhythm with peak values in the dark time and low values in the light time. NAT time profiles were measured in rats exposed to LD 14:10, to constant dark, and to acute (less than 48 hr) light-dark treatments. In all experiments, imposition of light suppressed NAT. The phase of the dark time NAT cycle was altered 2 hr or less by the following treatments: 3 hr light in the early subjective night, 3 hr light in the late subjective night, 2 hr or 6 hr light in the early subjective day, 4 hr early lights-on, 1 day of constant dark, or 1 day of constant light. When light was extended 4 hr into the dark time, NAT rose at lights out but fell again as the time of "expected" dawn approached. In contrast, the phase of the NAT cycle was shifted 12 hr (180 degrees) within 72 hr by reversing the phase of the light-dark cycle. NAT did not rise in the first dark period (coincident with the time of the subjective light time). The amplitude of the first shifted cycle was less than four control NAT profiles measured in rats kept in the original (unshifted) light-dark cycle.     

Daily serotonin rhythms in rat brain during postnatal development and aging

Aging is characterized by progressive decline in most physiological functions. The age-related sleep disturbances have been attributed to disturbances of circadian function. Neurotransmitter serotonin plays important role in the photic and non-photic regulation of circadian rhythms and is a precursor of melatonin, an internal zeitgeber. To understand the age induced changes in the functional integrity of circadian system, we studied daily serotonin rhythms in brain by measuring serotonin levels at variable time points in wide range of age groups such as 15 days, 1, 2, 3 (adult), 4, 6, 9, 12, 18 and 24-months old male Wistar rats. Animals were maintained under light-dark conditions (LD 12:12), 2 weeks prior to experiment. We report here, mean serotonin levels over 24 h period in brain is highest at 3 months and daily serotonin rhythmicity reliably begins at 3 months and disintegrates at middle age and beyond. The age induced changes in daily serotonin rhythmicity in brain obtained in present study will be a step towards understanding age induced disorders of circadian function.     

Ultrastructural Studies on the Pinealocyte Mitochondria During the Daytime and at Night

Rat pinealocytes were found to contain mitochondria in three configurational states and they were calculated during their maximum (11:00) and minimum (23:00) serotonin content under various conditions of lighting (LD 12:12 and D 24). Their proportions were found to change in the circadian rhythm. Analysis of these results indicated the existence of correlation between pinealocyte bioenergetics and melatonin biosynthesis and its lack in relation to serotonin. Cell groupings with mitochondria in the same configurational state were observed, which suggests the existence of functionally differentiated zones within the pineal gland. In this context, the biochemically demonstrated circadian rhythm in the pineal gland secretion results from the synchronization at the organ level arising from the resultant function of individual pinealocytes.     

Suppressing thyroid hormone signaling preserves cone photoreceptors in mouse models of retinal degeneration

Cone phototransduction and survival of cones in the human macula is essential for color vision and for visual acuity. Progressive cone degeneration in age-related macular degeneration, Stargardt disease, and recessive cone dystrophies is a major cause of blindness. Thyroid hormone (TH) signaling, which regulates cell proliferation, differentiation, and apoptosis, plays a central role in cone opsin expression and patterning in the retina. Here, we investigated whether TH signaling affects cone viability in inherited retinal degeneration mouse models. Retinol isomerase RPE65-deficient mice [a model of Leber congenital amaurosis (LCA) with rapid cone loss] and cone photoreceptor function loss type 1 mice (severe recessive achromatopsia) were used to determine whether suppressing TH signaling with antithyroid treatment reduces cone death. Further, cone cyclic nucleotide-gated channel B subunit-deficient mice (moderate achromatopsia) and guanylate cyclase 2e-deficient mice (LCA with slower cone loss) were used to determine whether triiodothyronine (T3) treatment (stimulating TH signaling) causes deterioration of cones. We found that cone density in retinol isomerase RPE65-deficient and cone photoreceptor function loss type 1 mice increased about sixfold following antithyroid treatment. Cone density in cone cyclic nucleotide-gated channel B subunit-deficient and guanylate cyclase 2e-deficient mice decreased about 40% following T3 treatment. The effect of TH signaling on cone viability appears to be independent of its regulation on cone opsin expression. This work demonstrates that suppressing TH signaling in retina dystrophy mouse models is protective of cones, providing insights into cone preservation and therapeutic interventions.Rod and cone photoreceptors degenerate under a variety of pathological conditions, including a wide array of hereditary retinal diseases, such as retinitis pigmentosa, macular degeneration, and cone–rod dystrophies. Defects in a large number of genes are linked to inherited retinal degenerative disorders (www.sph.uth.tmc.edu/RetNet/disease.htm), including those encoding enzymes involved in the recycling of 11-cis retinal in the retinal pigment epithelium (RPE), retinoid isomerase (RPE65), and lecithin retinol acyltransferase (LRAT), and the phototransduction-associated proteins (opsins, subunits of transducin, cGMP phosphodiesterase PDE6, guanylate cyclase, and cyclic nucleotide-gated channel). There are currently no treatments for human retinal dystrophies. Despite a high genetic heterogeneity, the degenerating photoreceptors show common cellular disorder features, including oxidative damage (1, 2), endoplasmic reticulum stress (3, 4), and apoptosis (5, 6).Thyroid hormone (TH) signaling regulates cell proliferation, differentiation, and apoptosis. The role of TH signaling in retina regarding its regulation of cone opsin expression and patterning has been well documented (7, 8). Most mammals possess dichromatic color vision that is mediated by two opsins with peak sensitivities to medium-long (M, green) and short (S, blue) wavelengths of light (9, 10). In mouse, M- and S-opsins are expressed in opposing gradients such that varying amounts of both opsins are coexpressed in cones in midretinal regions, whereas M-opsin predominates in dorsal (superior) regions and S-opsin predominates in ventral (inferior) regions (10, 11) (Fig. S1). During development and in the adult postmitotic retina, TH signaling via its receptor type β2 (TRβ2) suppresses expression of S-opsin, induces expression of M-opsin, and promotes the dorsal–ventral opsin patterning (7, 8). Importantly, TH signaling has been associated with cone viability. Triiodothyronine (T3) treatment was shown to cause cone death in mice and this effect was reversed by deletion of TRβ2 gene (12). Excessive TH signaling was also shown to induce auditory defects and cochlear degeneration in mice (13). TH signaling has been associated with apoptosis of a variety of human cell lines, including lymphocytes (14), breast cancer cells (15), HeLa cells (16), and pituitary tumor cells (17), and TH signaling has been well documented in apoptotic tissue remodeling during anuran metamorphosis (18, 19). To determine whether TH signaling affects cone viability in inherited retinal degeneration, we investigated cone death/survival in retinal degeneration mouse models following TH signaling suppression and stimulation. Retinol isomerase RPE65-deficient (Rpe65^−/−) (a model of Leber congenital amaurosis, LCA) (20, 21) and cone photoreceptor function loss type 1 (cpfl1) mice (PDE6C mutation, a model of achromatopsia) (22), displaying fast and severe cone degeneration, were used to determine whether suppressing TH signaling with antithyroid treatment reduces cone degeneration. Cone cyclic nucleotide-gated channel B subunit-deficient (Cngb3^−/−) (a model of achromatopsia) (23) and guanylate cyclase 2e-deficient (Gucy2e^−/−) (another model of LCA) mice (24), displaying relatively slow progressive and moderate cone degeneration, were used to determine whether stimulating TH signaling (with T3 treatment) deteriorates cones. We report here that cone survival was greatly improved in Rpe65^−/− and cpfl1 mice following TH signaling suppression, whereas cone degeneration was significantly increased in Cngb3^−/− and Gucy2e^−/− mice following TH signaling stimulation, demonstrating a protective role of suppressing TH signaling in cones.