Photoperiodism of Diapause Induction in the Silkworm, Bombyx mori.

The silkworm Bombyx mori exhibits a photoperiodic response (PR) for embryonic diapause induction. This article provides a comprehensive review of literature on the silkworm PR, starting from early works on population to recent studies uncovering the molecular mechanism. Makita Kogure (1933) conducted extensive research on the PR, presenting a pioneering paper on insect photoperiodism. In the 1970s and 80s, artificial diets were developed, and the influence of nutrition on PR was well documented. The photoperiodic photoreceptor has been investigated from organ to molecular level in the silkworm. Culture experiments demonstrated that the photoperiodic induction can be programmed in an isolated brain (Br)-subesophageal ganglion (SG) complex with corpora cardiaca (CC)-corpora allata (CA). The requirement of dietary vitamin A for PR suggests the involvement of opsin pigment in the photoperiodic reception, and a cDNA encoding an opsin (Boceropsin) was cloned from the brain. The effector system concerning the production and secretion of diapause hormone (DH) has also been extensively investigated in the silkworm. DH is produced in a pair of posterior cells of SG, transported to CC by nervi corporis cardiaci, and ultimately released into the hemolymph. Possible involvement of GABAergic and corazonin (Crz) signal pathways was suggested in the control of DH secretion. Knockout (KO) experiments of GABA transporter (GAT) and circadian clock genes demonstrated that GAT plays a crucial role in PR through circadian control. A model outlining the PR mechanism, from maternal photoperiodic light reception to DH secretion, has been proposed.

Three different seasonally expressed opsins are present in the brain of the Eared Dove, an opportunist breeder.

Birds living at high latitudes perceive the photoperiod through deep-brain photoreceptors (DBP) located in deep-brain neurons. During long photoperiods the information transmitted by these photoreceptors increases the activity of the hypothalamic-pituitary-gonadal (HPG) axis, leading to gonadal development. The presence of photopigments such as VA-Opsin, Opn4, Opn5 and Opn2 in brain areas implicated in reproductive behaviors has been firmly established in several avian species with seasonal breeding, whereas their existence in opportunistic breeding birds remains unconfirmed. The Eared Dove is an urban and peri-urban dove that breeds throughout the year. Males of this species do not exhibit the typical gonadal regression/recrudescence cycle, thus posing the question of what occurs upstream of the HPG axis. We addressed this issue by first studying the presence of diverse opsins located in DBP in the brains of Eared Dove males and whether these photopigments changed their expression throughout the year. We carried out an immunohistochemistry analysis on three different opsins: Opn2 (rhodopsin), Opn3 and Opn5. Our results demonstrate the discrete neuroanatomical distribution of these opsins in the brain of Eared Dove males and strongly indicate different seasonal expressions. In the anterior region of the hypothalamus, Opn2-positive cells were detected throughout the year. By contrast, Opn5 was found to be strongly and seasonally expressed during winter in the anterior and the hypothalamic region. Opn3 was also found to be significantly and seasonally expressed during winter in the hypothalamic region. We thus demonstrate for the first time that males of the Eared Dove, have three different deep-brain opsin-expressing photoreceptors with differential location/distribution in the anterior and hypothalamic region and differential seasonality. The persistence of Opn2 and the strong seasonal expression of nonvisual photopigments Opn3 and Opn5 in two areas of the avian brain, which are associated with reproduction, could be the primary distinction between seasonal and opportunistic breeders.

Diurnal regulation of metabolism by Gs-alpha in hypothalamic QPLOT neurons.

Neurons in the hypothalamic preoptic area (POA) regulate multiple homeostatic processes, including thermoregulation and sleep, by sensing afferent input and modulating sympathetic nervous system output. The POA has an autonomous circadian clock and may also receive circadian signals indirectly from the suprachiasmatic nucleus. We have previously defined a subset of neurons in the POA termed QPLOT neurons that are identified by the expression of molecular markers (Qrfp, Ptger3, LepR, Opn5, Tacr3) that suggest receptivity to multiple stimuli. Because Ptger3, Opn5, and Tacr3 encode G-protein coupled receptors (GPCRs), we hypothesized that elucidating the G-protein signaling in these neurons is essential to understanding the interplay of inputs in the regulation of metabolism. Here, we describe how the stimulatory Gs-alpha subunit (Gnas) in QPLOT neurons regulates metabolism in mice. We analyzed Opn5cre; Gnasfl/fl mice using indirect calorimetry at ambient temperatures of 22°C (a historical standard), 10°C (a cold challenge), and 28°C (thermoneutrality) to assess the ability of QPLOT neurons to regulate metabolism. We observed a marked decrease in nocturnal locomotion of Opn5cre; Gnasfl/fl mice at both 28°C and 22°C, but no overall differences in energy expenditure, respiratory exchange, or food and water consumption. To analyze daily rhythmic patterns of metabolism, we assessed circadian parameters including amplitude, phase, and MESOR. Loss-of-function GNAS in QPLOT neurons resulted in several subtle rhythmic changes in multiple metabolic parameters. We observed that Opn5cre; Gnasfl/fl mice show a higher rhythm-adjusted mean energy expenditure at 22°C and 10°C, and an exaggerated respiratory exchange shift with temperature. At 28°C, Opn5cre; Gnasfl/fl mice have a significant delay in the phase of energy expenditure and respiratory exchange. Rhythmic analysis also showed limited increases in rhythm-adjusted means of food and water intake at 22°C and 28°C. Together, these data advance our understanding of Gαs-signaling in preoptic QPLOT neurons in regulating daily patterns of metabolism.

Shedding new light on the generation of the visual chromophore.

The visual phototransduction cascade begins with a cis-trans photoisomerization of a retinylidene chromophore associated with the visual pigments of rod and cone photoreceptors. Visual opsins release their all-trans-retinal chromophore following photoactivation, which necessitates the existence of pathways that produce 11-cis-retinal for continued formation of visual pigments and sustained vision. Proteins in the retinal pigment epithelium (RPE), a cell layer adjacent to the photoreceptor outer segments, form the well-established "dark" regeneration pathway known as the classical visual cycle. This pathway is sufficient to maintain continuous rod function and support cone photoreceptors as well although its throughput has to be augmented by additional mechanism(s) to maintain pigment levels in the face of high rates of photon capture. Recent studies indicate that the classical visual cycle works together with light-dependent processes in both the RPE and neural retina to ensure adequate 11-cis-retinal production under natural illuminances that can span ten orders of magnitude. Further elucidation of the interplay between these complementary systems is fundamental to understanding how cone-mediated vision is sustained in vivo. Here, we describe recent advances in understanding how 11-cis-retinal is synthesized via light-dependent mechanisms.

The Spatial Extent of Optogenetic Silencing in Transgenic Mice Expressing Channelrhodopsin in Inhibitory Interneurons.

Optogenetic stimulation of inhibitory interneurons has become a commonly used strategy for silencing neuronal activity. This is typically achieved using transgenic mice expressing excitatory opsins in inhibitory interneurons throughout the brain, raising the question of how spatially extensive the resulting inhibition is. Here, we characterize neuronal silencing in VGAT-ChR2 mice, which express channelrhodopsin-2 in inhibitory interneurons, as a function of light intensity and distance from the light source in several cortical and subcortical regions. We show that light stimulation, even at relatively low intensities, causes inhibition not only in brain regions targeted for silencing but also in their subjacent areas. In contrast, virus-mediated expression of an inhibitory opsin enables robust silencing that is restricted to the region of opsin expression. Our results reveal important constraints on using inhibitory interneuron activation to silence neuronal activity and emphasize the necessity of carefully controlling light stimulation parameters when using this silencing strategy.

Chromophore-Independent Roles of Opsin Apoproteins in Drosophila Mechanoreceptors.

Rhodopsins, the major light-detecting molecules of animal visual systems [1], consist of opsin apoproteins that covalently bind a retinal chromophore with a conserved lysine residue [1, 2]. In addition to capturing photons, this chromophore contributes to rhodopsin maturation [3, 4], trafficking [3, 4], and stabilization [5], and defects in chromophore synthesis and recycling can cause dysfunction of the retina and dystrophy [6-9]. Indications that opsin apoproteins alone might have biological roles have come from archaebacteria and platyhelminths, which present opsin-like proteins that lack the chromophore binding site and are deemed to function independently of light [10, 11]. Light-independent sensory roles have been documented for Drosophila opsins [12-15], yet also these unconventional opsin functions are thought to require chromophore binding [12, 13, 15]. Unconjugated opsin apoproteins act as phospholipid scramblases in mammalian photoreceptor disks [16], yet chromophore-independent roles of opsin apoproteins outside of eyes have, to the best of our knowledge, hitherto not been described. Drosophila chordotonal mechanoreceptors require opsins [13, 15], and we find that their function remains uncompromised by nutrient carotenoid depletion. Disrupting carotenoid uptake and cleavage also left the mechanoreceptors unaffected, and manipulating the chromophore attachment site of the fly's major visual opsin Rh1 impaired photoreceptor, but not mechanoreceptor, function. Notwithstanding this chromophore independence, some proteins that process and recycle the chromophore in the retina are also required in mechanoreceptors, including visual cycle components that recycle the chromophore upon its photoisomerization. Our results thus establish biological function for unconjugated opsin apoproteins outside of eyes and, in addition, document chromophore-independent roles for chromophore pathway components.

Does blue light restore human epidermal barrier function via activation of Opsin during cutaneous wound healing?

BACKGROUND AND OBJECTIVE: Visible light has beneficial effects on cutaneous wound healing, but the role of potential photoreceptors in human skin is unknown. In addition, inconsistency in the parameters of blue and red light-based therapies for skin conditions makes interpretation difficult. Red light can activate cytochrome c oxidase and has been proposed as a wound healing therapy. UV-blue light can activate Opsin 1-SW, Opsin 2, Opsin 3, Opsin 4, and Opsin 5 receptors, triggering biological responses, but their role in human skin physiology is unclear. MATERIALS AND METHODS: Localization of Opsins was analyzed in situ in human skin derived from face and abdomen by immunohistochemistry. An ex vivo human skin wound healing model was established and expression of Opsins confirmed by immunohistochemistry. The rate of wound closure was quantitated after irradiation with blue and red light and mRNA was extracted from the regenerating epithelial tongue by laser micro-dissection to detect changes in Opsin 3 (OPN3) expression. Retention of the expression of Opsins in primary cultures of human epidermal keratinocytes and dermal fibroblasts was confirmed by qRT-PCR and immunocytochemistry. Modulation of metabolic activity by visible light was studied. Furthermore, migration in a scratch-wound assay, DNA synthesis and differentiation of epidermal keratinocytes was established following irradiation with blue light. A role for OPN3 in keratinocytes was investigated by gene silencing. RESULTS: Opsin receptors (OPN1-SW, 3 and 5) were similarly localized in the epidermis of human facial and abdominal skin in situ. Corresponding expression was confirmed in the regenerating epithelial tongue of ex vivo wounds after 2 days in culture, and irradiation with blue light stimulated wound closure, with a corresponding increase in OPN3 expression. Expression of Opsins was retained in primary cultures of epidermal keratinocytes and dermal fibroblasts. Both blue and red light stimulated the metabolic activity of cultured keratinocytes. Low levels of blue light reduced DNA synthesis and stimulated differentiation of keratinocytes. While low levels of blue light did not alter keratinocyte migration in a scratch wound assay, higher levels inhibited migration. Gene silencing of OPN3 in keratinocytes was effective (87% reduction). The rate of DNA synthesis in OPN3 knockdown keratinocytes did not change following irradiation with blue light, however, the level of differentiation was decreased. CONCLUSIONS: Opsins are expressed in the epidermis and dermis of human skin and in the newly regenerating epidermis following wounding. An increase in OPN3 expression in the epithelial tongue may be a potential mechanism for the stimulation of wound closure by blue light. Since keratinocytes and fibroblasts retain their expression of Opsins in culture, they provide a good model to investigate the mechanism of blue light in wound healing responses. Knockdown of OPN3 led to a reduction in early differentiation of keratinocytes following irradiation with blue light, suggesting OPN3 is required for restoration of the barrier function. Understanding the function and relationship of different photoreceptors and their response to specific light parameters will lead to the development of reliable light-based therapies for cutaneous wound healing. Lasers Surg. Med. © 2018 Wiley Periodicals, Inc.

Optical and thermal simulations for the design of optodes for minimally invasive optogenetics stimulation or photomodulation of deep and large cortical areas in non-human primate brain.

The use of optogenetics or photobiomodulation in non-human primate (NHP) requires the ability to noninvasively stimulate large and deep cortical brain tissues volumes. In this context, the optical and geometrical parameters of optodes are critical. Methods and general guidelines to optimize these parameters have to be defined. OBJECTIVE: We propose the design of an optode for safe and efficient optical stimulation of a large volume of NHP cortex, down to 3-5 mm depths without inserting fibers into the cortex. APPROACH: Monte Carlo simulations of optical and thermal transport have been carried out using the Geant4 application for tomographic emission (GATE) platform. Parameters such as the fiber diameter, numerical aperture, number of fibers and their geometrical arrangement have been studied. Optimal hardware parameters are proposed to obtain homogeneous fluence above the fluence threshold for opsin activation without detrimental thermal effects. MAIN RESULTS: The simulations show that a large fiber diameter and a large numerical aperture are preferable since they allow limiting power concentration and hence the resulting thermal increases at the brain surface. To obtain a volume of 200-500 mm3 of brain tissues receiving a fluence above the opsin activation threshold for optogenetics or below a phototocixity threshold for photobiomodulation, a 4 fibers configuration is proposed. The optimal distance between the fibers was found to be 4 mm. A practical implementation of the optode has been performed and the corresponding fluence and thermal maps have been simulated. SIGNIFICANCE: The present study defines a method to optimize the design of optode and the choice of stimulation parameters for optogenetics and more generally light delivery to deep and large volumes of tissues in NHP brain with a controlled irradiance dosimetry. The general guidelines are the use of silica fibers with a large numerical aperture and a large diameter. The combination of several fibers is required if large volumes need to be stimulated while avoiding thermal effects.

Age dependent variations in the deep brain photoreceptors (DBPs), GnRH-GnIH system and testicular steroidogenesis in Japanese quail, Coturnix coturnix japonica.

The complex physiology of aging involves a number of molecular and biochemical events, manifested as signs of senescence. Japanese quail is a very unique and advantageous model to study the signs and symptoms of senescence in the central and peripheral modules of HPG axis. In the present study, we have investigated the age dependent variations in hypothalamic deep brain photoreceptors (DBPs), central GnRH-I/II-GnIH-Mel1cR system, testicular GnRH-GnIH system, testicular steroidogenic genes and proteins, androgen receptor (AR) and serum testosterone level in quail of different age groups [3-wk (sexually immature), 6-wk (sexually mature and crossed the puberty), 16-wk (adult, sexually active and showing full breeding phase) and 144-wk (aged)]. Findings of our present study showed the differential expression of these genes/proteins in quail of different age groups. The low levels of the DBPs, GnRH-I, GnIH, Mel1cR in hypothalamus and GnRH-II in midbrain, significantly decreased testicular GnRH/GnRH-R-GnIH, steroidogenic genes/proteins and serum testosterone were observed in immature quail. The significantly increased expression of opsins in the DBPs, GnRH-I, GnIH, Mel1cR in hypothalamus and GnRH-II in midbrain influences the testicular GnRH-GnIH and stimulate the testicular steroidogenesis in mature and adult quail. In aged quail, the significantly decreased levels of hypothalamic DBPs, GnRH-I, GnIH, Mel1cR and midbrain GnRH-II modulates the testicular GnRH-GnIH and further suppresses the genes/proteins involved in steroidogenesis and results in reduced serum testosterone. Hence, it can be concluded from our findings that the testicular steroidogenesis and its neuroendocrine regulation varies with age, in Japanese quail.

Cone degeneration is triggered by the absence of USH1 proteins but prevented by antioxidant treatments.

Usher syndrome type 1 (USH1) is a major cause of inherited deafness and blindness in humans. The eye disorder is often referred to as retinitis pigmentosa, which is characterized by a secondary cone degeneration following the rod loss. The development of treatments to prevent retinal degeneration has been hampered by the lack of clear evidence for retinal degeneration in mutant mice deficient for the Ush1 genes, which instead faithfully mimic the hearing deficit. We show that, under normal housing conditions, Ush1g-/- and Ush1c-/- albino mice have dysfunctional cone photoreceptors whereas pigmented knockout animals have normal photoreceptors. The key involvement of oxidative stress in photoreceptor apoptosis and the ensued retinal gliosis were further confirmed by their prevention when the mutant mice are reared under darkness and/or supplemented with antioxidants. The primary degeneration of cone photoreceptors contrasts with the typical forms of retinitis pigmentosa. Altogether, we propose that oxidative stress probably accounts for the high clinical heterogeneity among USH1 siblings, which also unveils potential targets for blindness prevention.

Spectrally distinct channelrhodopsins for two-colour optogenetic peripheral nerve stimulation.

Technologies for peripheral nerve stimulation have conventionally relied on the anatomic placement of electrodes adjacent to subsets of sensory fibres or motor fibres that selectively target an end effector. Here, we demonstrate the use of optogenetics to directly target the innervating fibres of an end effector by relying on retrograde transfection of adeno-associated virus serotype 6 to restrict axonal opsin expression to the desired fibre targets. By using an in vivo screen in rats, we identify the first channelrhodopsins as well as a halorhodopsin that respond to red light in the peripheral nerve. Combining two channelrhodopsins with spectrally distinct activation profiles allowed us to drive opposing muscle activity via two-colour illumination of the same mixed nerve. We also show halorhodopsin-mediated reductions in electrically evoked muscle tremor spectrally optimized for deep peripheral nerves. Our non-invasive peripheral neurostimulator with targeted multi-fascicle resolution enables scientific and clinical exploration, such as motor control in paralysis, biomimetic sensation feedback for amputees and targeted inhibition of muscle tremor.

Testicular atrophy and reproductive quiescence in photorefractory and scotosensitive quail: Involvement of hypothalamic deep brain photoreceptors and GnRH-GnIH system.

Birds time their daily and seasonal activities in synchronization with circadian and annual periodicities in the environment, which is mainly provided by changes in photoperiod/day length conditions. Photoperiod appears to act at the level of eye, pineal and encephalic/deep brain photoperception and thus entrain the hypothalamic clock as well as reproductive circuitry in different avian species. In this article our focus of study is to elucidate out the underlying molecular mechanism of modulation of the hypothalamic reproductive circuitry following the photoperception through the hypothalamic photoreceptor cells and the subsequent alteration in the reproductive responses in quail, kept under different simulated photoperiodic conditions. Present study investigated the different simulated photoperiodic conditions induced hypothalamic DBP-GnRH-GnIH system mediated translation of photoperiodic information and subsequent exhibition of differential photosexual responses (scoto-/photo-sensitivity and refractoriness) in Japanese quail, Coturnix coturnix japonica. Paired testes weight and paired testicular volume increased 15.9 and 22.6-fold respectively in scotorefractory quail compare to that of scotosensitive phase and 12.8 and 24.3-fold in photosensitive quail compare to that of photorefractory phase. The pineal/eye melatonin (through melatonin receptor subtype Mel1cR) and hypothalamic deep brain photoreceptor (DBPs) cells directly modulate the hypothalamic GnRH-I/II and GnIH system and thus exhibit testicular stimulation or regression in response to different photoperiodic conditions (PS, PR, SS and SR). The hypothalamic alteration of DBP(s) and GnRH-GnIH system thus may induce the testicular stimulation in PS and SR quail and testicular regression in SS and PR quail.

Seasonal alterations in the daily rhythms in hypothalamic expression of genes involved in the photoperiodic transduction and neurosteroid-dependent processes in migratory blackheaded buntings.

The present study investigated seasonal alterations in the daily rhythms of hypothalamic expression of genes involved in the photoperiodic regulation of annual cycles in birds. We measured the 4-hourly mRNA expression of genes involved in the photoperiodic transduction (OPN5, EYA3, CGA, TSHß, DIO2, DIO3) and neurosteroid-dependent processes (AR, CYP19, ERα, ERß) in the hypothalamus of migratory blackheaded buntings photoinduced with photosensitive, photostimulated (early and late stimulated) and photorefractory seasonal states. There were significant differences in daily mRNA profiles between the photoperiodic states. Particularly, increased CGA, TSHß and DIO2 and decreased DIO3 mRNA levels in the early photostimulated state, compared to the photosensitive state, suggest that thyroid hormones have a role in photostimulation in buntings. Similar differences in the expression of genes coding for the aromatase enzyme (CYP19) and receptors for oestrogen (ERα, ERß) (but not androgen; AR) indicate that there is seasonal alteration in the neuro-oestrogen-mediated functions. Furthermore, peak expression times of CGA, TSHß and DIO2 genes at hours 14-15 of the day in the early stimulated state indicated molecular regulation of the daily rhythm of photoinducibility in buntings. Most significantly, however, we found an attenuated daily rhythm in thyroid hormone modulatory genes and a switch of peak expression time from day to night in CYP19 mRNA rhythm in the subsequent late photostimulated state, although testicular maturation still persisted. These alterations in daily rhythms may have signalled the initiation of processes underlying other seasonal phenologies in parallel with the gonadal response, such as a manifestation of the night-time flight in buntings. These results show alterations in daily rhythms underlying the transcriptional regulation of the photoperiod-induced seasonal states in migratory blackheaded buntings.

Cone Health and Retinoids.

Cones are photoreceptor cells used for bright light and color vision. Retinoids are vitamin A derivatives, one of which is the 11-cis aldehyde form that serves as the chromophore for both cone and rod visual pigments. In the visual disease, Type 2 Leber congenital amaurosis (LCA2), 11-cis-retinal generation is inhibited or abolished. Work by others has shown that patients with LCA2 have symptoms consistent with degenerating cones. In mouse models for LCA2, early cone degeneration is readily apparent: cone opsins and other proteins associated with the outer segment are delocalized and cell numbers decline rapidly within the first month. Rods would appear normal morphologically and functionally, if not for the absence of chromophore. Supplementation of mouse models of LCA2 with cis-retinoids has been shown to slow loss of cone photoreceptor cells if mice were maintained in darkness. Thus, 11-cis-retinal appears not only to have a role in the light response reaction but also to promote proper trafficking of the cone opsins and maintain viable cones.

Disruption of neuropsin mRNA expression via RNA interference facilitates the photoinduced increase in thyrotropin-stimulating subunit ß in birds.

It has long been known that the avian brain is capable of light detection independently of the eyes. The photoreceptive molecule neuropsin (OPN5) was identified in mammalian and avian brains, and shown to respond to biologically relevant light wavelengths. Whether OPN5 is functionally involved in light detection is unknown. Daylength plays a critical role in regulating the neuroendocrine control of reproduction in birds. The presence of light during a 'photoinducible' phase of the circadian cycle, which occurs 12-16 h after dawn, results in marked changes in hypothalamic gene expression. These changes ultimately control gonadotropin release from the pituitary gland that, in turn, stimulates gonadal development. In this study, we first measured OPN5 expression in the mediobasal hypothalamus (MBH) in border canaries during the photoinducible period in relation to thyrotropin (TSH) ß-subunit mRNA expression, which is implicated in the control of avian reproduction. Second, the knockdown of OPN5 via small interfering RNA antisense in the MBH revealed that there is an inhibitory input in the photoinduced regulation of TSHß mRNA expression. Our data indicate that a decrease in OPN5 mRNA expression is associated with the facilitation in TSHß mRNA expression in the MBH, a critical step for the light-induced increase in gonadal recrudescence. We hypothesise that the removal of an inhibitory input by OPN5 in birds may be a step that occurs during the photoinducible period. Given the distribution of OPN5 in the brain and periphery, this suggests a possible multifunctional role for light information in regulating other physiological processes.

Ceramide-1-phosphate, a new mediator of development and survival in retina photoreceptors.

PURPOSE. Simple sphingolipids control crucial cellular processes in several cell types. Previous work demonstrated that sphingolipids, such as ceramide, sphingosine, and sphingosine-1-phosphate, are key mediators in the regulation of survival, differentiation, and proliferation of retina photoreceptors. Ceramide-1-phosphate (C1P) regulates growth and survival in several cell types; however, little is known concerning its functions in the retina. Whether C1P also participates in controlling photoreceptor development was also explored. METHODS. Rat retina neuronal cultures were supplemented with 1 to 10 µM C1P. Proliferation was determined by evaluating 5-bromo-2-deoxyuridine (BrdU) uptake and the number of mitotic figures and differentiation by evaluating opsin and peripherin expression by immunocytochemistry and Western blot. Apoptosis was inhibited with the pan caspase inhibitor ZVADFMK and evaluated by TUNEL assay, propidium iodide/annexin V, and DAPI labeling. Preservation of mitochondrial membrane potential was evaluated. RESULTS. C1P enhanced BrdU uptake and increased mitosis in retinal progenitors. C1P addition advanced photoreceptor differentiation, enhancing opsin and peripherin expression and stimulating development of the apical processes in which these proteins were concentrated. In the absence of these trophic factors, photoreceptors degenerated after 4 days in vitro, and at day 6, almost 50% of photoreceptors were apoptotic. C1P decreased photoreceptor apoptosis, reducing this percentage by half. Inhibiting caspase activity reduced photoreceptor apoptosis in the controls, but did not increase opsin expression, implying that C1P has separate effects on differentiation and survival. CONCLUSIONS. These results suggest for the first time that C1P is a novel mediator that has multiple functions in photoreceptors, initially regulating their proliferation and then promoting their survival and differentiation.

Light prevents exogenous 11-cis retinal from maintaining cone photoreceptors in chromophore-deficient mice.

PURPOSE: To determine the effect of light/dark cycles on the cones of 11-cis retinal-treated RPE65/rhodopsin double knockout (Rpe65(-/-)Rho(-/-)) mice. Studies have shown that cones degenerate in chromophore-deficient mouse models for Leber Congenital Amaurosis (LCA), but exogenous supplementation of the native 11-cis retinal chromophore can inhibit this degeneration, suggesting that 11-cis retinal could be used as a therapeutic agent for preserving functional cones in patients with LCA. However, these treated mice were maintained in the dark. METHODS: 11-cis Retinal was introduced into Rpe65(-/-)Rho(-/-) mice at postnatal day 10 as a single subcutaneous injection mixed with a basement membrane matrix. The mice were maintained in either normal light/dark cycles or constant dark conditions. Fluorescence microscopy was used to assess retinal morphology. Cone cell survival was determined by counting cone opsin-containing cells on flat-mounted P30 retinas. Cross-sections of P21 mouse retina were used to assess cone cell integrity by visualizing opsin localization. Cone function was determined by electroretinography (ERG). RESULTS: Previous studies have shown that 11-cis retinal-treated mice lacking RPE65 and raised in constant dark have higher cone photoreceptor cell number, improved cone opsin localization, and enhanced cone ERG signals when compared with untreated mice. However, in this study the authors show that 11-cis retinal-treated Rpe65(-/-)Rho(-/-) mice raised in cyclic light did not show the improvements seen with the dark-reared mice. CONCLUSIONS: Thus, 11-cis retinal by itself, as well as other agents that form photosensitive pigments, will not be good therapeutic candidates for preserving cones in LCA.

G protein- and agonist-bound serotonin 5-HT2A receptor model activated by steered molecular dynamics simulations.

A 5-HT(2A) receptor model was constructed by homology modeling based on the ß(2)-adrenergic receptor and the G protein-bound opsin crystal structures. The 5-HT(2A) receptor model was transferred into an active conformation by an agonist ligand and a G(αq) peptide in four subsequent steered molecular dynamics (MD) simulations. The driving force for the transformation was the addition of several known intermolecular and receptor interhelical hydrogen bonds enforcing the necessary helical and rotameric movements. Subsquent MD simulations without constraints confirmed the stability of the activated receptor model as well as revealed new information about stabilizing residues and bonds. The active 5-HT(2A) receptor model was further validated by retrospective ligand screening of more than 9400 compounds, whereof 182 were known ligands. The results show that the model can be used in drug discovery for virtual screening and structure-based ligand design as well as in GPCR activation studies.

Assays for inverse agonists in the visual system.

Visual pigment proteins belong to the superfamily of G protein-coupled receptors and are the light-sensitive molecules in rod and cone photoreceptor cells. The protein moiety is known as opsin and the ligand in the dark is 11-cis retinal, which serves as both the photon detector and an inverse agonist. While much is known about properties of the rod pigment rhodopsin, much less is understood about cone visual pigments. Being able to identify ligands that effect opsins give an insight into structure-activity relationships. The action of some ligands indicates that there are differences between not only rod and cone opsins but also among the different classes of cone opsins. Furthermore, inverse agonists of cone opsins may have potential therapeutic uses under conditions when the native 11-cis retinal ligand is absent. A method for determining the effects of ligands on rod and cone opsin activity is described.

Cone outer segment morphology and cone function in the Rpe65-/- Nrl-/- mouse retina are amenable to retinoid replacement.

PURPOSE: RPE65, a major retinal pigment epithelium protein, is essential in generating 11-cis retinal, the chromophore for all opsins. Without chromophore, cone opsins are mislocalized and cones degenerate rapidly (e.g., Rpe65(-/-) mouse). Function, survival, and correct targeting of opsins is increased in Rpe65(-/-) cones on supplying 11-cis retinal. Here, we determine the consequences of 11-cis retinal withdrawal and supplementation on cone development in the all-cone Nrl(-/-) retina. METHODS: Rpe65(-/-) Nrl(-/-), Nrl(-/-), and wild-type mice were examined. Cone structure was analyzed by using TUNEL assay, electron microscopy, and cone-specific antibodies. Cone function was assessed with light-adapted single-flash ERGs. RESULTS: Rpe65(-/-)Nrl(-/-) mice had an increased number of TUNEL-positive photoreceptors during programmed cell death compared with Nrl(-/-) mice, in addition to accelerated age-related degeneration. Cone function in Rpe65(-/-)Nrl(-/-) mice was minimal, and opsins were mislocalized. Treatment with 11-cis retinal restored cone function, promoted outer segment formation, and enabled opsin trafficking to outer segments. Eliminating Rpe65 prevented rosette formation in Nrl(-/-) retinas; supplementation of Rpe65(-/-)Nrl(-/-) mice with 11-cis retinal resulted in their reoccurrence. CONCLUSIONS: Taken together, function and opsin trafficking in Nrl(-/-) and wild-type cones are comparable, confirming and extending our findings that cone maturation and outer segment development are dependent on the presence of chromophore. The data on age-related cone death in Rpe65(-/-)Nrl(-/-) mice and the reintroduction of rosettes after 11-cis retinal injections confirm that outer segments, which for steric reasons appear to introduce rosettes in an all-cone retina, are essential for cell survival. These results are important for understanding and treating chromophore-related cone dystrophies.