Optogenetic interrogation reveals separable G-protein-dependent and -independent signalling linking G-protein-coupled receptors to the circadian oscillator.

BACKGROUND: Endogenous circadian oscillators distributed across the mammalian body are synchronised among themselves and with external time via a variety of signalling molecules, some of which interact with G-protein-coupled receptors (GPCRs). GPCRs can regulate cell physiology via pathways originating with heterotrimeric G-proteins or ß-arrestins. We applied an optogenetic approach to determine the contribution of these two signalling modes on circadian phase. RESULTS: We employed a photopigment (JellyOp) that activates Gαs signalling with better selectivity and higher sensitivity than available alternatives, and a point mutant of this pigment (F112A) biased towards ß-arrestin signalling. When expressed in fibroblasts, both native JellyOp and the F112A arrestin-biased mutant drove light-dependent phase resetting in the circadian clock. Shifts induced by the two opsins differed in their circadian phase dependence and the degree to which they were associated with clock gene induction. CONCLUSIONS: Our data imply separable G-protein and arrestin inputs to the mammalian circadian clock and establish a pair of optogenetic tools suitable for manipulating Gαs- and ß-arrestin-biased signalling in live cells.

Human melanopsin forms a pigment maximally sensitive to blue light (λmax ≈ 479 nm) supporting activation of G(q/11) and G(i/o) signalling cascades.

A subset of mammalian retinal ganglion cells expresses an opsin photopigment (melanopsin, Opn4) and is intrinsically photosensitive. The human retina contains melanopsin, but the literature lacks a direct investigation of its spectral sensitivity or G-protein selectivity. Here, we address this deficit by studying physiological responses driven by human melanopsin under heterologous expression in HEK293 cells. Luminescent reporters for common second messenger systems revealed that light induces a high amplitude increase in intracellular calcium and a modest reduction in cAMP in cells expressing human melanopsin, implying that this pigment is able to drive responses via both Gq and Gi/o class G-proteins. Melanopsins from mouse and amphioxus had a similar profile of G-protein coupling in HEK293 cells, but chicken Opn4m and Opn4x pigments exhibited some Gs activity in addition to a strong Gq/11 response. An action spectrum for the calcium response in cells expressing human melanopsin had the predicted form for an opsin : vitamin A1 pigment and peaked at 479 nm. The G-protein selectivity and spectral sensitivity of human melanopsin is similar to that previously described for rodents, supporting the utility of such laboratory animals for developing methods of manipulating this system using light or pharmacological agents.

Melanopsin and inner retinal photoreception.

Over the last ten years there has been growing acceptance that retinal photoreception among mammals extends beyond rods and cones to include a small number of intrinsically photosensitive retinal ganglion cells (ipRGCs). These ipRGCs are capable of responding to light in the absence of rod/cone input thanks to expression of an opsin photopigment called melanopsin. They are specialised for measuring ambient levels of light (irradiance) for a wide variety of so-called non-image-forming light responses. These include synchronisation of circadian clocks to light:dark cycles and the regulation of pupil size, sleep propensity and pineal melatonin production. Here, we provide a review of some of the landmark discoveries in this fast developing field, paying particular emphasis to recent findings and key areas for future investigation.

Visual ecology of the Australian lungfish (Neoceratodus forsteri).

BACKGROUND: The transition from water to land was a key event in the evolution of vertebrates that occurred over a period of 15-20 million years towards the end of the Devonian. Tetrapods, including all land-living vertebrates, are thought to have evolved from lobe-finned (sarcopterygian) fish that developed adaptations for an amphibious existence. However, while many of the biomechanical and physiological modifications necessary to achieve this feat have been studied in detail, little is known about the sensory adaptations accompanying this transition. In this study, we investigated the visual system and visual ecology of the Australian lungfish Neoceratodus forsteri, which is the most primitive of all the lungfish and possibly the closest living relative to the ancestors of tetrapods. RESULTS: Juvenile Neoceratodus have five spectrally distinct retinal visual pigments. A single type of rod photoreceptor contains a visual pigment with a wavelength of maximum absorbance (lambdamax) at 540 nm. Four spectrally distinct single cone photoreceptors contain visual pigments with lambdamax at 366 (UVS), 479 (SWS), 558 (MWS) and 623 nm (LWS). No double cones were found. Adult lungfish do not possess UVS cones and, unlike juveniles, have ocular media that prevent ultraviolet light from reaching the retina. Yellow ellipsoidal/paraboloidal pigments in the MWS cones and red oil droplets in the LWS cones narrow the spectral sensitivity functions of these photoreceptors and shift their peak sensitivity to 584 nm and 656 nm, respectively. Modelling of the effects of these intracellular spectral filters on the photoreceptor colour space of Neoceratodus suggests that they enhance their ability to discriminate objects, such as plants and other lungfishes, on the basis of colour. CONCLUSION: The presence of a complex colour vision system based on multiple cone types and intracellular spectral filters in lungfishes suggests that many of the ocular characteristics seen in terrestrial or secondarily aquatic vertebrates, such as birds and turtles, may have evolved in shallow water prior to the transition onto land. Moreover, the benefits of spectral filters for colour discrimination apply equally to purely aquatic species as well as semi-aquatic and terrestrial animals. The visual system of the Australian lungfish resembles that of terrestrial vertebrates far more closely than that of other sarcopterygian fish. This supports the idea that lungfishes, and not the coelacanth, are the closest living relatives of the ancestors of tetrapods.

Visual pigments in a living fossil, the Australian lungfish Neoceratodus forsteri.

BACKGROUND: One of the greatest challenges facing the early land vertebrates was the need to effectively interpret a terrestrial environment. Interpretation was based on ocular adaptations evolved for an aquatic environment millions of years earlier. The Australian lungfish Neoceratodus forsteri is thought to be the closest living relative to the first terrestrial vertebrate, and yet nothing is known about the visual pigments present in lungfish or the early tetrapods. RESULTS: Here we identify and characterise five visual pigments (rh1, rh2, lws, sws1 and sws2) expressed in the retina of N. forsteri. Phylogenetic analysis of the molecular evolution of lungfish and other vertebrate visual pigment genes indicates a closer relationship between lungfish and amphibian pigments than to pigments in teleost fishes. However, the relationship between lungfish, the coelacanth and tetrapods could not be absolutely determined from opsin phylogeny, supporting an unresolved trichotomy between the three groups. CONCLUSION: The presence of four cone pigments in Australian lungfish suggests that the earliest tetrapods would have had a colorful view of their terrestrial environment.

Morphology, characterization, and distribution of retinal photoreceptors in the Australian lungfish Neoceratodus forsteri (Krefft, 1870).

The Australian lungfish Neoceratodus forsteri (Dipnoi) is an ancient fish that has a unique phylogenetic relationship among the basal Sarcopterygii. Here we examine the ultrastructure, histochemistry, and distribution of the retinal photoreceptors using a combination of light and electron microscopy in order to determine the characteristics of the photoreceptor layer in this living fossil. Similar proportions of rods (53%) and cones (47%) reveal that N. forsteri optimizes both scotopic and photopic sensitivity according to its visual demands. Scotopic sensitivity is optimized by a tapetum lucidum and extremely large rods (18.62 +/- 2.68 microm ellipsoid diameter). Photopic sensitivity is optimized with a theoretical spatial resolving power of 3.28 +/- 0.66 cycles degree(-1), which is based on the spacing of at least three different cone types: a red cone containing a red oil droplet, a yellow cone containing a yellow ellipsoidal pigment, and a colorless cone containing multiple clear oil droplets. Topographic analysis reveals a heterogeneous distribution of all photoreceptor types, with peak cone densities predominantly found in temporal retina (6,020 rods mm(-2), 4,670 red cones mm(-2), 900 yellow cones mm(-2), and 320 colorless cones mm(-2)), but ontogenetic changes in distribution are revealed. Spatial resolving power and the diameter of all photoreceptor types (except yellow cones) increases linearly with growth. The presence of at least three morphological types of cones provides the potential for color vision, which could play a role in the clearer waters of its freshwater environment.

Reproducible and sustained regulation of Gαs signalling using a metazoan opsin as an optogenetic tool.

Originally developed to regulate neuronal excitability, optogenetics is increasingly also used to control other cellular processes with unprecedented spatiotemporal resolution. Optogenetic modulation of all major G-protein signalling pathways (Gq, Gi and Gs) has been achieved using variants of mammalian rod opsin. We show here that the light response driven by such rod opsin-based tools dissipates under repeated exposure, consistent with the known bleaching characteristics of this photopigment. We continue to show that replacing rod opsin with a bleach resistant opsin from Carybdea rastonii, the box jellyfish, (JellyOp) overcomes this limitation. Visible light induced high amplitude, reversible, and reproducible increases in cAMP in mammalian cells expressing JellyOp. While single flashes produced a brief cAMP spike, repeated stimulation could sustain elevated levels for 10s of minutes. JellyOp was more photosensitive than currently available optogenetic tools, responding to white light at irradiances ≥1 µW/cm(2). We conclude that JellyOp is a promising new tool for mimicking the activity of Gs-coupled G protein coupled receptors with fine spatiotemporal resolution.

The optics of the growing lungfish eye: lens shape, focal ratio and pupillary movements in Neoceratodus forsteri (Krefft, 1870).

Lungfish (order Dipnoi) evolved during the Devonian period and are believed to be the closest living relatives to the land vertebrates. Here we describe the previously unknown morphology of the lungfish eye in order to examine ocular adaptations present in early sarcopterygian fish. Unlike many teleosts, the Australian lungfish Neoceratodus forsteri possesses a mobile pupil with a slow pupillary response similar to amphibians. The structure of the eye changes from juvenile to adult, with both eye and lens becoming more elliptical in shape with growth. This change in structure results in a decrease in focal ratio (the distance from lens center to the retina divided by the lens radius) and increased retinal illumination in adult fish. Despite a degree of lenticular correction for spherical aberration, there is considerable variation across the lens. A re-calculation of spatial resolving power using measured focal ratios from cryosectioning reveals a low ability to discriminate fine detail. The dipnoan eye shares more features with amphibian eyes than with most teleost eyes, which may echo the visual needs of this living fossil.

The number, morphology, and distribution of retinal ganglion cells and optic axons in the Australian lungfish Neoceratodus forsteri (Krefft 1870).

Australian lungfish Neoceratodus forsteri may be the closest living relative to the first tetrapods and yet little is known about their retinal ganglion cells. This study reveals that lungfish possess a heterogeneous population of ganglion cells distributed in a horizontal streak across the retinal meridian, which is formed early in development and maintained through to adult stages. The number and complement of both ganglion cells and a population of putative amacrine cells within the ganglion cell layer are examined using retrograde labelling from the optic nerve and transmission electron-microscopic analysis of axons within the optic nerve. At least four types of retinal ganglion cells are present and lie predominantly within a thin ganglion cell layer, although two subpopulations are identified, one within the inner plexiform and the other within the inner nuclear layer. A subpopulation of retinal ganglion cells comprising up to 7% of the total population are significantly larger (>400 microm2) and are characterized as giant or alpha-like cells. Up to 44% of cells within the retinal ganglion cell layer represent a population of presumed amacrine cells. The optic nerve is heavily fasciculated and the proportion of myelinated axons increases with body length from 17% in subadults to 74% in adults. Spatial resolving power, based on ganglion cell spacing, is low (1.6-1.9 cycles deg(-1), n = 2) and does not significantly increase with growth. This represents the first detailed study of retinal ganglion cells in sarcopterygian fish, and reveals that, despite variation amongst animal groups, trends in ganglion cell density distribution and characteristics of cell types were defined early in vertebrate evolution.