Maureen E. Estevez

Form and function of the M4 cell, an intrinsically photosensitive retinal ganglion cell type contributing to geniculocortical vision.

The photopigment melanopsin confers photosensitivity upon a minority of retinal output neurons. These intrinsically photosensitive retinal ganglion cells (ipRGCs) are more diverse than once believed, comprising five morphologically distinct types, M1 through M5. Here, in mouse retina, we provide the first in-depth characterization of M4 cells, including their structure, function, and central projections. M4 cells apparently correspond to ON α cells of earlier reports, and are easily distinguished from other ipRGCs by their very large somata. Their dendritic arbors are more radiate and highly branched than those of M1, M2, or M3 cells. The melanopsin-based intrinsic photocurrents of M4 cells are smaller than those of M1 and M2 cells, presumably because melanopsin is more weakly expressed; we can detect it immunohistochemically only with strong amplification. Like M2 cells, M4 cells exhibit robust, sustained, synaptically driven ON responses and dendritic stratification in the ON sublamina of the inner plexiform layer. However, their stratification patterns are subtly different, with M4 dendrites positioned just distal to those of M2 cells and just proximal to the ON cholinergic band. M4 receptive fields are large, with an ON center, antagonistic OFF surround and nonlinear spatial summation. Their synaptically driven photoresponses lack direction selectivity and show higher ultraviolet sensitivity in the ventral retina than in the dorsal retina, echoing the topographic gradient in S- and M-cone opsin expression. M4 cells are readily labeled by retrograde transport from the dorsal lateral geniculate nucleus and thus likely contribute to the pattern vision that persists in mice lacking functional rods and cones.

Intra-retinal visual cycle required for rapid and complete cone dark adaptation

Daytime vision is mediated by retinal cones, which, unlike rods, remain functional even in bright light and dark-adapt rapidly. These cone properties are enabled by rapid regeneration of their pigment. This in turn requires rapid chromophore recycling that may not be achieved by the canonical retinal pigment epithelium visual cycle. Recent biochemical studies have suggested the presence of a second, cone-specific visual cycle, although its physiological function remains to be established. We found that the Müller cells in the salamander neural retina promote cone-specific pigment regeneration and dark adaptation that are independent of the pigment epithelium. Without this pathway, dark adaptation of cones was slow and incomplete. Notably, the rates of cone pigment regeneration by the retina and pigment epithelium visual cycles were essentially identical, suggesting a possible common rate-limiting step. Finally, we also observed cone dark adaptation in the isolated mouse retina.

Breaking the Covalent Bond— A Pigment Property that Contributes to Desensitization in Cones

Retinal rod and cone pigments consist of an apoprotein, opsin, covalently linked to a chromophore, 11-cis retinal. Here we demonstrate that the formation of the covalent bond between opsin and 11-cis retinal is reversible in darkness in amphibian red cones, but essentially irreversible in red rods. This dissociation, apparently a general property of cone pigments, results in a surprisingly large amount of free opsin--about 10% of total opsin--in dark-adapted red cones. We attribute this significant level of free opsin to the low concentration of intracellular free 11-cis retinal, estimated to be only a tiny fraction (approximately 0.1 %) of the pigment content in red cones. With its constitutive transducin-stimulating activity, the free cone opsin produces an approximately 2-fold desensitization in red cones, equivalent to that produced by a steady light causing 500 photoisomerizations s-1. Cone pigment dissociation therefore contributes to the sensitivity difference between rods and cones.

Genetic dissection of retinal inputs to brainstem nuclei controlling image stabilization.

When the head rotates, the image of the visual world slips across the retina. A dedicated set of retinal ganglion cells (RGCs) and brainstem visual nuclei termed the “accessory optic system” (AOS) generate slip-compensating eye movements that stabilize visual images on the retina and improve visual performance. Which types of RGCs project to each of the various AOS nuclei remain unresolved. Here we report a new transgenic mouse line, Hoxd10–GFP, in which the RGCs projecting to all the AOS nuclei are fluorescently labeled. Electrophysiological recordings of Hoxd10–GFP RGCs revealed that they include all three subtypes of On direction-selective RGCs (On–DSGCs), responding to upward, downward, or forward motion. Hoxd10–GFP RGCs also include one subtype of On–Off DSGCs tuned for forward motion. Retrograde circuit mapping with modified rabies viruses revealed that the On–DSGCs project to the brainstem centers involved in both horizontal and vertical retinal slip compensation. In contrast, the On–Off DSGCs labeled in Hoxd10–GFP mice projected to AOS nuclei controlling horizontal but not vertical image stabilization. Moreover, the forward tuned On–Off DSGCs appear physiologically and molecularly distinct from all previously genetically identified On–Off DSGCs. These data begin to clarify the cell types and circuits underlying image stabilization during self-motion, and they support an unexpected diversity of DSGC subtypes.

Visual Cycle: Dependence of Retinol Production and Removal on Photoproduct Decay and Cell Morphology

The visual cycle is a chain of biochemical reactions that regenerate visual pigment following exposure to light. Initial steps, the liberation of all-trans retinal and its reduction to all-trans retinol by retinol dehydrogenase (RDH), take place in photoreceptors. We performed comparative microspectrophotometric and microfluorometric measurements on a variety of rod and cone photoreceptors isolated from salamander retinae to correlate the rates of photoproduct decay and retinol production. Metapigment decay rate was spatially uniform within outer segments and 50–70 times faster in the cells that contained cone-type pigment (SWS2 and M/LWS) compared to cells with rod-type pigment (RH1). Retinol production rate was strongly position dependent, fastest at the base of outer segments. Retinol production rate was 10–40 times faster in cones with cone pigments (SWS2 and M/LWS) than in the basal OS of rods containing rod pigment (RH1). Production rate was approximately five times faster in rods containing cone pigment (SWS2) than the rate in basal OS of rods containing the rod pigment (RH1). We show that retinol production is defined either by metapigment decay rate or RDH reaction rate, depending on cell type or outer segment region, whereas retinol removal is defined by the surface-to-volume ratio of the outer segment and the availability of retinoid binding protein (IRBP). The more rapid rates of retinol production in cones compared to rods are consistent with the more rapid operation of the visual cycle in these cells.

Mouse Ganglion-Cell Photoreceptors Are Driven by the Most Sensitive Rod Pathway and by Both Types of Cones

Intrinsically photosensitive retinal ganglion cells (ipRGCs) are depolarized by light by two mechanisms: directly, through activation of their photopigment melanopsin; and indirectly through synaptic circuits driven by rods and cones. To learn more about the rod and cone circuits driving ipRGCs, we made multielectrode array (MEA) and patch-clamp recordings in wildtype and genetically modified mice. Rod-driven ON inputs to ipRGCs proved to be as sensitive as any reaching the conventional ganglion cells. These signals presumably pass in part through the primary rod pathway, involving rod bipolar cells and AII amacrine cells coupled to ON cone bipolar cells through gap junctions. Consistent with this interpretation, the sensitive rod ON input to ipRGCs was eliminated by pharmacological or genetic disruption of gap junctions, as previously reported for conventional ganglion cells. A presumptive cone input was also detectable as a brisk, synaptically mediated ON response that persisted after disruption of rod ON pathways. This was roughly three log units less sensitive than the rod input. Spectral analysis revealed that both types of cones, the M- and S-cones, contribute to this response and that both cone types drive ON responses. This contrasts with the blue-OFF, yellow-ON chromatic opponency reported in primate ipRGCs. The cone-mediated response was surprisingly persistent during steady illumination, echoing the tonic nature of both the rod input to ipRGCs and their intrinsic, melanopsin-based phototransduction. These synaptic inputs greatly expand the dynamic range and spectral bandpass of the non-image-forming visual functions for which ipRGCs provide the principal retinal input.

Visual cycle and its metabolic support in gecko photoreceptors

Photoreceptors of nocturnal geckos are transmuted cones that acquired rod morphological and physiological properties but retained cone-type phototransduction proteins. We have used microspectrophotometry and microfluorometry of solitary isolated green-sensitive photoreceptors of Tokay gecko to study the initial stages of the visual cycle within these cells. These stages are the photolysis of the visual pigment, the reduction of all-trans retinal to all-trans retinol, and the clearance of all-trans retinol from the outer segment (OS) into the interphotoreceptor space. We show that the rates of decay of metaproducts (all-trans retinal release) and retinal-to-retinol reduction are intermediate between those of typical rods and cones. Clearance of retinol from the OS proceeds at a rate that is typical of rods and is greatly accelerated by exposure to interphotoreceptor retinoid-binding protein, IRBP. The rate of retinal release from metaproducts is independent of the position within the OS, while its conversion to retinol is strongly spatially non-uniform, being the fastest at the OS base and slowest at the tip. This spatial gradient of retinol production is abolished by dialysis of saponin-permeabilized OSs with exogenous NADPH or substrates for its production by the hexose monophosphate pathway (NADP+glucose-6-phosphate or 6-phosphogluconate, glucose-6-phosphate alone). Following dialysis by these agents, retinol production is accelerated by several-fold compared to the fastest rates observed in intact cells in standard Ringer solution. We propose that the speed of retinol production is set by the availability of NADPH which in turn depends on ATP supply within the outer segment. We also suggest that principal source of this ATP is from mitochondria located within the ellipsoid region of the inner segment.

Chromophore Supply Rate-Limits Mammalian Photoreceptor Dark Adaptation

Efficient regeneration of visual pigment following its destruction by light is critical for the function of mammalian photoreceptors. Here, we show that misexpression of a subset of cone genes in the rd7 mouse hybrid rods enables them to access the normally cone-specific retina visual cycle. The rapid supply of chromophore by the retina visual cycle dramatically accelerated the mouse rod dark adaptation. At the same time, the competition between rods and cones for retina-derived chromophore slowed cone dark adaptation, indicating that the cone specificity of the retina visual cycle is key for rapid cone dark adaptation. Our findings demonstrate that mammalian photoreceptor dark adaptation is dominated by the supply of chromophore. Misexpression of cone genes in rods may represent a novel approach to treating visual disorders associated with mutations of visual cycle proteins or with reduced retinal pigment epithelium function due to aging.

The 9-methyl group of retinal is essential for rapid Meta II decay and phototransduction quenching in red cones.

Cone photoreceptors of the vertebrate retina terminate their response to light much faster than rod photoreceptors. However, the molecular mechanisms underlying this rapid response termination in cones are poorly understood. The experiments presented here tested two related hypotheses: first, that the rapid decay rate of metarhodopsin (Meta) II in red-sensitive cones depends on interactions between the 9-methyl group of retinal and the opsin part of the pigment molecule, and second, that rapid Meta II decay is critical for rapid recovery from saturation of red-sensitive cones after exposure to bright light. Microspectrophotometric measurements of pigment photolysis, microfluorometric measurements of retinol production, and single-cell electrophysiological recordings of flash responses of salamander cones were performed to test these hypotheses. In all cases, cones were bleached and their visual pigment was regenerated with either 11-cis retinal or with 11-cis 9-demethyl retinal, an analogue of retinal lacking the 9-methyl group. Meta II decay was four to five times slower and subsequent retinol production was three to four times slower in red-sensitive cones lacking the 9-methyl group of retinal. This was accompanied by a significant slowing of the recovery from saturation in cones lacking the 9-methyl group after exposure to bright (>0.1% visual pigment photoactivated) but not dim light. A mathematical model of the turn-off process of phototransduction revealed that the slower recovery of photoresponse can be explained by slower Meta decay of 9-demethyl visual pigment. These results demonstrate that the 9-methyl group of retinal is required for steric chromophore–opsin interactions that favor both the rapid decay of Meta II and the rapid response recovery after exposure to bright light in red-sensitive cones.

Turning Cones Off: the Role of the 9-Methyl Group of Retinal in Red Cones

Our ability to see in bright light depends critically on the rapid rate at which cone photoreceptors detect and adapt to changes in illumination. This is achieved, in part, by their rapid response termination. In this study, we investigate the hypothesis that this rapid termination of the response in red cones is dependent on interactions between the 9-methyl group of retinal and red cone opsin, which are required for timely metarhodopsin (Meta) II decay. We used single-cell electrical recordings of flash responses to assess the kinetics of response termination and to calculate guanylyl cyclase (GC) rates in salamander red cones containing native visual pigment as well as visual pigment regenerated with 11-cis 9-demethyl retinal, an analogue of retinal in which the 9-methyl group is missing. After exposure to bright light that photoactivated more than ∼0.2% of the pigment, red cones containing the analogue pigment had a slower recovery of both flash response amplitudes and GC rates (up to 10 times slower at high bleaches) than red cones containing 11-cis retinal. This finding is consistent with previously published biochemical data demonstrating that red cone opsin regenerated in vitro with 11-cis 9-demethyl retinal exhibited prolonged activation as a result of slowed Meta II decay. Our results suggest that two different mechanisms regulate the recovery of responsiveness in red cones after exposure to light. We propose a model in which the response recovery in red cones can be regulated (particularly at high light intensities) by the Meta II decay rate if that rate has been inhibited. In red cones, the interaction of the 9-methyl group of retinal with opsin promotes efficient Meta II decay and, thus, the rapid rate of recovery.