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

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

Cyclic Nucleotide-gated Ion Channels in Rod Photoreceptors Are Protected from Retinoid Inhibition

In vertebrate rods, photoisomerization of the 11-cis retinal chromophore of rhodopsin to the all-trans conformation initiates a biochemical cascade that closes cGMP-gated channels and hyperpolarizes the cell. All-trans retinal is reduced to retinol and then removed to the pigment epithelium. The pigment epithelium supplies fresh 11-cis retinal to regenerate rhodopsin. The recent discovery that tens of nanomolar retinal inhibits cloned cGMP-gated channels at low [cGMP] raised the question of whether retinoid traffic across the plasma membrane of the rod might participate in the signaling of light. Native channels in excised patches from rods were very sensitive to retinoid inhibition. Perfusion of intact rods with exogenous 9- or 11-cis retinal closed cGMP-gated channels but required higher than expected concentrations. Channels reopened after perfusing the rod with cellular retinoid binding protein II. PDE activity, flash response kinetics, and relative sensitivity were unchanged, ruling out pharmacological activation of the phototransduction cascade. Bleaching of rhodopsin to create all-trans retinal and retinol inside the rod did not produce any measurable channel inhibition. Exposure of a bleached rod to 9- or 11-cis retinal did not elicit channel inhibition during the period of rhodopsin regeneration. Microspectrophotometric measurements showed that exogenous 9- or 11-cis retinal rapidly cross the plasma membrane of bleached rods and regenerate their rhodopsin. Although dark-adapted rods could also take up large quantities of 9-cis retinal, which they converted to retinol, the time course was slow. Apparently cGMP-gated channels in intact rods are protected from the inhibitory effects of retinoids that cross the plasma membrane by a large-capacity buffer. Opsin, with its chromophore binding pocket occupied (rhodopsin) or vacant, may be an important component. Exceptionally high retinoid levels, e.g., associated with some retinal degenerations, could overcome the buffer, however, and impair sensitivity or delay the recovery after exposure to bright light

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

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

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

Evidence for ON channel input to ipRGCs from both M-cones and S-cones.

<p><b>A</b>, <b>B</b>: Raster plots of responses to spectrally narrowband stimuli for ten ipRGCs recorded simultaneously in a Cx36 knockout mouse retina. These brisk ON responses were presumably exclusively cone-driven because Cx36 KO mice lack the primary and secondary rod ON pathways and because the flashes were subthreshold for activation by intrinsic melanopsin phototransduction. The response to a 400 nm stimulus (A) closely resembled that to a 500 nm stimulus (B), despite the fact that the former would be more effective in stimulating S-cones and the latter much more effective for M-cones. The two stimuli were approximately matched for photon flux density (400 nm: ∼5×10¹¹ photons cm⁻² s⁻¹; 500 nm: ∼7×10¹¹ photons cm⁻² s⁻¹). <b>C</b>: synaptic blockade eliminated these responses (in this case, the 400 nm response), confirming their origin in the outer retina. <b>D</b>: intrinsic, melanopsin-driven responses revealed in the same cells under synaptic blockade by increasing the intensity, spectral bandwidth and duration of the light. <b>E</b>, <b>F</b>: Group data summarizing the irradiance-response behavior at 400nm and 500nm for these cells (n  =  10), with response amplitudes measured either in terms of the number of evoked spikes (E) or the mean firing rate during the stimulus (F). The curves for the two wavelengths are nearly identical. This is inconsistent with a pure M-cone input, which would predict sensitivity at 500 nm ∼1 log unit greater than that at 400 nm. It is also inconsistent with a pure S-cone input, which would predict >4 log unit greater sensitivity at 400 nm than at 500 nm. We confirmed in a larger sample (25 ipRGCs from three Cx36 KO retinas) that 400nm and 500nm stimuli of matched irradiance evoked comparable responses. <b>G</b>: Whole-cell voltage clamp recordings from a representative example of an M1 cell (top panels) and an M2 cell (bottom panels) from the dorsal retina, where middle and long-wavelength cone opsins are expressed in distinct cone types. Responses are shown to a 1 s step of 360 nm (left) or 500 nm (right) light (horizontal black line). Different colored traces represent responses to different light intensities (in photons•cm⁻²•s⁻¹) for the M1 cell: 3×10¹¹−2×10¹⁴ (360 nm) and 2×10¹²−6×10¹⁴ (500 nm); and for the M2 cell: 7×10¹⁰−5×10¹³ (360 nm) and 1×10¹¹−2×10¹⁴ (500 nm). Similar results were obtained in all M1 and M2 cells recorded, whether in the dorsal retina (six M2 cells and one M1 cell) or the ventral retina (four M2 cells and one M1 cell).</p

ipRGCs with distinct types of intrinsic light response exhibit similar synaptically driven photoresponses.

<p>Upper panel: raster plots of two representative ipRGCs with distinct kinetics of melanopsin-driven intrinsic photoresponse, apparently corresponding to two types defined by Tu et al., (2005) in adult mice. Type II cell (blue) has long onset latency and shorter post-stimulus discharge; Type III has short latency and prolonged afterdischarge. Stimulus was a 60 sec light step (yellow bar; 480 nm, ∼6×10¹² photons/cm²·s). Recordings were performed under synaptic blockade. Lower panel: intensity-response series of the same two cells, but without synaptic blockade. The light stimuli were one second flashes 500 nm flashes ascending in intensity within the series. Both cells showed the same irradiance-response properties, with thresholds approximately 0.02 Rh*/rod/s.</p

Light-evoked synaptic inputs to ipRGCs are less sensitive in Cx36 knockout mice.

<p><b><i>A</i></b>: Raster displays comparing responses of a single representative ipRGC in a Cx36 knockout mouse (right column) with those of an ipRGC in a wildtype littermate (left column). Responses of each cell are shown for a series of 500 nm flashes of increasing intensity. The dimmest stimulus evoking a clear response was approximately three log units higher in the Cx36 KO mouse (∼22.7 Rh^*/rod/s) than it was in the wildtype control (0.02 Rh*/rod/s; see also the data for the C57Bl76 control mice; Fig. 2). <b><i>B</i></b>: Group data comparing irradiance-response curves for ipRGCs recorded in the Cx36 knockout (black squares; n = 31) and wildtype animals (open squares; n = 24). Data come from 4 pairs of littermates consisting of one wildtype and one knockout animal. The triangles near the abscissa indicate the response thresholds (5% of maximum; open triangle, Cx36^+/+; filled triangle, Cx36^−/−), which are about 3 orders of magnitude higher in the knockouts than in the wildtype mice. This reduction in sensitivity presumably reflects the loss of input from the primary rod pathway.</p

Input from the most sensitive rod pathway to ipRGCs.

<p><b><b><i>A</i></b><b>:</b></b> Raster plots of synaptically driven spike responses of a single typical ipRGC in response to a series of flashes increasing in intensity (500 nm; 1 sec). Recordings were extracellular, obtained on a multielectrode array in a dark-adapted wildtype (C57Bl6) mouse retina. <b><i>B</i></b>: Irradiance-response curve plotted in semi-log coordinates and based on data averaged from 23 wildtype mouse ipRGCs. For each cell, response amplitude at a given intensity was expressed as the firing rate during that stimulus normalized to the maximal light-evoked firing rate at any intensity. These values were averaged across all cells for each intensity and plotted, with error bars representing the standard error of the mean. The smooth curve represents the Michaelis-Menten fit to these points and the single triangle along the abscissa indicates the response threshold (5% of maximum, calculated from the fit). This irradiance-response profile closely resembles those of ON type RGCs receiving input from the primary rod pathway <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0066480#pone.0066480-Volgyi1" target="_blank">[50]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0066480#pone.0066480-Deans2" target="_blank">[51]</a>. <b><i>C</i></b>: Distribution of the thresholds of the ON responses in ipRGCs (left; n = 8) in comparison with those of non-ipRGCs (right; n = 22) recorded in the same piece of retina. The thresholds of ipRGCs all located in the lower part of this plot, indicating they are among the most light-sensitive mouse RGCs.</p