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. 2015 Mar 4;85(5):1043-55.
doi: 10.1016/j.neuron.2015.02.011.

Melanopsin Tristability for Sustained and Broadband Phototransduction

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Free PMC article

Melanopsin Tristability for Sustained and Broadband Phototransduction

Alan Joseph Emanuel et al. Neuron. .
Free PMC article

Abstract

Mammals rely upon three ocular photoreceptors to sense light: rods, cones, and intrinsically photosensitive retinal ganglion cells (ipRGCs). Rods and cones resolve details in the visual scene. Conversely, ipRGCs integrate over time and space, primarily to support "non-image" vision. The integrative mechanisms of ipRGCs are enigmatic, particularly since these cells use a phototransduction motif that allows invertebrates like Drosophila to parse light with exceptional temporal resolution. Here, we provide evidence for a single mechanism that allows ipRGCs to integrate over both time and wavelength. Light distributes the visual pigment, melanopsin, across three states, two silent and one signaling. Photoequilibration among states maintains pigment availability for sustained signaling, stability of the signaling state permits minutes-long temporal summation, and modest spectral separation of the silent states promotes uniform activation across wavelengths. By broadening the tuning of ipRGCs in both temporal and chromatic domains, melanopsin tristability produces signal integration for physiology and behavior.

Conflict of interest statement

A.J.E. and M.T.H.D. have no conflicts of interest.

Figures

Figure 1
Figure 1. Persistent Responses and Temporal Integration of IpRGCs
(A) Membrane voltage of an ipRGC in response to a series of 10-sec pulses of white light (xenon at an intensity of 2.5×10−5 μW μm−2, equivalent to 3.3×104 lux). The corresponding spike rate is shown below in 10-sec bins. Light monitor at bottom. Activation that continues beyond the period of illumination is referred to as the persistent response. (B) Excerpts of the trace in A illustrating the response to the first and sixth light pulses. Dashed line represents the baseline (−60 mV). (C) Subthreshold membrane voltage, averaged from 30–40 sec after each light pulse, is displayed for individual cells (connected markers, n = 6 cells) and the population mean (bars) for this protocol. The difference from baseline, normalized to the maximum difference, is displayed for each cell. Closed markers represent the cell shown in A. (D) Subthreshold membrane voltage after the last pulse for the cell shown in A (in 5-sec bins). Time point 0 corresponds to the end of illumination. Fit is a single exponential (τ = 107 sec). All experiments were performed at 35 °C with synaptic transmission intact. See also Figure S1.
Figure 2
Figure 2. Persistent Responses are Inherent to IpRGCs and Bi-directionally Modulated by Light
(A) A persistent response evoked during pharmacological block of synaptic transmission. A flash of monochromatic, short-wavelength light was followed by darkness (~20 min) and then a pulse of long-wavelength light. (B) An example of wavelength- and timing-dependent modulations of the persistent response in another ipRGC. (C) Protocol for quantifying persistent responses in current clamp displayed with a representative recording. Periods of measurement are indicated by bars. Dashed lines indicate the average voltages measured following each stimulus (with lines extended for clarity). Persistent responses following short-wavelength stimuli are sufficiently large, in this cell, to cause depolarization block. Passive electrical properties were tested with hyperpolarizing current injections (5 pA, 1 sec; bottom trace). (D) Population data from the experiment illustrated in C with the difference in membrane voltage (from darkness) plotted for individual cells (connected markers) and the population mean (bars). Closed markers indicate the cell in C. Groups marked with asterisks and daggers differ significantly from each other (n = 8 cells, p < 0.001). All experiments were performed at 23 °C with synaptic transmission blocked. Light stimuli were 50-ms flashes or 10-sec pulses (monitors below traces). See Figure S2 for spectra and intensities of light.
Figure 3
Figure 3. Wavelength-dependence of Persistent Responses Measured in Voltage Clamp
(A) Example of a persistent response (i.e., the light-evoked current that continues to flow beyond the period of illumination) modulated by successive pulses of short- and long-wavelength light. This response does not show temporal integration because it is saturated with each short-wavelength pulse at the intensity used here, and there is negligible dark regeneration between pulses. Light monitors shown below trace. See Figure S2 for spectra and intensities of light. (B) Protocol for quantifying the magnitude of the persistent response as a function of wavelength. Test wavelengths were alternated with a “reset” wavelength of 560 nm to establish a baseline. The intensities of all stimuli (1×109 – 2×109 photons μm−2 sec−1) were sufficient to produce a saturated persistent response at each wavelength tested. (C) Population data from protocol in B for difference in current from baseline (left), difference in current normalized to minimum and maximum for each cell (middle), and current noise in the same period (standard deviation normalized to minimum and maximum; right). Connected markers are individual cells. All experiments were in voltage clamp (−80 mV) at 23 °C with synaptic transmission blocked.
Figure 4
Figure 4. Two Silent States of Melanopsin Detected in IpRGCs
Left: The spectral sensitivity (i.e., action spectrum) of a single ipRGC measured in darkness as well as during a 600-nm background light (black and red markers, respectively). Plotted is the sensitivity of the cell to dim-flashes of each test wavelength (SF, in pA photons−1 μm2) normalized to the maximum for this cell (SFmax). Continuous curves are single-state nomograms fit to the data with λmax = 471 nm (darkness) and 454 nm (600-nm background). Right: Population averages for the same conditions (mean ± SEM; n = 6 cells) with λmax = 471 nm (darkness, the “cyan” state) and 453 nm (600-nm background, the “violet” state). 600-nm background was delivered at 4×106 – 7×108 photons μm−2 s−1. Measurements were made at 35 and 23 °C with no detectable variation in λmax with temperature. Synaptic transmission was blocked and the holding voltage was −80 mV. See also Figure S3.
Figure 5
Figure 5. Broadened Spectral Sensitivity of IpRGCs Due to Activation of Melanopsin from Two Silent States
(A) Action spectrum of a single ipRGC (left) and the population (right) on a background of short-wavelength light (440 nm). Dashed lines are single-state nomograms used to fit the spectra in Figure 4 (black, representing the cyan state and red, the violet state). Insets: Same data but the curves are weighted sums of the same two nomograms (Left: 53% cyan and 47% violet; Right: 69% cyan and 31% violet). (B) Action spectrum measured from a single ipRGC (left) and the population (right) on a background of white (xenon) light. Black and red dashed lines represent the cyan and violet nomograms, respectively. Insets: same data but each curve is the weighted sum of the nomograms for the cyan and violet states (Left: 60% cyan and 40% violet; Right: 59% cyan and 41% violet). Background lights were 1×105 – 7×105 photons μm−2 sec−1 (440 nm) and 1.5×10−7 μW μm−2 (equivalent to 50 lux; xenon). Synaptic transmission was blocked. Experiments were performed at 35 and 23 °C, with no difference between these conditions detected. Holding voltage was −80 mV. All error bars are SEM.
Figure 6
Figure 6. Indistinguishable Activation from the Two Silent States of Melanopsin
(A) Schematic of the protocol used to compare responses evoked from the cyan and violet states. A 440-nm background generates a majority fraction of the cyan state and a 600-nm background generates a dominant fraction of the violet state. Backgrounds were matched in intensity to produce equivalent activation of ipRGCs and presented in random order (3 and 2 cells with 440 and 600 nm first, respectively). (B) Dim-flash responses evoked on 440- and 600-nm backgrounds (blue and red traces, respectively). Traces are the average of seven trials. Fits are convolutions of two exponentials, A (et/τ1et/τ2), shown normalized to their peaks in the bottom panel (time constants for blue and red traces: τ1, 2.3 and 1.7 sec; τ2, 5.7 and 7.5 sec). Light monitors are below. (C) Time constants from the population of cells (closed markers for the cell in B). “n.s.” is lack of statistical significance. Background lights were 9×105 photons μm−2 s−1 (440 nm) and 1×108 photons μm−2 s−1 (600 nm). Synaptic transmission was blocked in all experiments, which were performed at 23 °C. Holding voltage was −80 mV.
Figure 7
Figure 7. A State Model for Tristable Melanopsin
(A) State diagram of melanopsin (top) based on parameters measured biochemically from purified pigment (Matsuyama et al., 2012). Shown are melanopsin (R), metamelanopsin (M), and extramelanopsin (E) with chromophores designated. Below are plotted the relative photosensitivities (i.e., products of the extinction coefficients and quantum efficiencies) of these states as a function of wavelength. Only two model parameters are experimentally undefined: the quantum efficiency of the E state and the fraction of M isomerizations that yields the R versus E state (set at 0.4 and 0.5, respectively; Experimental Procedures). Only the latter parameter is not depicted here. Direct photoconversion between the R and E states is unlikely given energetic constraints of chromophore isomerization. (B) Predicted equilibrium fraction of each pigment state as a function of wavelength. Lines show the R state (black), M state (blue), and E state (red). See also Figure S4.
Figure 8
Figure 8. Melanopsin Tristability under Diverse Lighting Conditions
(A) Measured spectra of various, common light sources (in photons μm−2 sec−1 nm−1 prior to normalization). (B) Left: State diagram and relative photosensitivities as displayed in Figure 7A. Middle: Predicted equilibrium fractions of melanopsin states for monochromatic illumination at two wavelengths (440 and 560 nm). Right: Predicted equilibrium fractions of melanopsin states for broadband illumination by the sources shown in A. (C) Same as B but for a hypothetical bistable melanopsin with only the ground state (R) and metamelanopsin (M). (D) Same as B but for Drosophila rhodopsin (R) and metarhodopsin (M). Midday and sunset spectra in A are courtesy of Sönke Johnson (Johnsen et al., 2006).

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