Intrinsically photosensitive retinal ganglion cells

Abstract

Life on earth is subject to alternating cycles of day and night imposed by the rotation of the earth. Consequently, living things have evolved photodetective systems to synchronize their physiology and behavior with the external light-dark cycle. This form of photodetection is unlike the familiar "image vision," in that the basic information is light or darkness over time, independent of spatial patterns. "Nonimage" vision is probably far more ancient than image vision and is widespread in living species. For mammals, it has long been assumed that the photoreceptors for nonimage vision are also the textbook rods and cones. However, recent years have witnessed the discovery of a small population of retinal ganglion cells in the mammalian eye that express a unique visual pigment called melanopsin. These ganglion cells are intrinsically photosensitive and drive a variety of nonimage visual functions. In addition to being photoreceptors themselves, they also constitute the major conduit for rod and cone signals to the brain for nonimage visual functions such as circadian photoentrainment and the pupillary light reflex. Here we review what is known about these novel mammalian photoreceptors.

Conflict of interest statement

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the authors.

Figures

FIG. 1

Photoreception without rods and cones. A: suppression of plasma melatonin by light (lighter region) in a patient who was blind from Leber’s congenital amaurosis and lacked a detectable electroretinogram. B: the sleep-wake pattern of the blind patient in A. Solid horizontal lines represent periods of sleep, and open triangles (red) indicate times of peak plasma melatonin level (a marker of the patient’s endogenous circadian period). This patient’s activity patterns were entrained to the environmental 24-h cycle. [A and B modified from Czeisler et al. (34).] C: suppression of melatonin synthesis in wild-type and rodless/coneless mice by light of different irradiances. [Modified from Lucas et al. (115).] D: shift of circadian phase in wild-type, rodless/coneless (rdta/cl), and rodless (rdta) mice by light of different irradiances. E: retinal cross-sections of wild-type and rodless/coneless (rdta/cl) mice stained with antibodies recognizing rod pigment (top), rod and green cone pigments (middle), and ultraviolet cone pigment (bottom) to demonstrate that no outer retinal photoreceptors are detectable in rodless/coneless mice. The same was observed for retinas of rd/rd cl mice used for C. OS, outer segments; IS, inner segments; ONL, outer nuclear layer; INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer; RPE, retinal pigment epithelium. Scale bar is 40 μm. [D and E modified from Freedman et al. (52).]

FIG. 2

Intrinsic photosensitivity of retinal ganglion cells expressing melanopsin. A: predicted secondary structure of melanopsin cloned from Xenopus laevis. [Modified from Provencio et al. (155).] B: immunostaining for melanopsin in a flattened mouse retina revealing a population of retinal ganglion cells with extensive dendritic arbors. Scale bar is 100 μm. [Modified from Provencio et al. (157).] C, top: an ipRGC in the rat identified by retrograde-labeling from the suprachiasmatic nucleus (SCN) and studied with patch-clamp electrophysiological recording. Even with pharmacological block of synaptic transmission, this cell depolarized to light and fired action potentials. Inset shows camera lucida drawing of the recorded cell. [Modified from Berson et al. (17).] Bottom: microelectrode recording from a macaque ipRGC also showing intrinsic photosensitivity. Inset shows tracing of a macaque melanopsin-expressing retinal ganglion cell. Scale bars in top and bottom are 100 and 200 μm, respectively. D: total number of spikes elicited by a 10-s light stimulus rising monotonically with light intensity for a macaque ipRGC recorded with synaptic transmission blocked. [C and D modified from Dacey et al. (35).]

FIG. 3

Brain targets of ipRGCs. A schematic of the mouse brain in sagittal view showing a sampling of regions innervated by ipRGCs. [Modified from Hattar et al. (87).] PO, preoptic area; SCN, suprachiasmatic nucleus; SPZ, subparaventricular zone; pSON, peri-supraoptic nucleus; AH, anterior hypothalamic nucleus; LH, lateral hypothalamus; MA, medial amygdaloid nucleus; LGv, ventral lateral geniculate nucleus; IGL, intergeniculate leaflet; BST, bed nucleus of the stria terminalis; LGd, dorsal lateral geniculate nucleus; LHb, lateral habenula; SC, superior colliculus; OPN, olivary pretectal nucleus; PAG, periaqueductal gray. A: flat-mount retina of a mouse with the tau-lacZ marker gene targeted into the melanopsin gene locus (opn4+/−tauLacZ+/−). Blue color shows X-gal staining of the β-galactosidase activity coded by tau-lacZ in the ipRGCs. IpRGC axons can be seen coursing to the optic disc. Scale bar is 100 μm. [Modified from Hattar et al. (89).] B: ventral view of the opn4+/−tauLacZ+/− brain showing ipRGC axons running in the optic nerve and innervating the suprachiasmatic nuclei (SCN). Scale bar is 1 mm. C: coronal section of the opn4+/−tauLacZ+/− mouse brain showing dense innervation of the SCN. Scale bar is 100 μm. D: prominent innervation of the intergeniculate leaflet (IGL) by ipRGC axons. Coronal section, scale bar is 100 μm. DLG, dorsal lateral geniculate nucleus. [B–D modified from Hattar et al. (88).] E: olivary pretectal nucleus (OPN) is a major target of ipRGCs. Coronal section, scale bar is 100 μm. [Modified from Lucas et al. (116).]

FIG. 4

Morphological diversity and distribution of ipRGCs. A: stacked confocal micrographs demonstrating three morphological subclasses of ipRGCs in mouse. IpRGCs were filled with neurobiotin (green) and the retinas processed for choline acetyltransferase (red) to visualize a population of amacrine cells as anatomical markers. Scale bar is 50 μm. [Modified from Schmidt et al. (174).] B, left: distribution of melanopsin-expressing retinal ganglion cells (dots) in the macaque retina. Superior (S), nasal (N), temporal (T), and inferior (I) directions are indicated. Small open circle is the fovea. Right: higher power views of melanopsin cells in the peripheral retina (top, scale bar is 100 μm) and near the fovea (bottom, scale bar is 200 μm). Note that melanopsin-cell dendrites and somata encircle but do not enter the fovea. C: stacked confocal images of vertical sections through the macaque retina immunostained with melanopsin (green) and counterstained with propidium iodide (red). Note a melanopsin cell displaced to the INL and stratifying its dendrites in the OFF sublamina of the IPL, and another in the GCL and stratifying in the ON sublamina of the IPL (arrows). Scale bar is 50 μm. [B and C modified from Dacey et al. (35).]

FIG. 5

Physiological diversity of ipRGCs. A: simultaneous voltage-clamp recordings from an M1 (gray arrow) and an M2 (white arrow) ipRGC in the flat-mount retina, identified by reporter-gene expression in a melanopsin BAC-transgenic mouse. Synaptic transmission is blocked pharmacologically. The M1 cell shows a much larger intrinsic photocurrent (gray trace on right) than does the M2 cell (black trace) in response to the same light stimulus. Scale bar is 50 μm. B: light-evoked depolarization (recorded in current clamp) in the two ipRGC subtypes as a function of light intensity. M1 cells (gray curve) are roughly 10-fold more sensitive than M2 cells (black curve) at 32–34°C. [A and B modified from Schmidt and Kofuji (173).] C: an early demonstration of physiological diversity of ipRGCs using multielectrode-array recording from retinas of rod/cone-degenerated mice. The total number of light-induced spikes is plotted for two populations of cells in the adult mouse discriminated by cluster analysis at 35°C. [Modified from Tu et al. (205).] D: M1 and M2 ipRGCs also differ in intrinsic membrane properties, with the former firing spikes at a lower rate and being more prone to depolarization block. Whole cell, current-clamp recordings from ipRGCs in flat-mount retinas with synaptic transmission blocked are shown at room temperature. [Modified from Schmidt and Kofuji (173).].

FIG. 6

Whole-animal photic functions involving ipRGCs. A: actogram showing circadian-phase shifting of a wild-type mouse in complete darkness. Dark vertical blips represent wheel-running activity, plotted on a double 24-h time cycle. The endogenous circadian period of this mouse is slightly under 24 h, producing an earlier initiation of activity on each successive day. A 15-min pulse of light given soon after the initiation of activity (open circle) results in a phase shift, with activity beginning with a delay on the next day. B: the extent of phase-shifting for wild-type and melanopsin-null (opn4−/−) mice, with light of different intensities. [A and B modified from Panda et al. (145).] C: negative masking (arrest of locomotor activity) for wild-type (left) and melanopsin-null (right) mice. Melanopsin-null mice can initiate, but not sustain, negative masking by light. Solid circles represent wheel-running activity in darkness. Open circles represent wheel-running activity in light, switched on at time 0. [Modified from Mrosovsky and Hattar (133).] D: pupillary light reflex (PLR) of wild-type mouse and mouse lacking rods/cones and melanopsin (“triple-null” mouse). Triple-null mouse shows virtually no PLR to a light that drives a maximum pupil constriction in the wild-type mouse (top). [Modified from Hattar et al. (89).] E: PLR of wild-type mouse and mouse with ipRGCs largely genetically ablated by targeted, conditional expression of diphtheria-toxin receptor followed by administration of diphtheria toxin. In mice with intact, functioning rods and cones, conditional ablation of ipRGCs eliminated the PLR. [Modified from Hatori et al. (85).].

FIG. 7

Flash response and absolute sensitivity of ipRGCs. A: responses of an ipRGC in the flat-mount retina to diffuse 50-ms flashes at different intensities. Stimulus timing is shown below. B: three smallest responses from A, elicited by successive approximate doublings of flash intensity, on expanded ordinate and longer time base, to demonstrate linearity. Responses fit with the same convolution of two single-exponential decays but scaled by the relative flash intensities (red). C: intensity-response relationships plotted from A. Black circles, peak response-intensity relationship fit with Michaelis equation; red circles, instantaneous intensity-response relationship at 200 ms from flash onset, fit with a saturating exponential function. Dashed curve is a Michaelis fit aligned for comparison with saturating-exponential fit. D: comparison of flash intensity-response relations for rods, cones, and ipRGCs. Rod and cone relations are saturating exponentials (not very different from Michaelis in shape or, for rods and cones, the half-saturating flash intensity), and ipRGC relation is Michaelis. E: partial series of responses of an in situ ipRGC to repeated identical flashes, mostly too dim to elicit a response, at 35°C. The unitary response, by fluctuation analysis, was 2.3 pA. Red traces are identical and represent the expected profile of the unitary response (a brighter flash within the linear range of the cell, scaled to 2.3 pA). Apparent failures (marked by *) and an ambiguous trial (marked by +) were judged according to several detection algorithms. [A–E modified from Do et al. (39).]

FIG. 8

Step response and adaptation of ipRGCs. A: an ipRGC recorded in the flat-mount rat retina under current clamp, showing sustained firing during long steps of light. Numbers to the left of each trace represent the log irradiance (500 nm photons·cm−2·s−1). [Modified from Berson et al. (17).] B: adaptation to light in spike-firing by ipRGCs in current clamp. C: adaptation of intrinsic photocurrent, recorded in voltage clamp, to light. [B and C modified from Wong et al. (227).] All recordings with synaptic transmission blocked and at room temperature.

FIG. 9

Melanopsin phototransduction. A: heterologous expression of mouse melanopsin conferring photosensitivity to a HEK293 cell. Traces show voltage responses to two light intensities (stimulus marked by horizontal bar below the traces). B: spectral sensitivity of light response of HEK293 cells expressing melanopsin heterologously. Curve is opsin-based pigment nomogram with λmax = ~480 nm, similar to that of ipRGCs. [A and B modified from Qiu et al. (161).] C: spectral sensitivity of macaque ipRGC with nomogram fit, together with those (faint curves) of macaque rods as well as macaque short- (S), medium- (M), and long-wavelength (L) cones. [Modified from Dacey et al. (35).] D: block of intrinsic photosensitivity of cultured rat ipRGCs by GDPβS (general G protein blocker) and by GPAnt-2 (Gq subfamily blocker) but not following prolonged exposure to pertussis and cholera toxins (which affect Gi and Gs subfamilies, respectively). E: block of intrinsic photosensitivity of cultured rat ipRGCs by U73122 (phospholipase C blocker) but not by its inactive analog. [D and E modified from Graham et al. (65).] F: current-voltage relation of the intrinsic photocurrent. The membrane currents of an ipRGC elicited by voltage ramps delivered in darkness (black) and light (blue). Difference gives the light-sensitive current (red). [Modified from Warren et al. (219).]

FIG. 10

Schematic of ipRGCs and retinal circuitry. A schematic of the retinal cross-section, illustrating the reported circuitry of M1 (left) and M2 (right) ipRGCs. Downward-pointing arrows indicate transmission to ipRGCs, and upward-pointing arrows indicate transmission from ipRGCs. Red arrows indicate excitation, and blue arrowheads indicate inhibition. Question marks indicate connections/interactions awaiting confirmation from electrophysiology. M1, M1 ipRGC; M2, M2 ipRGC; R, rod bipolar cell; C, cone bipolar cell; II, AII amacrine cell; MA, monostratified amacrine cell; DA, dopaminergic amacrine cell; PL. photoreceptor layer; IPL, inner plexiform layer; INL, inner nuclear layer; GCL, ganglion cell layer. IpRGCs are also coupled electrically to cells in the GCL (blue cells). The retinal circuitry of M3 cells has not been reported and thus is not illustrated here.

FIG. 11

ON synapses in the OFF sublamina to ipRGCs. A: vertical section through a mouse retina expressing GFP under control of the mGluR6 promoter, active in ON cone bipolar cells. Scale bar is 20 μm. B: high-power view of section in A to show axonal swellings, which are en passant synapses, and lateral extensions from the axons terminating in swellings, which are ectopic synapses. Scale bar is 10 μm. [A and B modified from Dumitrescu et al. (43).] C: melanopsin immunostaining of a flat-mounted rabbit retina (red). Descending axons of calbindin-expressing bipolar cells (green) forming ectopic, en passant synapses on ipRGC dendrites in the OFF sublamina of the IPL. Scale bar is 20 μm. D: axon terminals of calbindin-expressing bipolar cells in rabbit (green) contacting ipRGC dendrites in the ON sublamina of the IPL. Scale bar is 10 μm. E: synaptic ribbons (positive for RIBEYE-immunostaining) colocalized with junctions between calbindin-positive axons (blue) and ipRGC dendrites (green). Scale bar is 1 μm. [C–E modified from Hoshi et al. (94).]

FIG. 12

Synaptic drive to ipRGCs. A: macaque ipRGCs driven at light onset by long- and medium-wavelength cone signals, and at light offset by short-wavelength cone signals. B: receptive fields of ON and OFF responses are virtually coextensive and match the dendritic fields of the ipRGCs. Shown are Gaussian fits to the receptive fields. C: response of a macaque ipRGC to a 470-nm light step as a function of retinal illumination for 3 stimulus conditions: dark-adapted (blue circles), light-adapted (black circles), and light-adapted with synaptic block (open circles). Boxed area below plot shows melanopsin-associated, rod and cone response ranges in relation to scotopic, mesopic, and photopic ranges of human vision and pupil diameter. [A–C modified from Dacey et al. (35).] D: normalized, population spike rates of ipRGCs and conventional RGCs recorded by multielectrode array, with synaptic transmission intact. Signaling by ipRGCs is sustained, even at light intensities that are too low to drive intrinsic photosensitivity, suggesting that synaptic input to these neurons is also specialized for long-duration signaling. Numbers at left are log attenuations of the broadband tungsten-halogen light stimulus (unattenuated stimulus sampled at 480 nm, 2.3 × 1013 photons·cm−2·s−1). [Modified from Wong et al. (228).]

FIG. 13

IpRGCs during development. A: calcium imaging of a retina taken from wild-type mouse at birth. Two of three cells studied in this field of view (left) showed a light-driven rise in calcium (right). Timing of the light stimulus shown by shaded region. [Modified from Sekaran et al. (178).] B: increase in photosensitivity of ipRGCs with age. Whole cell, current-clamp recordings from mouse ipRGCs in the flat-mount retina with synaptic transmission intact. Responses to four different light intensities are shown for three developmental times. [Modified from Schmidt et al. (174).]

FIG. 14

Potential clinical relevance of ipRGCs. A: conventional RGCs in rod/cone-degenerated mice, driven to express melanopsin by viral transduction. Whole cell current-clamp recording in flat-mount retina to study their physiology (at 32–35°C) and Lucifer Yellow injection to study their morphology. Light responses from two cells consisting of a long-lasting depolarization and action-potential firing are shown (stimulus timing and duration indicated by a short black bar beneath the recorded trace). Conventional RGCs expressing melanopsin responded to light even in rod/cone-degenerate retina and under pharmacological block of synaptic transmission. Scale bars are 50 μm. B: visual discrimination task. Mice swam down a water-filled alley toward an illuminated or dark compartment, with the rewarded stimulus being paired with the location of a submerged platform. C: melanopsin-transduced (open circles) mice outperforming control-transduced (triangles) rod/cone-degenerated mice in the visual detection task over an 8-day trial. [A–C modified from Lin et al. (111).]