Using the photopigment melanopsin, intrinsically photosensitive retinal ganglion cells (ipRGCs) respond directly to light to drive circadian clock resetting and pupillary constriction. We now report that ipRGCs are more abundant and diverse than previously appreciated, project more widely within the brain, and can support spatial visual perception. A Cre-based melanopsin reporter mouse line revealed at least five subtypes of ipRGCs with distinct morphological and physiological characteristics. Collectively, these cells project beyond the known brain targets of ipRGCs to heavily innervate the superior colliculus and dorsal lateral geniculate nucleus, retinotopically organized nuclei mediating object localization and discrimination. Mice lacking classical rod-cone photoreception, and thus entirely dependent on melanopsin for light detection, were able to discriminate grating stimuli from equiluminant gray and had measurable visual acuity. Thus, nonclassical retinal photoreception occurs within diverse cell types and influences circuits and functions encompassing luminance as well as spatial information.

Melanopsin-expressing ipRGCs were previously considered to be a homogenous cell population, with sparsely branched dendritic arbors stratifying in the outermost sublamina of the inner plexiform layer (IPL) (). Subsequent work revealed morphological and functional diversity among ganglion cells expressing melanopsin or exhibiting intrinsic photosensitivity (). A second type of melanopsin-expressing ipRGCs has a monostratified dendritic arbor in the inner part of the IPL. First discovered in primate retina (), these cells have also been observed in rodents, where they are termed M2 or type II cells to distinguish them from the originally characterized, outer-stratifying M1 or type I cells (). Indirect evidence suggests that M2 ipRGCs share some central targets with M1 cells, including the suprachiasmatic nucleus (SCN) and olivary pretectal nucleus (OPN) (). Other variants of ipRGCs have been reported, some with cell bodies displaced to the inner nuclear layer and others with bistratified dendritic arbors (sometimes called “type III cells” and here termed M3 cells) (). The photosensitivity and brain projections of these newer cell types are less well characterized than those of M1 ipRGCs. The identification of new subtypes of melanopsin cells raises the prospect that the ipRGC retinal diversity extends to new innervation of brain targets for supporting other light-dependent physiological functions.

Photoreceptive net in the mammalian retina. This mesh of cells may explain how some blind mice can still tell day from night.

Photoreceptive net in the mammalian retina. This mesh of cells may explain how some blind mice can still tell day from night.

Photoreceptive net in the mammalian retina. This mesh of cells may explain how some blind mice can still tell day from night.

A small percentage of mammalian retinal ganglion cells contain the photopigment melanopsin and are capable of autonomous phototransduction (). These intrinsically photosensitive retinal ganglion cells (ipRGCs) combine their direct, melanopsin-based photoresponses with signals derived from rods and cones and convey these directly to a subset of retinal targets in the brain. The ipRGCs mediate a variety of physiological responses to ambient light, such as circadian photoentrainment and the pupillary light reflex ().

Photoreceptive net in the mammalian retina. This mesh of cells may explain how some blind mice can still tell day from night.

Results

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Berson D.M. Synaptic influences on rat ganglion-cell photoreceptors. Figure 2 Diversity of Morphology and Intrinsic Light Responses of Ganglion Cells GFP in the Opn4Cre/+; Z/EG Mouse Show full caption (A–C) Intracellular dye filling of representative examples of three subtypes of ipRGCs targeted by their GFP fluorescence in vitro, an M1 cell (A), an M2 cell (B), and an M4 cell (C). All three of these cells were intrinsically photosensitive, as shown by the whole-cell voltage-clamp recordings below (J–L), obtained during pharmacological blockade of retinal synapses. Light pulse was 20 s. Each trace in a given panel shows the response to a different light intensity. Values at left are the number of log units of attenuation in stimulus intensity from the maximal (“0 log”; 2.3 × 1013 photons cm−2 s−1). Light-evoked currents were much larger in the M1 cell (J) than in the M2 cell (K) or M4 cell (L); they also returned more quickly to baseline after the stimulus. Fast downward deflections are presumed action currents resulting from incomplete voltage clamp. (D–I) Immunofluorescence (dotted circle) for melanopsin (E, G, and I) and Lucifer yellow-injected cells (D, F, and H) show that M1 and M2 cells that are used for recording are melanopsin positive, whereas the M4 cell (C, H, I, and L) despite showing an intrinsic photoresponse lacked detectable melanopsin immunofluorescence. Note that this figure is optimized to highlight the recorded cells and hence some melanopsin-positive cells appear to lack GFP labeling (>95% of melanopsin-positive cells express GFP). Top trace in (J) slightly retouched to eliminate electrical artifact from series resistance test conducted well after the light response. Scale bars in (A)–(C), 100 μm; (D)–(I), 20 μm. Whole-cell patch-clamp recordings of EGFP-positive ganglion cells in Opn4Cre/+; Z/EG mice, in the presence of synaptic blockers, revealed that nearly all (46 of 51 cells tested; 90%) were intrinsically photosensitive ( Figures 2 and S2 ), even when they lacked detectable melanopsin immunoreactivity ( Figures 2 H and 2I). Under pharmacological blockade of retinal synapses, these cells exhibited sluggish, persistent light responses, characteristic of melanopsin-based phototransduction (). A small minority of EGFP-labeled cells lacked demonstrable intrinsic photosensitivity (5 of 51 cells; 10%), but exhibited brisk synaptically driven light responses (data not shown). Such cells either may have leaky expression or may have transiently expressed melanopsin during development; this would have triggered permanent expression of the marker proteins, since after Cre-mediated excision of the stop codon, marker protein expression is regulated solely by the promoter of the reporter transgene ( Figures S1 C and S1D).

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Cooper H.M. Immunohistochemical evidence of a melanopsin cone in human retina. We also detected the marker proteins in a small percentage of rods and cones; these were melanopsin immunonegative ( Figures 1 C–1H). Their labeling by the marker proteins could be either due to leaky or transient expression of Cre or because the Cre system is more sensitive than immunohistochemistry for detecting melanopsin expression. The finding may be linked to melanopsin-like immunoreactivity in a very few human cones ().

Schmidt and Kofuji, 2009 Schmidt T.M.

Kofuji P. Functional and morphological differences among intrinsically photosensitive retinal ganglion cells. Figure 3 Differences in Soma Size, Dendritic Field Diameter, Total Branchpoints, and Dendritic Length of M2 and M4 ipRGCs Show full caption (A) Six Lucida drawings of representative M2 and M4 ipRGCs. (B) Total dendritic branch length (TDBL) versus soma size of M2 and M4 cells (numbers are in micrometers, TDBL: M2; 1553 ± 428, M4; 4584 ± 1465, soma size: M2; 15.7 ± 2.2, M4 20.1 ± 2.2). (C) Dendritic field diameter versus total dendritic branchpoints of M2 and M4 cells (total branchpoints: M2; 14.3 ± 4.4, M4; 37.8 ± 9.6, dendritic field diameter (μm): M2; 243 ± 39.9, M4; 301 ± 35.4). Open circles are M2 ipRGCs while black diamonds are M4 ipRGCs. Range is provided as average ± standard deviation. We used dye filling to visualize the morphology of the reporter-labeled cells that were intrinsically photosensitive. Labeled cells included not only the previously characterized M1 and M2 ipRGCs, but also several new morphologically distinct ganglion cell types ( Figure 2 and S2 ). M1 ipRGCs had sparsely branching monostratified dendritic ramifications in the outermost IPL, had an average dendritic field diameter of 350 ± 87 μm (mean ± SD; n = 12), and cell body diameter of 15.6 ± 2.4 μm (n = 7; Figure 2 A). M2 ipRGCs had relatively large radiate dendritic arbors ( Figure 2 B) stratifying within the inner half of the IPL (ON sublayer). Dendritic field diameters (324 ± 30 μm; mean ± SD; n = 4) were similar in size to those of M1 ipRGCs, but the arbors were more orderly, with more regular branching angles and more uniform dendritic density within the field ( Figure 3 A ). The cell bodies of M2 ipRGCs were slightly larger on average than those of M1 cells (17.4 ± 1.7 μm; n = 5). All M1 and M2 ipRGCs tested were melanopsin immunopositive ( Figures 2 D–2G; n = 15 M1 and 11 M2). Though both subtypes invariably exhibited an intrinsic light response ( Figures 2 J and 2K), the response in M1 ipRGCs was an order of magnitude larger than that of the M2 ipRGCs with a shorter latency to peak ( Figures 2 J, S3 A, and S3B), confirming an earlier report ().

We encountered at least two additional subtypes of intrinsically photosensitive cells among the EGFP-positive ganglion cells that were melanopsin immunonegative ( Figures 2 H and 2I and Figure S2 ; data not shown). One type resembled an ON alpha ganglion cell ( Figure 2 C, n = 6), with a large soma (17.1–22.3 μm diameter; n = 3) and a large radiate dendritic arbor (302–444 μm diameter; n = 3) monostratifying in the ON sublayer. These cells exhibited weak intrinsic light responses ( Figure 2 L; peak photocurrent of 18.5 ± 11.4 pA; n = 4). We provisionally term these alpha-like cells “M4,” (reserving “M3” for the bistratified ipRGCs). The second type of EGFP-positive but melanopsin-immunonegative ipRGC also stratified in the ON sublayer of the IPL but could be distinguished from both M2 and alpha-like M4 ipRGCs by its relatively compact, highly branched dendritic arbor ( Figure S2 A; diameter: 149–217 μm; n = 3). Of the eight cells of this group that were tested, seven were intrinsically photosensitive, although as with the M2 and M4 ipRGCs, these responses were much weaker (peak photocurrents 12.9 ± 4 pA, Figure S2 B) than those of M1 ipRGCs. We provisionally term these smaller field bushy type neurons “M5 cells.”

The M2 and M4 ipRGCs are the least readily distinguished, because both have large radiate dendritic arbors stratifying in the ON sublayer. To clarify the morphological distinctions between them, we filled dozens of EGFP-positive RGCs by intracellular dye injection with sharp electrodes, an efficient method for high-quality filling. We reconstructed the somadendritic profiles of all well-filled cells with wide-field dendritic arbors limited to the inner IPL (thus excluding M1, M3 [bistratified], and M5 cells). Dye filled cells could be readily divided into M2 and M4 subtypes ( Figure 3 ). Drawings of representative examples show that M2 cells had dendritic arbors that were sparser and slightly smaller than those of M4 cells ( Figure 3 ). By comparison with M4 cells, M2 cells had fewer branchpoints ( Figure 3 C), less total dendritic length ( Figure 3 B), and smaller dendritic-field diameters ( Figure 3 C). Their axons also appeared consistently finer than those of M4 cells.

We encountered three additional EGFP-positive cells that were intrinsically photosensitive but lacked detectable melanopsin immunoreactivity. These were not easily grouped with any of the subtypes described above; all had relatively weak intrinsic light responses (<20 pA) and bushy, highly branched dendrites. Our failure to detect melanopsin in these unclassified cells or in the M4 and M5 ipRGCs is presumably because their expression of the pigment is very low and because both the Cre labeling system and electrophysiological recordings of photoresponses detect it with greater sensitivity than does immunofluorescence.

All of the ipRGC cell types also exhibited synaptically mediated excitatory influences from rods and/or cones, as reflected by brisk light responses that were abolished by blockade of ionotropic and metabotropic glutamate receptors ( Figures S3 C–S3F).

Figure 4 Comparison of Retinofugal Projections of Presumed Melanopsin-Expressing Ganglion Cells as Revealed by Two Lines of Reporter Mice in Representative Coronal Sections Show full caption (Left column) Axons of ipRGCs revealed by alkaline phosphatase histochemical labeling in Opn4Cre/+; Z/AP mice. (Right column) Axons of a subset of M1 melanopsin ganglion cells by X-gal staining in Opn4tau-LacZ/+ mice. (A and B) The suprachiasmatic nucleus (SCN) (A) and lateral geniculate nucleus (LGN) (B) showing the intergeniculate leaflet (IGL, dotted lines) flanked by the dorsal LGN (dLGN, upper solid white outline) and ventral LGN (vLGN, lower solid white outline). Labeling of the dLGN and vLGN is much more prominent in Opn4Cre/+; Z/AP sections (left). (C) Olivary pretectal nucleus (OPN). Whereas fiber labeling is largely restricted to the shell of the nucleus in Opn4tau-LacZ/+ mice (right), the core of the nucleus is also strongly labeled in the Opn4Cre/+; Z/AP model (left). (D) The posterior pretectal nucleus (PPN) contains minimal fiber labeling in Opn4tau-LacZ/+ brains, but exhibits strong, patchy labeling in the Opn4Cre/+; Z/AP mouse. (E) The superior colliculus (SC) contains only a few labeled fibers in the Opn4tau-LacZ/+ mouse, but much more extensive labeling in the Opn4Cre/+; Z/AP animal, especially in the stratum opticum. Dotted line marks approximate boundary between the superficial gray layer and stratum opticum. Scale bars, 200 μm. In order to trace the axons of the new types of melanopsin cells to their brain targets, we used the Opn4Cre/+; Z/AP mouse, which uses the same promoter as the Z/EG line ( Figures S1 C and S1D). Placental alkaline phosphatase (AP) is expressed in the plasma membrane of tagged cells, including their axons, where it can be visualized by histochemical staining. In the retina, the AP staining was similar to that observed with EGFP labeling in the Opn4Cre/+; Z/EG ( Figure 1 ), with similar cell numbers and morphological subtypes. We examined the distribution of AP-positive axons in the brain to obtain an overview of the central projections of all ipRGCs. Labeled axons were evident in the optic nerve, chiasm and tract as well as in a number of central visual nuclei ( Figures 4 A–4E ). Before attributing all such axonal labeling to retinofugal fibers, however, we had to consider nonretinal sources, since AP was expressed in a limited number of neurons distributed in the cerebral cortex, diencephalon, and brainstem ( Figures S4 F and S4G). We examined brain sections from mice in which both eyes had been removed 3 weeks earlier, sufficient time to ensure virtually complete degeneration of retinofugal axons ( Figure S4 ). Essentially no AP staining remained within the retinorecipient nuclei in these animals ( Figures S4 A–S4E), whereas cellular and axonal labeling persisted in most nonvisual areas ( Figures S4 F and S4G). Therefore, all fiber labeling within visual nuclei arises from AP-positive retinal ganglion cells.

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Yau K.W. Melanopsin and rod-cone photoreceptive systems account for all major accessory visual functions in mice. Figure 7 Mice in which ipRGCs Are the Sole Functional Photoreceptor (Gnat1−/−; Cnga3−/− Animals, MO: “Melanopsin Only” Animals) Can Discriminate Patterns Show full caption (A) Optokinetic tracking (OKT). Spatial frequency threshold for C57BL/6 mice (WT, n = 5; 0.392 c/d [SEM = 0.001]) was comparable to previously reported values (C57 mice [n = 7]; 0.397 c/d [SEM = 0.001]), but no tracking was observed at any spatial frequency in Gnat1−/−; Cnga3−/− mice or in mice lacking any functional photoreceptors (triple knockouts Gnat1−/−; Cnga3−/−; Opn4−/−; TKO). (B) Individual movement trajectories of an Gnat1−/−; Cnga3−/− (MO) animal performing the Visual Water Task (VWT). Green trajectories indicate successful attempts to locate the platform under the monitor displaying the grating (+); red trajectories are failures. (C) Spatial frequency thresholds (acuity) measured in the Visual Water Task. Acuity of C57BL/6 mice (WT, n = 5; 0.55 c/d [SEM = 0.006]) was similar to previously reported values (C57 mice [n = 7]; 0.54 c/d [SEM = 0.0005]). Acuity of Gnat1−/−; Cnga3−/− animals was lower but measurable at (0.16 c/d [SEM = 0.002]; n = 9). Triple-knockout animals (n = 7) could not perform the task, so no threshold could be obtained. (D) Mean number of trials required to reach criterion performance in the Visual Water Task on a discrimination between a sine wave grating (0.12 c/d) and uniform gray of the same mean luminance. Wild-type mice (C57BL/6; n = 5) averaged 71 (SEM = 2.4) trials to achieve criterion performance, while Gnat1−/−; Cnga3−/− mice (n = 9) reached the criterion in an average of 148 (SEM = 9.2) trails. The triple-knockout animals (n = 7) failed to reach criterion in 405 trials. (E) Raw performance as a function of spatial frequencies for four individual C57 wild-type and Gnat1−/−; Cnga3−/− animals. Error bars in (A), (C), and (D) are standard error of the mean. Figure 8 Pattern-Induced Activation of cfos in the Visual Cortex in WT and Gnat1−/−; Cnga3−/− Mice, but Not in Triple-Knockout Animals Show full caption (A) Fos-positive cells were observed in the V1 region of the visual cortex in WT and Gnat1−/−; Cnga3−/− (MO) mice that were exposed to a pattern for 10 min under a 450 lux white light (Pattern), but not in animals exposed to the same light intensity without a pattern (Control). Fos-positive cells were not observed in triple-knockout (TKO) mice exposed to either condition. Panels to the right are magnification of each cortical region showing either lack or presence of nuclear cfos staining. (B) Quantification of the data by counting the number of cfos-positive cells in V1. Note that only WT and MO animals show significant increases compared to control levels. Statistical analysis was carried out using unpaired student's t test. Error bars are standard error of the mean. The extensive projections of reporter-labeled axons to the dLGN and SC are surprising because these nuclei mediate spatial and discriminative visual functions very different from the non-image-forming mechanisms to which ipRGCs are traditionally linked. This prompted us to examine whether melanopsin-based phototransduction might support pattern vision in the absence of functioning rods and cones. In the Visual Water Task (), we assessed the visual performance of a mouse strain in which rod and cone phototransductions are silenced (), leaving ipRGCs as the only functional photoreceptors (Gnat1−/−; Cnga3−/− double knockout animals;). The Gnat1−/−; Cnga3−/− double knockout animals and WT animals have similar melanopsin expression in the retina as revealed by immunohistochemistry ( Figure S5 A) and real-time quantitative PCR (qPCR; Figure S5 B). Gnat1−/− is a rod transducin knockout line that eliminates rod phototransduction (), whereas the Cnga3−/− line eliminates the cone cyclic nucleotide gated channel causing the absence of cone phototransduction (). Gnat1−/−; Cnga3−/− mice in the absence of the melanopsin protein (triple-knockout animals; Gnat1−/−; Cnga3−/−; Opn4−/−) lack circadian photoentrainment, sustained pupillary light reflex, or direct light effects (masking responses) on wheel running activity (). Gnat1−/−; Cnga3−/− mice, which contain only ipRGCs as functional photoreceptors, were indeed able to discriminate high-contrast, sinusoidally modulated gratings from uniform gray stimuli of the same mean luminance, although they needed roughly twice as many trials as control mice to reach criterion performance (70% correct, measured at 0.12 cycles/degree [c/d]; Figure 7 D ). The acuity in the Gnat1−/−; Cnga3−/− mice was measurable at 0.16 ± 0.002 c/d (mean ± standard error) ( Figures 7 C and 7E), although it was much lower than that of wild-type animals (C57BL/6; 0.55 ± 0.006 c/d). In addition, using cfos immunostaining in the visual cortex, we show that Gnat1−/−; Cnga3−/− mice have higher cfos expression in the cortex when they are exposed to a pattern ( Figures 8 A and 8B ).