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. 2010 Jul 15;67(1):49-60.
doi: 10.1016/j.neuron.2010.05.023.

Melanopsin-expressing retinal ganglion-cell photoreceptors: cellular diversity and role in pattern vision

Jennifer L Ecker  1 , Olivia N Dumitrescu, Kwoon Y Wong, Nazia M Alam, Shih-Kuo Chen, Tara LeGates, Jordan M Renna, Glen T Prusky, David M Berson, Samer Hattar
Affiliations

Melanopsin-expressing retinal ganglion-cell photoreceptors: cellular diversity and role in pattern vision

Jennifer L Ecker et al. Neuron. .

Abstract

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.

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Figures

Fig. 1
Fig. 1. Cre-mediated recombination with Z/AP and Z/EG reporters labels melanopsin-expressing ganglion cells
(A) Alkaline phosphatase staining of a vertical retinal section from an Opn4Cre/+; Z/AP mouse reveals ganglion cells and their dendrites in both the ON and OFF sublaminae of the IPL. (B) Opn4Cre/+; Z/EG retinal whole mount showing intrinsic GFP fluorescence signal in several retinal ganglion cells. (C–E) Double immunofluorescent labeling of ganglion cells with antibodies against alkaline phosphatase (C) and melanopsin (D). (F–H) Double immunolabeling of ganglion cells by antibodies against green fluorescent protein (F) and melanopsin (G). A few rod and cone cells labeled by the Cre reporter are visible in the ONL in C, E, F, and H, but these cells lack melanopsin immunoreactivity. ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer. Scale bars, 20 μm.
Fig. 2
Fig. 2. Diversity of morphology and intrinsic light responses of ganglion cells GFP in the Opn4Cre/+; Z/EG mouse
(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, K, L), obtained during pharmacological blockade of retinal synapses. Light pulse was 20 seconds. 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 (more than 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.
Fig. 3
Fig. 3. Differences in soma size, dendritic field diameter, total branchpoints and dendritic length of M2 and M4 ipRGCs
(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 μm, 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.
Fig. 4
Fig. 4. Comparison of retinofugal projections of presumed melanopsin-expressing ganglion cells as revealed by two lines of reporter mice in representative coronal sections
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) The suprachiasmatic nucleus (SCN), (B) Lateral geniculate nucleus (LGN), 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.
Fig. 5
Fig. 5. Charting of the synaptic input from ipRGCs to the LGN complex
Charting of alkaline-phosphatase positive retinal fibers in the lateral geniculate nucleus, as seen at five coronal levels, shows substantial innervation of the dLGN from ipRGCs. The section in A is most rostral, E most caudal. Abbreviations: dLGN, dorsal lateral geniculate; eml, external medullary lamina; fi, fimbria; H, hippocampus; ic, internal capsule; IGL, intergeniculate leaflet; LP, lateral posterior nucleus; MG, medial geniculate nucleus; ot, optic tract; Po, posterior nuclei; Rt, thalamic reticular nucleus; st, stria terminalis; vLGN, ventral lateral geniculate (including parvocellular [pc] and magnocellular [mc] subdivisions); VP, ventral posterior nucleus; ZI, zona incerta.
Fig. 6
Fig. 6. Direct comparison in Opn4Cre/tau-LacZ; Thy-1-Brainbow-1.0 mice labeling of retinal neurons and retinofugal axons by the two reporters of melanopsin expression
Anti-β-galactosidase immunoreactivity (red fluorescence) marks M1 ipRGCs and axons; GFP-like immunofluorescence (green) labels fluorescent proteins expressed in all subtypes of ipRGCs. (A) Retinal whole mount. Antibody labeling reveals that the Cre-mediated reporter (green) is present in most β-galactosidase immunoreactive cells (red) but is much more widely expressed. (B–D) Fiber labeling in selected coronal brain sections. In the olivary pretectal nucleus (B), M1 axons, expressing β-galactosidase (red) are largely restricted to the shell of the nucleus, whereas the fluorescent proteins (green) heavily label axons in the core as well as more weakly double-labeling the β-gal-positive axons in the shell. In the LGN (C), the intergeniculate leaflet contains a mixture of double-labeled axons (yellow; M1 afferents) and axons labeled only with fluorescent proteins (green; presumably from M2, M4 and/or M5 ipRGCs). The latter are also visible in parts of the vLGN and in the dLGN. (D) The suprachiasmatic nucleus (SCN) contains mainly double labeled fibers, indicating that most retinal afferents originate in M1 cells, though at higher magnification some fibers singly labeled for fluorescent proteins (green) can be seen (not shown).
Fig. 7
Fig. 7. Mice in which ipRGCs are the sole functional photoreceptor (Gnat1−/−; Cnga3−/− animals, MO: “melanopsin only” animals) can discriminate patterns
(A) Optokinetic tracking (OKT). Spatial frequency threshold for in C57/Bl6 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 C57/Bl6 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. Wildtype mice (C57/Bl6; 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 wildtype and Gnat1−/−; Cnga3−/− animals. Error bars in A, C and D are standard error of the mean.
Fig. 8
Fig. 8. Pattern induced activation of cfos in the visual cortex in WT and Gnat1−/−; Cnga3−/− mice, but not in triple knockout animals
(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 ten minutes 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.
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