Melanopsin-containing retinal ganglion cells: architecture, projections, and intrinsic photosensitivity

Abstract

The primary circadian pacemaker, in the suprachiasmatic nucleus (SCN) of the mammalian brain, is photoentrained by light signals from the eyes through the retinohypothalamic tract. Retinal rod and cone cells are not required for photoentrainment. Recent evidence suggests that the entraining photoreceptors are retinal ganglion cells (RGCs) that project to the SCN. The visual pigment for this photoreceptor may be melanopsin, an opsin-like protein whose coding messenger RNA is found in a subset of mammalian RGCs. By cloning rat melanopsin and generating specific antibodies, we show that melanopsin is present in cell bodies, dendrites, and proximal axonal segments of a subset of rat RGCs. In mice heterozygous for tau-lacZ targeted to the melanopsin gene locus, beta-galactosidase-positive RGC axons projected to the SCN and other brain nuclei involved in circadian photoentrainment or the pupillary light reflex. Rat RGCs that exhibited intrinsic photosensitivity invariably expressed melanopsin. Hence, melanopsin is most likely the visual pigment of phototransducing RGCs that set the circadian clock and initiate other non-image-forming visual functions.

Fig. 1

Immunocytochemistry of melanopsin-containing RGCs in the flat-mounted rat retina. (A) Confocal images at the level of the ganglion cell layer showing labeling with the melanopsin NH₂-terminal specific antibody. The fluorescent immunolabeling is in green, and the nuclei are stained by red fluorescent propidium iodide. Arrows 1 and 2 indicate axons associated with the indicated RGC cell bodies heading toward the optic disc. Note the beaded appearance of the dendrites. Because the image is at a particular focal plane, some dendrites and axons are not visible. (B) Nonstacked (1) and stacked (2) confocal images of the same retinal field from another preparation, but without nuclear counterstaining. The stacked picture combined all focal planes containing labeled processes. Note the peripheral localization of the melanopsin-labeling in the cell bodies in (B1). Because the stacking increased background, the sensitivity of the camera was reduced, making some faint processes not clearly visible. (C) Camera-lucida drawings of several melanopsin-positive RGCs, obtained from stacked images. The beaded appearance of the dendrites is not shown. The left and right panels show nondisplaced and displaced RGCs, respectively. The displaced cells have smaller and apparently more sparse dendritic fields. Arrows indicate axons. (D) Soma-size distribution of (a sample of) nondisplaced melanopsin-positive RGCs, which account for >95% of all labeled RGCs. (E) Overall distribution of melanopsin-positive RGCs on the flat-mounted right and left retinas of the same rat. Dozens of local dark-field images were taken separately at low magnification, and the montage was assembled with Adobe Photoshop. Each cell body is represented by a dot of about the appropriate size. Note the higher cell density in the superior and temporal quadrants. Only nondisplaced RGCs (>95% of total) are included. S, superior; I, inferior; N, nasal; T, temporal.

Fig. 2

Melanopsin-positive RGCs in cross sections of rat retina. All are single (non-stacked) confocal images from 30-μm retinal sections, with a depth of field of only a few micrometers. Melanopsin fluorescent immunolabeling is in green and nuclear counterstaining is in blue. In all images, the brightness of the nuclear staining has been reduced to show the processes in or around the INL; as a result, the nuclear staining in the GCL is quite faint. ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer. (A1 to A4) Nondisplaced RGCs. Arrows in (A3) and (A4) indicate processes in the INL; the contrast and brightness of these two images have been enhanced to show these processes. (B1 and B2) Displaced RGCs. The contrast and brightness in (B2) have also been enhanced to show the axon (arrow). (C1 and C2) Double immunostaining of melanopsin and the presynaptic protein synaptophysin (red Cy3 fluorescence). The punctate melanopsin labeling of the processes (see arrows) did not colocalize or juxtapose with synaptophysin labeling. The cell in (C2) is a displaced RGC. (D) Preadsorption of antibody with the peptide-BSA conjugate abolished all immunostaining.

Fig. 3

Targeting of tau-lacZ into the mouse melanopsin gene locus. (A) (Left) Targeting strategy. In the wild-type (WT) schema, the boxes represent partial fragments of exons 1 and 9 of the melanopsin gene, with ATG indicating the start site of the melanopsin protein. The ATG in the targeting vector (TV) corresponds to the start site of the tau-lacZ fusion protein. The pgk-neo is flanked by loxP sites (open, inverted triangles) for specific cre-recombinase removal. Upon homologous recombination, the tau-lacZ fusion protein in the targeted locus (TL) retains its own start site, whereas the melanopsin start site together with the rest of the gene was eliminated. The dark bar underneath wild-type schema represents the outer probe used for screening ES cells and subsequently generated animals, by Southern (DNA) blotting. X, Xho I; B, Bgl II; BM, Bam HI; P, Pac I. The squiggles in the wild-type schema indicate a size difference of 2.6 kb between the melanopsin gene and the tau-lacZ construct. The four dotted lines indicate the homologous recombination arms used for targeting melanopsin. Primers c and d were used in PCR for screening site-specific integration of the tau-lacZ construct in electroporated ES cells. Primers a and b were used for genotyping of heterozygous animals. (Right) Genomic DNA from wild-type and +/− mice digested with Bam HI and Pac I and hybridized with the outer probe, producing a 4.4-kb fragment in the wild-type locus and a 3.7-kb fragment in the targeted locus, as expected. (B) Colocalization of β-galactosidase and melanopsin immunoreactivities in a flat-mounted retina from a +/− mouse. (Left) β-Galactosidase labeling. (Middle) Melanopsin labeling. (Right) Merged image. (C) Flat-mount view of a +/− mouse retina stained with X-gal. Labeled axons (blue), which converge to the optic disc, are visible. The brown coloration near the optic disc is from remnants of the retinal pigment epithelium overlying the retina. (D) Magnified view of an X-gal– labeled RGC. (E) Ventral view of a +/− mouse brain stained with X-gal, showing bilateral blue staining of the SCN (arrow). (F) Magnified view of the blue-labeled axons in an optic nerve. (G) Coronal section of the +/− brain showing blue optic nerve fibers converging to, and innervating, the SCN bilaterally. (H) Coronal section of the +/− brain showing uniform blue-labeled innervation of the left IGL and scattered innervation of the VLG. The DLG shows no labeling. (I) Coronal section of the +/− brain showing innervation of the pretectal region. The blue staining corresponds approximately to the OPN, demarcated in red on the right.

Fig. 4

Presence of melanopsin in intrinsically light-responsive RGCs that innervate the SCN. Sample data from three cells (A to C), all identified by retrograde transport of fluorescent rhodamine beads from the SCN before whole-cell recording from the flat-mounted retina. In each panel, the voltage trace at left shows the cell’s response to a long light stimulus indicated below by the step. Broad-band tungsten light source; irradiance at retina of 6 × 10¹⁵ photons s⁻¹ cm⁻² at 500 nm. Bathing Ames medium contained agents to block Ca²⁺-mediated synaptic transmission: either (A and B) 2-mM CoCl₂ alone or (C) in combination with pharmacological blockers of the glutamatergic signaling between conventional photoreceptors and RGCs [100-μM L(+)-2-amino-4-phosphonobutyric acid (APB), 20-μM 6,7-dinit-roquinoxaline-2,3-dione (DNQX), and 50-μM DL-2-amino-5-phosphonovaleric acid (APV)] (12). Large-amplitude, fast depolarizing events are action potentials. Fluorescence images of the recorded cells are shown at right. (Left) Intracellular staining (green) with LY introduced from the whole-cell recording pipette. (Middle) Antibody to melanopsin immunofluorescence [Cy-3 fluorophore, red, in (A) and (B); Alexa Fluor 647, white, in (C)]. (Right) Superimposed fluorescence images. Far left in (C), Fluorescence of rhodamine latex beads (red), the retrograde tracer. All micrographs are stacked, pseudocolored confocal images, except for the LY image in (C), which is a montage of optimized epifluorescence (nonconfocal, hence somewhat blurred) micrographs at different focal planes. Arrow in (A) marks the melanopsin-positive dendrite of a second ganglion cell neighboring the one recorded. In (B) and (C), other strongly melanopsin-immunopositive cells are visible near the recorded cell; these cells were not recorded from and hence showed no LY labeling.