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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, β-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.
Retinal rods and cones, with their light-sensitive, opsin-based pigments, are the primary photoreceptors for vertebrate vision. Visual signals are transmitted to the brain through RGCs, the output neurons whose axons form the optic nerve. This system, through its projections to the lateral geniculate nucleus and the midbrain, is responsible for interpreting and tracking visual objects and patterns. A separate visual circuit, running in parallel with this image-forming visual system, encodes the general level of environmental illumination and drives certain photic responses, including synchronization of the biological clock with the light-dark cycle (1), control of pupil size (2), acute suppression of locomotor behavior (3), melatonin release (4), and others (5–7). Surprisingly, this non–image-forming system does not appear to originate from rods and cones. For example, rods and cones are not required for photoentrainment of circadian rhythms (8), a function mediated by the retinohypothalamic tract (9, 10) and its target, the SCN, the brain’s circadian pacemaker (1). Nor are rods and cones necessary for the pupillary light reflex, mediated by the retinal projection to the pretectal region of the brainstem (2). At present, the best candidate for a photopigment is an opsin-like protein called melanopsin, which is expressed by a subset of mouse and human RGCs (11). The accompanying report (12) shows that RGCs projecting to the SCN are directly sensitive to light. Thus, melanopsin may be the photopigment responsible for this intrinsic photosensitivity, and it may also trigger other non–image-forming visual functions.
We cloned the full-length cDNA for rat melanopsin (13), on the basis of homology to mouse melanopsin (11). The predicted amino acid sequence lacks the last 43 residues of mouse melanopsin but otherwise shows 92% identity (14). Polyclonal antibodies were generated against its NH2- and COOH-terminal sequences (15). Fluorescent immunocytochemistry (16) of flat-mounted rat retina with the antibody to melanopsin labeled a small percentage of RGCs, including cell bodies, dendrites, and axons (Fig. 1A). Somatic immunoreactivity appeared mainly at the cell surface (Fig. 1B1), suggestive of melanopsin being targeted to the plasma membrane. Every labeled retinal cell was a ganglion cell, on the basis of the presence of an axon coursing into the optic fiber layer and toward the optic disc. Axonal labeling disappeared beyond the optic disc and was not visible in the innervated targets (see below). More than 95% of labeled cell bodies were in the ganglion cell layer, the remainder being displaced to the inner nuclear layer. Dendrites from adjacent cells overlapped extensively, forming a reticular network (Fig. 1B2). The stained dendrites and proximal axons had a beaded appearance, showing punctate, dense labeling. The complete dendritic fields of labeled cells, visualized from stacked confocal images (e.g., Fig. 1B2), had varied sizes and shapes (Fig. 1C). Labeled displaced RGCs (Fig. 1C, right three cells) had similar soma sizes but less extensive dendritic arborizations than nondisplaced cells (Fig. 1C, left three cells). The mean somatic diameter of labeled non-displaced RGCs was 16 μm (Fig. 1D), but the limited sample of dendritic-field measurements precluded any statistics. Morphologically, these neurons fit within the type III group of rat RGCs (17), especially those shown to be intrinsically photosensitive (12). The density of melanopsin-positive cells was slightly higher in the superior and temporal quadrants of the rat retina (Fig. 1E). A complete count of these cells in the two retinas of Fig. 1E gave 2320 and 2590, respectively, although some faintly labeled cells could have been missed. Assuming 100,000 RGCs in the rat retina (18), these numbers represent about 2.5% of the total. The corresponding numbers for mouse melanopsin-immunoreactive RGCs were 680 and 780 from two eyes of one animal, or about 1% of the total [assuming 60,000 ganglion cells in a mouse retina (19)].
To locate more precisely the cell bodies and dendritic arborizations of the melanopsin-positive RGCs, we examined the rat retina in cross section (Fig. 2). Whether in the ganglion cell layer (Fig. 2A) or displaced to the inner nuclear layer (Fig. 2B), the melanopsin-expressing RGCs extended dendrites into the inner plexiform layer, where they arborized most extensively at the border with the inner nuclear layer. Some arborizations invaded and often terminated in the inner nuclear layer (arrows in Fig. 2A3 and 2A4). The displaced RGCs had dendritic arborizations that were more planar and sparse (Figs. 2B1 and and1C,1C, right). Melanopsin-immunoreactive puncta were present throughout the dendrites, and they showed no correlation with the retinal laminae and no colocalization or juxtaposition with the presynaptic protein synaptophysin (Fig. 2C), suggesting that the puncta did not correspond to synaptic sites.
To examine the axonal projections of the melanopsin-positive RGCs, we targeted tau-lacZ into the melanopsin gene locus in mouse (20) (Fig. 3A). Tau-lacZ codes for a protein composed of the β-galactosidase enzyme fused to a signal sequence from tau (an actin-associated protein), which allows the fusion protein to be preferentially transported down the axon to the presynaptic terminal (21). In retinas from heterozygous animals, which have one copy each of the melanopsin and tau-lacZ genes, β-galactosidase and melanopsin immunoreactivities colocalized in the ganglion cells (Fig. 3B). The morphology of the melanopsin-immunopositive cells in mouse was similar to that in rat. In X-gal labeling (22) of β-galactosidase activity (Fig. 3, C and D; blue color), the axons, cell bodies, and proximal dendrites were well labeled. In one heterozygous mouse retina, the total number of labeled cells was about 600, similar to the number for melanopsin-immunopositive cells in the wild-type mouse retina mentioned above. A ventral view of the brain from a heterozygous mouse showed blue-labeled axons coursing in the optic nerves and targeting the SCN bilaterally (Fig. 3, E and F). A coronal section of the brain at the SCN showed dense axonal terminations in the paired nuclei (Fig. 3G). Labeled axons continued caudally in the optic tract to the lateral geniculate complex, terminating throughout the intergeniculate leaflet (IGL) (Fig. 3H). Stained fibers also sparsely innervated the ventral lateral geniculate (VLG) but did not invade the dorsal lateral geniculate (DLG). Labeled axons also formed a terminal field in the pretectum, in the vicinity of the olivary pretectal nucleus (OPN) (Fig. 3I). Neither melanopsin immunoreactivity nor β-galactosidase staining labeled neuronal somata in these or other brain regions, indicating that the axons containing melanopsin all originated from the retina.
To address whether melanopsin could be the photopigment responsible for the intrinsic light sensitivity of rat RGCs that project to the SCN (12), we injected these photosensitive RGCs intracellularly with Lucifer Yellow (LY) (23) and then stained them for melanopsin immunoreactivity. Intrinsically photosensitive ganglion cells were invariably melanopsin-positive (n = 18; Fig. 4), whereas conventional ganglion cells lacking intrinsic light responses were melanopsin-negative (n = 4). Thus, melanopsin is most likely the photopigment that confers the intrinsic light sensitivity to this subset of RGCs. In some photosensitive ganglion cells, individual dendrites were smoothly filled with LY but still exhibited punctate melanopsin immunoreactivity, suggesting that there may be local clusters of the opsin.
Taken together, our findings suggest that melanopsin may indeed be a photopigment, consistent with an opsin-based action spectrum demonstrated for the SCN-projecting RGCs (12). The presence of melanopsin throughout the dendritic arbors of these cells may permit spatial integration of retinal irradiance and is consistent with the extended receptive fields of these cells (12). It is unclear whether these photosensitive dendrites simultaneously serve the more conventional function of receiving synaptic inputs from rod- and cone-driven networks. There is evidence for rod and cone influences on neurons in both the SCN (24) and the OPN (2), but whether these reflect convergence at the photosensitive RGCs or convergence within the brain remains unclear. In both distribution and morphology, the melanopsin-positive RGCs described here broadly match those identified in rat and mouse retina that project to the SCN [(25–28); see also (29, 30)], although most of these other studies labeled presumably only a fraction of the cells.
Although the innervation of the SCN is the densest, the IGL, VLG, and OPN were also innervated by β-galactosidase–positive RGC axons in heterozygous tau-lacZ mice. Some of the fibers innervating the IGL and OPN could be collaterals of axons in the retinohypothalamic tract (31). Neurons in both IGL and OPN encode ambient light levels (32–34), a property almost certainly conferred by the photosensitive RGCs, which faithfully report retinal irradiance (12). Like the SCN, the IGL and the VLG are implicated in circadian photoentrainment (32), whereas the OPN is a key node in the circuit mediating the pupillary light reflex (33, 34), another non–image-forming visual function. Indeed, rodless and coneless mice retain a pupillary light response (2), with the spectral tuning and high thresholds expected for a response driven by the intrinsically photosensitive RGCs (12). Thus, it appears that melanopsin-containing RGCs are generally involved in non–image-forming visual functions.
We thank R. R. Reed, and S. S. Wang and H. Zhao in his laboratory, for the tau-lacZ construct and invaluable advice. We also thank I. Provencio for providing the melanopsin BAC clone, J. Nathans and H. Sun for discussions, K. Takamiya, Y. Zhang, S. A. Ralls, M. Dehoff, B. E. Lonze, and especially M. Cowan and the Johns Hopkins Transgenic Facility for technical help/advice in the generation of the tau-lauZ knock-in mice as well as providing the ES cells. We are grateful to R. Richardson and S. Carlson for technical assistance with the experiments on colocalization of melanopsin and intrinsic photic sensitivity. Finally, we thank R. Masland and members of the Yau laboratory, especially J. Bradley, W.-H. Xiong, and H. Zhong, for help/critique on the experiments. This work was supported by grants from the U.S. National Eye Institute to D.M.B. and K.-W.Y.