central projections of melanopsin-expressing retinal ganglion...

Central Projections of Melanopsin-Expressing Retinal Ganglion Cells in the

Mouse

SAMER HATTAR,1 MONICA KUMAR,3 ALEXANDER PARK,1 PATRICK TONG,2

JONATHAN TUNG,3 KING-WAI YAU,1,2AND DAVID M. BERSON3*

1Department of Neuroscience, Johns Hopkins University School of Medicine,Baltimore, Maryland 21205-2105

2Department of Ophthalmology, Johns Hopkins University School of Medicine,Baltimore, Maryland 21205-2105

3Department of Neuroscience, Brown University, Providence, Rhode Island 02912

ABSTRACTA rare type of ganglion cell in mammalian retina is directly photosensitive. These novel

retinal photoreceptors express the photopigment melanopsin. They send axons directly to thesuprachiasmatic nucleus (SCN), intergeniculate leaflet (IGL), and olivary pretectal nucleus(OPN), thereby contributing to photic synchronization of circadian rhythms and the pupillarylight reflex. Here, we sought to characterize more fully the projections of these cells to the brain.By targeting tau-lacZ to the melanopsin gene locus in mice, ganglion cells that would normallyexpress melanopsin were induced to express, instead, the marker enzyme �-galactosidase. Theiraxons were visualized by X-gal histochemistry or anti-�-galactosidase immunofluorescence. Es-tablished targets were confirmed, including the SCN, IGL, OPN, ventral division of the lateralgeniculate nucleus (LGv), and preoptic area, but the overall projections were more widespreadthan previously recognized. Targets included the lateral nucleus, peri-supraoptic nucleus, andsubparaventricular zone of the hypothalamus, medial amygdala, margin of the lateral habenula,posterior limitans nucleus, superior colliculus, and periaqueductal gray. There were also weakprojections to the margins of the dorsal lateral geniculate nucleus. Co-staining with the choleratoxin B subunit to label all retinal afferents showed that melanopsin ganglion cells provide mostof the retinal input to the SCN, IGL, and lateral habenula and much of that to the OPN, but thatother ganglion cells do contribute at least some retinal input to these targets. Staining patternsafter monocular enucleation revealed that the projections of these cells are overwhelminglycrossed except for the projection to the SCN, which is bilaterally symmetrical. J. Comp. Neurol.497:326–349, 2006. © 2006 Wiley-Liss, Inc.

Indexing terms: melanopsin; circadian; retinofugal; pupil; suprachiasmatic nucleus; retinal

ganglion cell; retinohypothalamic tract; pretectum; superior colliculus

Melanopsin is an opsin protein originally identified infrog dermal melanophores (Provencio et al., 1998, 2000)and now firmly established as a functional sensory pho-topigment (Newman et al., 2003; Melyan et al., 2005;Panda et al., 2005; Qiu et al., 2005; Fu et al., 2005). Inmammals, it is expressed in the eye, almost exclusively ina small percentage of retinal ganglion cells (RGCs; Prov-encio et al., 2000, 2002; Hannibal et al., 2002; Hattar etal., 2002). These melanopsin-expressing RGCs (mRGCs)are directly sensitive to light and thus constitute a thirdclass of mammalian retinal photoreceptors, in addition tothe rods and cones (Berson et al., 2002; Berson, 2003;Warren et al., 2003).

Grant sponsor: the National Eye Institute; Grant number: 5R01EY014596 (to K.-W.Y.); Grant number: 5 R01 EY012793 (to D.B.).

Dr. Hattar’s current address: Department of Biology, Johns HopkinsUniversity, Baltimore, MD 21218-2685.

*Correspondence to: David M. Berson, Department of Neuroscience, Box1953, Brown University, Providence, RI 02912. E-mail: [email protected]

Received 1 June 2005; Revised 6 December 2005; Accepted 11 January2005

DOI 10.1002/cne.20970Published online in Wiley InterScience (www.interscience.wiley.com).

THE JOURNAL OF COMPARATIVE NEUROLOGY 497:326–349 (2006)

© 2006 WILEY-LISS, INC.

Among their many striking structural and functionaldifferences from the classical photoreceptors is that, asganglion cells, they project directly to the brain. One tar-get of these projections is the suprachiasmatic nucleus ofthe hypothalamus (Gooley et al., 2001; Berson et al., 2002;Hannibal et al., 2002; Hattar et al., 2002), a link thathelps to synchronize this master pacemaker of circadianrhythms to the solar cycle (Berson, 2003; Hirota andFukada, 2004). A second target, the intergeniculate leaflet(Hattar et al., 2002, Morin et al., 2003), also participatesin circadian photoentrainment (Harrington, 1997). A thirdoutput reaches the olivary pretectal nucleus (Hattar et al.,2002; Morin et al., 2003), a key element in the circuitmediating the pupillary light reflex (Trejo and Cicerone,1984; Clarke and Ikeda, 1985; Young and Lund, 1994).

Other targets of the mRGCs have begun to emerge,implying their participation in diverse functions. Thesetargets include the preoptic region, hypothalamic sub-paraventricular zone, ventral lateral geniculate nucleus,and superior colliculus (Hattar et al., 2002; Gooley et al.,2003; Morin et al., 2003; Hannibal and Fahrenkrug, 2004).Past studies of these projections have relied mainly onretrograde transport of tracers injected into candidatetargets combined with immunohistochemistry or in situhybridization to identify retinal cells expressing melanop-sin. This has limited the number of possible targetsprobed, and little information has been available aboutthe form, trajectory, or distribution of the axons. In thisstudy, we set out to describe the full extent of centralprojections of mRGCs, exploiting a gene-targeted mousestrain engineered to express the �-galactosidase markerenzyme selectively in mRGCs (Hattar et al., 2002).

A second goal of the present study was to characterizethe relative strength of ipsilateral and contralateral pro-jections from the mRGCs. A striking feature of the retino-hypothalamic projection in the mouse is that the crossedand uncrossed components are about equally strong(Youngstrom and Nunez, 1986; Cassone et al., 1988;Miura et al., 1997; Abrahamson and Moore, 2001),whereas more than 95% of retinal fibers are crossed in therest of the visual system (Drager and Olsen, 1980; Miuraet al., 1997). Because most retinal afferents to the SCNare derived from mRGCs, we wondered whether these

ganglion cells may also have unusually robust ipsilateralprojections to their other targets. To pursue this issue, weexamined in the same knock-in mice the effects of monoc-ular enucleation on the relative density of �-galactosidase-positive fibers on the two sides of the brain.

We also sought further evidence on the degree to whichinputs from mRGCs converged with those of other RGCs(non-mRGCs) in specific target nuclei. All photic re-sponses to which mRGCs are known to contribute persistin some form when the melanopsin gene is knocked out(Panda et al., 2002; Ruby et al., 2002; Lucas et al., 2003;Mrosovsky and Hattar, 2003). Because direct photosensi-tivity is lost in mRGCs when melanopsin is deleted (Lucaset al., 2003), the residual responses presumably reflect theinfluences of rods and/or cones (Hattar et al., 2003; Pandaet al., 2003). Indeed, there is electrophysiological evidencefor rod and cone input to the SCN (Aggelopoulos andMeissl, 2000). The routes by which these rod/cone signalsreach the critical brain nuclei are unknown. The mRGCsthemselves are a likely conduit, because these cells areknown to be excited by rod- or cone-driven synaptic net-works (Dunn and Berson, 2002; Belenky et al., 2003;Dacey et al., 2005) and because they retain their normalaxonal outputs in the face of melanopsin deletion (Lucaset al., 2003). Alternatively, conventional (non-melanopsin)ganglion cells may relay rod and cone signals to thesebrain regions (Gooley et al., 2001; Morin et al., 2003;Sollars et al., 2003; Hannibal and Fahrenkrug, 2004).We assessed the relative contribution of mRGCs andnon-mRGCs to the retinal input to individual nuclei bydouble-immunofluorescence experiments in which antero-grade transport labeled all retinal afferents while�-galactosidase marked the axons of melanopsin RGCs.

MATERIALS AND METHODS

Animals

All procedures were conducted in accordance with NIHguidelines and approved by the Institutional Animal Careand Use Committees of Brown University and Johns Hop-kins University. The studies used 24 male and femaleadult mice of the B6/129 strain genetically modified to

Abbreviations

AH anterior hypothalamic nucleusAHP anterior hypothalamic area, posteriorAP anterior pretectal nucleusAV anteroventral thalamic nucleusBST bed nucleus of the stria terminalisCL central lateral thalamic nucleusCP cerebral pedunclef fornixfr fasciculus retroflexushc habenular commissureic internal capsuleIGL intergeniculate leafletLD lateral dorsal thalamic nucleusLGd lateral geniculate nucleus, dorsal divisionLGv lateral geniculate nucleus, ventral divisionLH lateral hypothalamusLHb lateral habenulaLP lateral posterior thalamic nucleusLPO lateral preoptic areaMA medial amygdaloid nucleusMHb medial habenula

MPO medial preoptic areaOPN olivary pretectal nucleusot optic tractox optic chiasmPAG periaqueductal graypc posterior commissurePO preoptic areapSON peri-supraoptic nucleusSC superior colliculusSCN suprachiasmatic nucleusSGS stratum griseum superficiale of the superior colliculusSI substantia innominatasm stria medullarisSO stratum opticum of the superior colliculusst stria terminalisSPZ subparaventricular zoneSZ stratum zonale of the superior colliculusst stria terminalisVLPO ventrolateral preoptic areaVMH ventromedial hypothalamusvSPZ ventral subparaventricular zone

The Journal of Comparative Neurology. DOI 10.1002/cne

327PROJECTIONS OF MELANOPSIN GANGLION CELLS

replace the melanopsin gene opn4 with tau-lacZ (Hattaret al., 2002). The tau-lacZ gene codes for a protein consist-ing of the �-galactosidase enzyme fused to a signal se-quence from tau to promote axonal transport of themarker enzyme (Mombaerts et al., 1996). Except wherespecifically stated, all data are based on 22 mice that arehomozygous for the tau-lacZ knock-in (tau-lacZ�/�) andare therefore completely devoid of melanopsin (opn4�/�).Animals were not backcrossed to an inbred strain. Ani-mals were housed under a 14:10-hour light-dark cycle andwere killed during the light phase.

X-gal labeling

To visualize the �-galactosidase marker enzyme, micewere anesthetized by intraperitoneal injection of tribro-moethanol (Winthrop Laboratories, New York, NY) (0.2ml/g) and arterially perfused for 10 minutes with 25 ml ofcold 4% paraformaldehyde in phosphate-buffered saline(PBS; pH 7.4). Brains were removed, cryoprotected in 30%sucrose in PBS overnight, and cut coronally on a slidingmicrotome at 50 �m. X-gal staining followed Mombaertset al. (1996). Sections were washed twice for 10 minuteseach in buffer B (100 mM phosphate buffer at pH 7.4, 2mM MgCl2, 0.01% Na-desoxycholate, and 0.02% [octylphe-noxy] polyethoxyethanol [IGEPAL]). They were then incu-bated in staining solution [buffer B plus potassium ferri-cyanide (5 mM), potassium ferrocyanide (5 mM), andX-gal1 (5-bromo-4-chloro-3-indolyl-�-D-galactopyranoside;1 mg/ml] at room temperature in darkness. Incubationslasted for 6–8 hours for homozygotes and 12–18 hours forheterozygotes. For retinal labeling, eyes were removedfrom animals, fixed as described above, and hemisectednear the ora serrata. The lens and vitreous were removed,the remaining eyecup was stained with X-gal as above for24–30 hours at 37°C in darkness, and the retina wasisolated. Retinas and brain sections were mounted onglass slides and coverslipped in glycerol. For X-gal stain-ing of whole brains, animals were perfused first with fix-ative, as above, and then with 60 ml of the X-gal stainingsolution. The brain was then removed and immersed inthe same staining solution for 3–5 days at 36°C. The brainwas dehydrated and cleared by immersion in methanol (2days) and then in a 2:1 solution of benzyl benzoate andbenzyl alcohol (2 hours) and then immersed in light min-eral oil for photography.

Enucleation

A total of seven mice were subjected to monocular (n �6) or binocular (n � 1) enucleation. They were anesthe-tized by intramuscular ketamine (50 mg/kg ip), and theeyes were topically anesthetized with a drop of 0.5% tet-racaine (Wilson Ophthalmics, Mustang, OK). One or botheyes were proptosed by gentle lid retraction and enucle-ated with Vannas scissors. Pressure applied with cottongauze provided hemostasis. The mice were placed in awarm chamber (37°C) for 1 hour during recovery fromanesthesia. Mice were sacrificed 15–21 days after the enu-cleation.

Cholera toxin tracing experiments

Mice were anesthetized with ketamine (60 mg/kg ip) andmedetomidine (1.0 mg/kg ip) and placed in a stereotaxicdevice. One eye was incised just behind the ora serrata witha 30G hypodermic needle and injected intravitreally withcholera toxin B subunit (CTB; 1 �l of a 1% w/v solution indistilled water) either in the unconjugated form (List Biolog-ical Lab., Campbell, CA; polyclonal ) or conjugated to AlexaFluor 488 (C-22841; Molecular Probes, Eugene, OR). For thedata shown in Figure 9F–J, the CTB injection was binocularbut failed in one eye, producing negligible anterograde label-ing, so that the overall labeling pattern was effectively likethat in cases of monocular injection. Anesthesia was thenreversed with atipamezole (2 mg/kg ip; Pfizer, Exton, PA).Three days after surgery, mice were killed with an overdoseof Nembutal and perfused transcardially with PBS followedby 4% paraformaldehyde in PBS. The brain was placed inthe same fixative for 1 hour and then cryoprotected over-night in 30% sucrose in PBS.

Single- and double-labelimmunofluorescence processing

For all immunohistochemical studies, coronal sectionsof the brain were cut as for the X-gal studies, washed (3 �5 minutes) in PBS, and blocked with 6% normal donkeyserum (NDS) in PBS containing 0.3% Triton X-100. Fordouble-immunofluorescence visualization of CTB and�-galactosidase, sections were incubated (72 hours; 4°C)in a mixture of the two primary antibodies, a polyclonalgoat anti-choleragenoid antibody (List #703, 1:5,000) anda polyclonal rabbit antibody raised against highly purified�-galactosidase from E. coli [Cortex (San Leandra, CA)#CR7001RP5, 1:2,000], containing 0.3% Triton X-100 and2% NDS. Sections were washed in PBS, incubated in Alexa-488-tagged donkey anti-rabbit secondary antibody (Molecu-lar Probes A-21206, 1:400), washed again, and incubated inAlexa-568-tagged donkey anti-goat secondary (MolecularProbes A-11057, 1:400). When CTB was visualized insteadby direct conjugation to Alexa 488 (see above), �-galactosidase immunofluorescence was achieved with a poly-clonal rabbit anti-�-galactosidase primary antibody(Eppendorf-5 Prime, Boulder, CO; 1:1,000; overnight at 4°C)and a goat anti-rabbit secondary antibody tagged with Alexa568 (Molecular Probes, A11011; 1:400). Secondary antibodysolutions included 0.3% Triton X-100 and 2% NDS, andincubations were carried out for 1–2 hours at room temper-ature. Sections were washed, mounted on glass slides, andcoverslipped with Vectashield (Vector, Burlingame, CA).

Anti-CTB immunolabeling was evident only in knownretinofugal tracts or targets, matched the distributionof transported CTB directly tagged with Alexa488, andwas absent in brains from mice not injected with thetracer. Anti-�-galactosidase immunoreactivity with ei-ther antibody corresponded closely to the pattern ofhistochemical staining for the enzyme by X-gal and wasundetectable in wild-type brains. For anti-VIP immuno-fluorescence, we followed Abrahamson and Moore (2001),using the same primary antibody [Immunostar (Hudson,WI) polyclonal rabbit anti-VIP IgG raised against porcineVIP conjugated to bovine thyroglobulin, #20077;1:20,000], but substituting the Alexa-488-labeled donkeyanti-rabbit secondary antibody described above (Molecu-lar Probes). The distribution of VIP-like immunofluores-cence in the SCN closely matched earlier descriptions

1X-gal is from VWR, Bridgeport, NJ. All other reagents are from Sigma(St. Louis), unless otherwise stated.


328 S. HATTAR ET AL.

(Abrahamson and Moore, 2001). Such immunostaining isabolished by preadsorption with VIP but not with variousother neuropeptides (manufacturer’s technical informa-tion).

A few retinas were immunostained for �-galactosidase.Retinas were postfixed overnight in 4% paraformaldehyde

(in PBS) at 4°C. Immunohistochemical protocols followedthose used for the brain sections except that the anti-�-galactosidase primary antibody (Cortex #CR7001RP5) wasused at 1:1,000 dilution and the secondary antibody wastagged with Alexa Fluor 488, used at 1:250 dilution, over-night at 4°C.

Fig. 1. X-gal labeling of melanopsin ganglion cells in a retinal wholemount. Composite image derivedfrom a stack of brightfield photomicrographs locally masked to reveal all labeled processes in sharp focus.Arrowheads mark axons in the optic fiber layer. Inset: Schematic charting of distribution of X-gal-labeled cell bodies in one retinal wholemount. Scale bar � 50 �m.

Fig. 2. Distribution of axons of mRGCs in optic nerve and tract.A: Confocal image of axonal anti-�-galactosidase immunoreactivity ina transverse section of the optic nerve. B,C: X-gal staining of axons inthe optic tract contralateral (B) and ipsilateral (C) to a monocular

enucleation. Axonal degeneration is almost complete in B but nearlyabsent in C, indicating that, beyond the SCN, melanopsin RGCs havepredominantly crossed projections. Scale bar � 100 �m in A; 200 �min C (applies to B,C).



Fig. 3. Overview of axonal projections of mRGCs. A,B: Wholebrains stained by X-gal. A: Dorsal view, with rostral at the top.B: Lateral view with rostral at the left and dorsal at the top. C: Sche-matic drawing illustrating nuclear targets of melanopsin RGCs. Prin-

ciple targets are shown in dark gray and secondary targets in lightgray. Minor targets are indicated by small lettering and dots. Forabbreviations, see list. Scale bar � 500 �m in A (applies to A,B).

Microscopy and image processing

Brightfield images of X-gal staining and epifluorescenceimages of immunostaining were acquired on a NikonE-600 compound microscope equipped with a 100-W mer-cury arc lamp and a Spot RT Slider camera (DiagnosticsInstruments; Sterling Heights, MI) coupled to a Dell Pen-tium III computer running Windows 2000 and SPOT soft-ware. Confocal images were obtained at 16-bit resolutionon a Zeiss 410 laser scanning microscope by using akrypton-argon laser. Images acquired were imported intoAdobe (San Jose, CA) Photoshop 6.0, and global adjust-ments were made to optimize brightness and contrast. Insome cases, depth of field was enhanced in Photoshop byselectively masking portions of each image in a stack ofimages obtained at a series of focal planes so as to displayonly those portions of each image in which the labeledelements were in optimal focus. For double immunofluo-rescence, care was taken in making adjustment of sensi-tivity to ensure separation of the signals from the twofluorophores. Except where noted, boundaries and fullnames of nuclei follow the atlas of Paxinos and Franklin(2001), although some abbreviations have been changedfor consistency with those widely used in the literature onnon-image-forming visual networks. The plates of thatatlas also served as the model for the schematic coronalsections of Figure 4.

RESULTS

The distribution and form of �-galactosidase-positivecells and processes were virtually identical whether visu-alized by X-gal staining or by anti-�-galactosidase immu-nofluorescence. The marker enzyme was detectable onlyin a small population of inner retinal neurons and in theiraxons in the optic pathway. No �-galactosidase-positivesomata were observed anywhere in the brain, and axonallabeling in the brain was abolished by binocular enucle-ation (data not shown). The �-galactosidase-positive fiberswere essentially restricted to a subset of targets withinthe overall retinofugal projection as revealed by CTB la-beling, although the superior signal-to-noise properties ofthe �-galactosidase methods permitted detection of iso-lated fibers and sparse terminal fields that could havebeen missed had we relied on the CTB method alone.Thus, we conclude that all axons labeled by X-gal histo-chemistry or anti-beta galactosidase immunostaining de-rive from retinal ganglion cells that would normally ex-press melanopsin (mRGCs).

Retina

Labeled cell bodies were observed exclusively in theinner retina. The great majority occupied the ganglion celllayer, but roughly 5% lay in the inner nuclear layer nearits inner border. The latter were presumably displacedganglion cells rather than amacrine cells, because whenthey were well stained, their axons could be traced intothe optic fiber layer. In well-stained material, approxi-mately 750 cells in each retina were X-gal positive. Thesewere distributed relatively uniformly over the retina, al-though in some retinas there appeared to be an elevateddensity of cells at the retinal margin (Fig. 1, inset). Theextent of dendritic labeling was highly variable bothwithin and among retinas. In the best stained cells, den-

drites could be followed for several hundred micrometers(Fig. 1), and these invariably arborized within the outer-most stratum of the IPL, adjacent to the inner nuclearlayer. With few exceptions, labeled axons could be seenemerging from well-stained cells and coursing toward theoptic disk (Fig. 1, arrowheads).

Optic nerve and tract

Labeled fibers were distributed throughout the opticnerve as it approached the chiasm. Figure 2A illustrates aconfocal image of a transverse cross section of the nerveimmunostained for �-galactosidase. A total of 777 labeledaxons were counted in this section, which correspondsclosely to the counts of X-gal-labeled somas in the retina.Beyond the optic chiasm, X-gal positive fibers becameclearly segregated within the optic tract, occupyingmainly the periphery of the tract’s dorsal half and largelyavoiding its core or ventral aspect (Fig. 2C). After monoc-ular enucleation, nearly all X-gal-positive fibers in thecontralateral optic tract degenerated, leaving only weaklystained granular debris (Fig. 2B) and a smattering ofintact labeled axons, which presumably arose from theintact ipsilateral eye. A complementary pattern was evi-dent in the tract ipsilateral to the enucleation (Fig. 2C),with very little evidence of degenerating labeled fibers.Thus, beyond the hypothalamus, the projections ofmRGCs are overwhelmingly crossed.

Overview of projections to the brain

The distributions of labeled fibers were highly consis-tent from brain to brain, whether visualized by X-galstaining or anti-�-galactosidase immunofluorescence, al-though the sparser terminal fields were sometimes unde-tectable in cases of suboptimal staining. An overview ofthe central projections of these RGCs appears in Figures 3and 4. Figure 3 shows the trajectories and terminations ofX-gal-stained fibers in a stained brain wholemount,viewed from its dorsal (Fig. 3A) or lateral (Fig. 3B) aspect.The most prominent targets are summarized in the sche-matic diagram of Figure 3C. Figure 4 illustrates the dis-tribution of labeled fibers after monocular (right) enucle-ation in schematic chartings of coronal sections and thusprovides an indication of the relative weighting of crossedand uncrossed projections from these ganglion cells tospecific brain nuclei.

Several major regions of termination are apparent inthese figures. The first lies near the optic chiasm and in-cludes not only the dense projections to the suprachiasmaticnucleus but also several other hypothalamic nuclei and theamygdala. The second lies in and near the lateral geniculatecomplex and can be traced rostrally as far as the vicinity ofthe anterior thalamic nuclei and bed nucleus of the striaterminalis. Fibers pass medially from the geniculate com-plex and course over the dorsal surface of the brain to inner-vate a third, chevron-shaped terminal zone, the rostral armof which abuts the habenula. The other arm runs caudola-terally along the pretectothalamic border in what may be theposterior limitans nucleus. Just caudal to this, a fourth, verydense terminal field marks the olivary pretectal nucleus, andsparser terminations are also apparent in the superior col-liculus and periaqueductal gray.

There was a marked difference in appearance betweenpassing fibers, such as those in the optic tract, and fibers inpresumed terminal zones. Passing fibers were generally verythin, relatively straight, sparsely varicose, and more lightly



stained, whereas those in terminal zones appeared thickerand more densely stained. When fiber density was lowenough to resolve individual fibers, they appeared tortuousand often exhibited branching and prominent terminalswellings.

Suprachiasmatic nucleus

The SCN is the most densely innervated target of�-galactosidase-positive fibers in these mice. After monoc-ular enucleation, labeling was remarkably bilaterally

Fig. 4. 1–22: Chartings of schematic coronal sections, modified from the atlas of Paxinos and Franklin(2001), summarizing the distribution of X-gal-labeled fibers as seen in cases of monocular enucleation.The right side of the brain, shown on the right of each charting, is contralateral to the intact eye. Forabbreviations, see list.



symmetric, indicating that mRGCs in each eye projectsequally strongly to the two SCNs (Figs. 4, 5A). In double-immunofluorescence experiments, most CTB-labeled reti-nal afferents in the SCN (red) were also �-galactosidaseimmunopositive (green), so most retinal afferents to thisnucleus apparently arise from mRGCs (Fig. 5B–E). How-ever, at least a few CTB-positive fibers and puncta lacked�-galactosidase (filled arrows in Figs. 5D,E), and thesepresumably arose from non-mRGCs. X-gal-positive fibersappeared to fill the entire nucleus including both its“shell” and “core” subregions, as defined either cytoarchi-tecturally or by the distribution of fibers immunoreactivefor vasoactive intestinal peptide (VIP; Abrahamson andMoore, 2001; Yan and Silver, 2002; Fig. 5F–I).

Other hypothalamic and basal limbic targets

Labeled fibers were present in a variety of hypothalamicnuclei in addition to the SCN. Rostrolateral to the SCN,scattered fibers reached the lateral and ventrolateral preop-tic area and in some brains encroached on the nucleus of thehorizontal limb of the diagonal band (Fig. 4, sections 1 and 2;Fig. 6A,B). Fibers extended dorsally and caudally from theSCN to infiltrate the ventral part of the subparaventricularzone (vSPZ; Lu et al., 2001; Fig. 5H). These were almostentirely limited to the ventral part of the vSPZ as defined bythe distribution of VIP-immunoreactive fibers (Fig. 5H,I),whereas the dorsal SPZ and paraventricular nucleus were

essentially devoid of labeling. Double-immunofluorescencematerial indicated that the majority of retinal afferents inthe vSPZ come from mRGCs (Fig. 6D–F) but that at leastsome appear to originate in non-mRGCs. Monocular enucle-ation revealed that the uncrossed input from mRGCs to thisregion is nearly as strong as the crossed input (Fig. 5A).

Labeled fibers coursed dorsally and laterally from theSCN to weakly innervate the lateroanterior hypothalamicand medial preoptic nuclei. There was relatively densefiber labeling in the retrochiasmatic region, includingparts of the lateroanterior nucleus and central division ofthe anterior hypothalamic nucleus (Fig. 4, sections 7 and8; Fig. 6C). This labeling was continuous with that in thevSPZ and may lie within the caudal part of the SPZ (Lu etal., 2001). As the optic tract emerged from the optic chi-asm, fibers peeled off dorsally to form a moderately densenest in the vicinity of the supraoptic nucleus (SON; Fig. 4,sections 5–7; Fig. 6A,C). Most of these fibers appeared toterminate just dorsal to the nucleus itself, in a region thathas been termed the peri-supraoptic nucleus (pSON).Double-immunofluorescence material revealed that sub-stantial numbers of CTB-labeled retinal afferents in thisregion lacked �-galactosidase and thus presumably origi-nate from non-mRGCs (data not shown). In well-stainedbrains, a few �-galactosidase-positive fibers could be seeninnervating the medial amygdaloid nucleus (Fig. 4, sec-tion 8; Fig. 6C). One or two labeled fibers were also de-

Figure 4 (Continued)



Fig. 5. Axonal terminations of melanopsin ganglion cells in thesuprachiasmatic nucleus of the hypothalamus. A: X-gal staining ofmelanopsin afferents to the SCN after right monocular enucleation.Although most labeled fibers in the contralateral (left) optic tract havedegenerated, leaving only weak, granular labeling, labeling of theSCN is bilaterally symmetric. B: Confocal image of SCN showingdouble immunostaining for all contralateral retinal inputs (red; anti-cholera toxin B [CTB] immunofluorescence) and for all bilateral mela-nopsin RGC inputs (green; anti-�-galactosidase immunofluorescence).Most red, CTB-labeled afferents exhibit green anti-�-galactosidaselabeling also, giving them a yellow appearance in the image, but a fewcontain little if any green labeling and presumably arise from non-melanopsin RGCs. Green axons lacking red immunofluorescence arepresumably melanopsin afferents from the ipsilateral eye, which wasnot injected with CTB. Large circular dark profiles are unlabeled

somata of SCN neurons. Projected stack of seven 1-�m confocal opti-cal sections. C–E: Confocal image (optic section thickness 1 �m)grazing the caudal margin of the SCN from the same brain as in B andlabeled by the same double-immunofluorescence method. Reducedafferent density permits individual axons to be resolved. Filled arrowsindicate crossed retinal afferents (red) that are �-galactosidase neg-ative (no green). Open arrows denote presumptive ipsilateral affer-ents from melanopsin RGCs (green, no red). F–I: Comparison of thedistribution within the SCN and vSPZ of axons of melanopsin gan-glion cells (X-gal staining in F and H) with those of VIP-immunoreactive processes in serially adjacent sections. F and G arefrom the rostral pole of the SCN; H and I are from the mid-caudalSCN. Wisps of fiber labeling dorsal to the main SCN field in H liewithin the vSPZ. Scale bar � 100 �m in A, C (applies to C–E), and F(applies to F–I); 10 �m in B.



tected in the zona incerta and substantia innominata insome brains.

We scanned the piriform cortex with particular carebecause this region is sparsely innervated by retinal fibersin rodents and primates (Pickard and Silverman, 1981;Mick et al., 1993; Ling et al., 1998) and because in micewith advanced degeneration of rods and cones light evokesbiochemical responses in this region (Alvarez-Lopez et al.,2004), suggesting a possible input from mRGCs. However,neither �-galactosidase-positive fibers nor CTB-labeledretinal afferents were observed in this region in any brain.Other than the SCN and vSPZ, all the basal limbic nucleiappeared to have stronger mRGC input from the con-tralateral retina than from the ipsilateral retina, althoughall these regions appeared to receive at least a weak un-crossed retinal input.

Intergeniculate leaflet

Beyond the amygdala, labeled fibers coursed within theoptic tract without obvious terminations until theyreached the lateral geniculate complex. By far the densestterminal labeling within the geniculate complex was inthe intergeniculate leaflet (IGL; Figs. 3A, 7, 9). In coronalsections through most of the length of the geniculate com-plex, this zone of dense labeling corresponded closely tothe IGL as shown in the atlas of Paxinos and Franklin(2001), a thin band separating the dorsal and ventraldivisions of the lateral geniculate nucleus (LGd and LGv).However, this distinctively dense plexus of labeled fiberscontinued both rostrally and caudally beyond the confinesof the IGL as shown in the atlas. Because this comportswith the extent of the nucleus as revealed by other mark-ers for the IGL in rodents (Morin et al., 1992), we interpretthe entire zone of dense fiber labeling as the IGL, depart-ing in this respect from the Paxinos and Franklin atlas.Caudally, the IGL thus defined is found nearly as far backas the caudal pole of the medial geniculate nucleus. There,labeling consisted of a small group of fibers nestled be-tween the dorsolateral margin of the cerebral peduncle,the caudalmost part of the optic tract, and the medialgeniculate (Fig. 9D; see also Fig. 4, section 18), in a regiontermed the parapeduncular nucleus by Paxinos andFranklin (2001).

Rarely, fibers could be detected in the adjacent part ofthe zona incerta. Rostrally, the labeling in the IGL ex-tended at least as far as the rostral pole of the LGd (Fig.7B). Indeed, fibers in this terminal field extended stillfarther forward, albeit thinning progressively, where theyoccupied the dorsolateral margin of the dorsal thalamus atthe level of the lateral dorsal thalamic nucleus, just dor-somedial to the stria terminalis (Figs. 3A,B, 4, sections8–10). In optimally stained brains, at least a few fiberscould be traced nearly to the rostral pole of the dorsalthalamus, at which point they turned ventrally to end inthe vicinity of the bed nucleus of the stria terminalis andanterior thalamic nuclei (Fig. 4, sections 4 and 5; Fig. 7C).

Monocular enucleation showed that ipsilateral-eye in-puts from mRGCs to the IGL were far weaker thancontralateral-eye inputs (Fig. 7A,D). The ipsilateral in-puts were present to some degree throughout the IGL buttended to be heaviest in the dorsolateral half of the nu-cleus. Double-immunofluorescence staining showed abun-dant co-localization of �-galactosidase and CTB tracer inretinal afferents to the IGL (Fig. 7E,F), suggesting thatmost retinal inputs to this nucleus arise from mRGCs.

However, at least some �-galactosidase-negative retinalterminals were apparent in the IGL both contralateraland ipsilateral to the monocular tracer injection (redpuncta in Fig. 7E,F).

Ventral lateral geniculate nucleus

The most conspicuous terminal labeling in the LGv wasfound in a compact part of the parvocellular division. Thiswas found ventromedially in the caudal part of the nu-cleus and was directly contiguous with the dense labelingof the IGL (Fig. 7A). In fact, in some brains, at least partof this terminal field appeared to be a small rostral exten-sion of the caudoventral IGL. Tortuous fibers with appar-ent terminal swellings were scattered throughout theLGv, especially rostrally and medially in the parvocellulardivisions, but these were far sparser than in the IGL.Elsewhere within the LGv, including within the externaldivision, which is the most prominent target of retinalinput, many of the labeled fibers were thin, took relativelystraight, parallel courses, and lacked extensive branchingor swelling. They thus appeared to be passing fibers enroute to the IGL and more medial diencephalic regions.Enucleation experiments showed that the great bulk ofthe melanopsin RGC input to the LGv derives from thecontralateral eye (Fig. 4, sections 13–17). Double-immunofluorescence analysis indicated that even thestronger crossed input from mRGCs accounts for only asmall fraction of the overall retinal input to the LGv (Fig.7E).

Dorsal lateral geniculate nucleus

Cursory examination revealed little obvious terminallabeling within the dorsal division of the lateral geniculatenucleus (LGd). Much of the nucleus was entirely devoid offiber labeling, and the few readily discerned labeled axonsappeared to be passing fibers (Fig. 7A, white arrow). Thesefibers, presumably constituents of the fascicles of the “in-ternal optic tract” (see, e.g., Frost et al., 1986), paralleledthose in the overlying optic tract and appeared to beheading for more medial targets such as the pretectalregion and the habenula. In optimally stained brains,however, a thin sheet of sparse, tortuous fibers with ap-parent terminal swellings could be detected at the ventro-medial and rostral margins of the LGd. From examinationof double-labeled material, it is clear that these fibers liewithin the LGd proper as defined by dense, CTB-labeledretinogeniculate input. In coronal sections through themiddle of the LGd, this sheet of terminal labeling ap-peared as a thin band paralleling the external medullarylamina. This terminal field was apparent not only in thecrossed projection of mRGCs (Fig. 7A, black arrow) butalso in the ipsilateral projection (Fig. 7D, black arrow),which appeared to terminate within the main ipsilateral-eye segment of the LGd. This band of labeling could befollowed to the rostral pole of the LGd, where it curledupward so that it occupied the full cross section of thenucleus at this level (Fig. 7B; see also Fig. 3A,B).

Habenular region and posterior limitansnucleus

Labeled fibers coursed medially over the LGd in thesuperficial optic tract to terminate in a chevron-shapedfield near the habenular complex and rostral border of thepretectum. The form of this terminal plexus is best appre-



Figure 6


ciated in the dorsal view of the X-gal-reacted wholemount(Fig. 3A). The caudal arm of this field occupied the rostro-dorsal margin of the pretectal region, at its boundary withthe dorsal thalamus (Fig. 4, sections 13 and 14). In coronalsections, this zone of labeling was visible at the dorsalsurface of the anterior pretectal nucleus, where labeledaxons of the superficial optic tract took on the darker,thicker appearance typical of terminal fibers (Fig. 8B).This region is considered an extension of the posteriorlimitans nucleus (PLi) in some rodents (e.g., Morin andBlanchard, 1997), and though this is not consistent withthe mouse atlas of Paxinos and Franklin (2001), we ten-tatively adopt that nomenclature here. The main body ofthe rodent PLi is generally depicted as a thin shell abut-ting the lateral margin of the anterior pretectal nucleus.We were able to trace a very few �-galactosidase-positiveaxons to this region. These lay rostral to the PLi as drawnby Paxinos and Franklin (2001) and within what theyconsider the mediorostral division of the lateral posteriornucleus.

At the level of the habenular commissure, the terminalfield assumed a purely rostrocaudal orientation (Fig. 3A),forming a very dense terminal plexus at the dorsolateralmargin of the lateral habenular nucleus. This plexus layin a cell-sparse region on the surface of the diencephalon,in the shallow groove where the habenula abuts the cen-tral lateral nucleus and lateral nuclear group of the thal-amus (Figs. 3A, 4, sections 10–12; Fig. 8A). The plexuswas most prominent at the caudal end of the habenularcomplex and contained progressively fewer axons at morerostral levels. Nonetheless, it was readily apparent inmost brains at least as far rostral as the rostral pole of theLGd, and in some brains an isolated fiber or two could betraced to the rostral limit of the habenula, just lateral tothe stria medullaris. Although most of the fibers in thisplexus lay within about 100 �m of the surface, a few fiberspenetrated more deeply. These terminated at the lateralor, rarely, the ventral margins of the lateral habenula,within the fiber capsule that bounds the nucleus (Fig. 8A,arrowheads). Labeled fibers were sometimes found in as-sociation with the stria medullaris, but they never in-vaded the core of the lateral habenula. Nearly all CTB-labeled optic afferents in this region were �-galactosidasepositive, indicating that melanopsin ganglion cells providenearly all retinal input to this area (data not shown).These fibers were almost entirely crossed, although thereis an extremely sparse ipsilateral input, as indicated bythe one or two labeled fibers still evident contralateral to

a removed eye (Fig. 4, section 12). Because mRGCs arepresumed to exert substantial influence over the release ofmelatonin, we carefully examined the region of the pinealstalk and the pineal itself, but we observed no labeling ineither structure.

Pretectal region

Except for the labeling in the posterior limitans nucleus,noted above, labeling in the pretectal region was almostentirely confined to a dense, well-defined terminal fieldthat we consider to be the olivary pretectal nucleus (OPN;Fig. 4, sections 15–20). The terminal field was complex inshape, with a large, hollow head rostromedially and atapering body that extended caudolaterally and typicallysplit into two or three tails. Its rostral pole was foundslightly behind the habenular commissure and just underthe pial surface at the medial edge of the pretectal region(Fig. 9A). Here, it was ovoid in coronal cross-section andabout 400 �m across. Although it lay fairly close to thecaudal end of the terminal field in the posterior limitansnucleus, it was clearly separated from the PLi by a shortspan containing only passing fibers (Fig. 3A). Near therostral pole, the terminal zone appeared hollowed out,taking on the form of an annulus in coronal sections (Fig.9A,B,F,I). Caudally, the nucleus became horizontally elon-gated (roughly 300 �m wide and 50–100 �m thick) and insome sections appeared to be broken into two or threepatches (Figs. 3A, 9C,D,H,J). Most of these were elongatedparallel to the tectal surface, 100–200 �m in width, andwere separated by largely terminal-free gaps of about thesame size. At more caudal levels, the terminal labelingassumed a progressively more lateral and ventral posi-tion, terminating near the dorsal terminal nucleus (DTN)of the accessory optic system.

Although rostrally this terminal zone correspondedclosely to the olivary pretectal nucleus as defined by Paxi-nos and Franklin (2001), caudally it extended well beyondtheir OPN to encroach on what they designate as thecaudal part of the posterior pretectal nucleus and therostrolateral portions of the superior colliculus (stratumopticum and stratum griseum intermediale). The field ofmRGC terminations corresponds more closely to the de-scription of the anterior olivary pretectal nucleus of Pak etal. (1987), which they defined in wild-type mice on thebasis of retinal input. However, the correspondence isimperfect because near the rostral pole of the nucleus,mRGC input was limited to a shell marking the margins ofthe circular field of overall retinal input (Fig. 9F,I). Morecaudally, the terminations of mRGCs lay at the base of themuch broader zone of retinopretectal terminations thatincludes the nucleus of the optic tract and posterior pre-tectal nucleus (Fig. 9G,H,J; Scalia, 1972; Scalia andArango, 1979; Pak et al., 1987). At its caudal limit, themRGC terminal field lay just beneath the DTN. The DTNitself was generally devoid of mRGC axons, although astray fiber or two could be detected in the best stainedbrains. The small nest of retinal input in the caudomedialpretectum that Pak et al. (1987) termed the posteriorolivary pretectal nucleus was clearly evident in our CTBmaterial (Fig. 9G) but apparently lacked any input frommRGCs.

The uncrossed input to the OPN from melanopsin RGCswas much weaker than the crossed input, as reported forother rodents (Muscat et al., 2003; Hannibal and Fahren-krug, 2004) and appeared more restricted in its distribu-

Fig. 6. Hypothalamic and other basal limbic targets beyond theSCN. A: Basal view of X-gal-stained whole brain. Top of image isrostral and the right margin is near the midline. The optic chiasm andthe right optic nerve and tract (left in image) have been retractedcaudally to expose the right SCN and pSON. A few fibers are alsoevident in the preoptic region. B,C: X-gal staining in coronal sectionsjust rostral (B) or caudal (C) to the SCN, showing scattered fibers inthe preoptic area including the VLPO (B), dense input to the peri-SON(C), and scattered labeling in the anterior and lateral hypothalamusand medial amygdala (C). D–F: Confocal immunofluorescence imagesof the ventral SPZ illustrating relationship between crossed retinalafferents (red; D) and afferents from melanopsin RGCs (green, E).Format as in Figure 5C–E; F is the merged image. Many but not all ofthe crossed retinal afferents arise from �-galactosidase-positive(melanopsin) afferents. For abbreviations, see list. Scale bar � 500�m in A and C; 200 �m in B; 25 �m in D (applies to D–F).



Figure 7

tion (Fig. 9O), being essentially absent from the caudalthird of the terminal field and from its ventral half atrostral levels. Double-immunofluorescence studies showedthat even within the zone of densest termination of mRGCaxons, many retinal terminals lacked the �-galactosidasemarker enzyme and thus arose from non-mRGCs (Fig.9K–N).

A small number of X-gal-labeled fibers coursed ventro-medially from the OPN toward the posterior commissure(Fig. 9O). Some penetrated the commissure itself, runningperpendicular to its fibers, and could be traced into theunderlying rostral periaqueductal gray. Others appearedto terminate more laterally, in and near the nucleus of theposterior commissure. This retinal terminal field appearsto correspond to the commissural pretectal area in the rat(Prichard et al., 2002) and to the commissural pretectalnucleus in hamsters (Morin and Blanchard, 1997, 1999).Very rarely, a few fibers could be traced caudally from thissystem as far as the periaqueductal gray beneath thecaudal superior colliculus and to the vicinity of the dorsalraphe nucleus.

Superior colliculus

The superior colliculus was a significant target of la-beled axons (Fig. 3A). These appeared to reach the SCprimarily through the medial part of the brachium of thesuperior colliculus, passing caudally from the terminalfield in the OPN. The fibers continued caudally in thestratum opticum of the SC, giving off dorsally directedbranches at every coronal level through the nucleus.These represented a tiny fraction of overall retinal inputto the SC (data not shown), and the sparseness of the fiberlabeling made it possible to observe the morphology ofindividual fibers in considerable detail. Extensive branch-ing, looping, and terminal swelling were seen in the su-perficial gray layer, and branches were often seen ascend-ing as far as the stratum zonale, where they terminatedjust beneath the pia (Fig. 10). We rarely observed exten-sive clusters of terminal boutons on single axons, butwhen we did these could appear either in the upper orlower part of the superficial gray layer. In one brain, wedetected two labeled axonal branches in the deeper col-licular layers and underlying tegmentum.

Crossed projections to the tectum were much moreprominent than uncrossed ones. Both crossed and un-

crossed inputs were typically densest in the medial half ofthe SC, followed by its lateral margin (Fig. 10A), and werepresent throughout the rostrocaudal extent of the nucleus(Figs. 3A, 10C). The ipsilateral input was not confined tothe deeper part of the superficial layers, as has generallyreported for overall uncrossed retinal input (Godement etal., 1984), but instead clearly invaded the upper superfi-cial gray layer and even reached the pial surface in somesections (Fig. 10C).

Lack of terminations in accessory opticsystem

All components of the accessory optic system were easilyidentifiable on the basis of their CTB-labeled retinal in-put. Labeling was entirely absent within the medial ter-minal nucleus, by far the largest accessory optic nucleusin the mouse (Pak et al., 1987). The lateral terminal nuclei(LTN) and inferior and superior fasciculi of the accessoryoptic tract were also devoid of �-galactosidase-positivefibers. Although labeled fibers terminating in the caudalpole of the OPN approached the ventral margin of theDTN, the DTN itself was typically free of labeling.

DISCUSSION

This study builds on recent work detailing the brainregions contacted directly by the axons of melanopsin-expressing, intrinsically photosensitive retinal ganglioncells (Gooley et al., 2001, 2003; Hannibal et al., 2002,2004; Hattar et al., 2002; Morin et al.. 2003; Sollars et al.,2003; Dacey et al., 2005). The findings expand the range oftarget structures to include a diverse set of nuclei in thehypothalamus and other subcortical forebrain limbiczones, several divisions of the lateral geniculate complex,the habenular region, the superior colliculus, and the peri-aqueductal gray. Crossed inputs from mRGCs to theseregions greatly outnumber uncrossed inputs except in theSCN and vSPZ, which get balanced inputs from the twoeyes. Nearly all these terminal fields receive at least someinput from RGCs that do not express melanopsin, al-though mRGCs appear to provide the dominant input tothe SCN, IGL, and habenular region.

Anatomical and functional identity ofmRGCs: are there multiple types?

There is no doubt that the fiber projections describedhere arise from retinal ganglion cells that normally ex-press melanopsin. The retina was the only part of thecentral nervous system that contained �-galactosidase-positive somata. Furthermore, all �-galactosidase-containing axons in the brain could be traced back to theoptic pathway, and all of them degenerated after bilateraleye removal.

Most, if not all, of these ganglion cells are presumably ofthe type shown to be intrinsically photosensitive (Bersonet al., 2002), because these cells express melanopsin (Hat-tar et al., 2002; Dacey et al., 2005) and require it for theirintrinsic photosensitivity (Lucas et al., 2003). The mor-phology of intrinsically photosensitive RGCs (Berson etal., 2002; Warren et al., 2003) closely matches that of cellsexpressing melanopsin in wild-type rodents (Hattar et al.,2002; Provencio et al., 2002; Hannibal et al., 2002) orexpressing �-galactosidase in this study, especially intheir predominant dendritic stratification in the outer-

Fig. 7. Distribution of afferents from melanopsin ganglion cells inlateral geniculate complex. A–D: Coronal sections reacted with X-gal.A and D are from middle of rostrocaudal extent of LGd. A is contralat-eral and D is ipsilateral to the remaining eye in a monocularly enu-cleated mouse. Black arrows indicate sparse terminal labeling nearthe ventromedial margin of LGd. White arrow in A indicates axon ofpassage in one of the fascicles traversing the LGd. B shows substan-tial terminal labeling at the rostral margin of the LGd as well asstrong labeling of the IGL at this level. C shows the rostral limit ofthis terminal field in or near the BST. E,F: Confocal merged immu-nofluorescence images of the IGL and adjacent geniculate nuclei il-lustrating relationship between afferents from mRGCs of both eyes(green) and total retinal afferents (red) from one eye, either thecontralateral eye (E) or the ipsilateral eye (F). Most retinal afferentsto the IGL are from melanopsin RGCs (green and yellow puncta), butmany are not (red puncta). Green profiles (without red) representterminations of axons from mRGCs in the eye not injected with CTB.Most retinal afferents to the LGv do not arise from melanopsin RGCs.For abbreviations, see list. Scale bar � 100 �m in A–F.



most IPL. Two of the densest projections evident in thepresent data, those to the SCN and OPN, have been di-rectly confirmed for photosensitive ganglion cells in stud-ies combining retrograde transport and intracellular re-cording (Berson et al., 2002; Dacey et al., 2005). Together,these data support the view that inputs from ganglion-cellphotoreceptors sustain photic effects on circadian phaseand pupil diameter in mice lacking functional rods andcones (Yoshimura and Ebihara, 1996; Lucas et al., 2001;Hattar et al., 2003). That view is further supported by thecorrespondence in spectral tuning among these residualbehavioral photoresponses, the intrinsic light responses of

photosensitive ganglion cells, and the light responses ofcells heterologously expressing melanopsin (Panda et al.,2005; Qiu et al., 2005; but see Melyan et al., 2005).

Although it is thus reasonable to conclude that most�-galactosidase-positive fibers in this study arise fromintrinsically photosensitive RGCs, it is not certain that alldo because there is some evidence for a second type ofmelanopsin-expressing ganglion cell. In the mouse retina,a sensitive melanopsin antibody (UF006) labels two tiersof immunopositive dendrites in the IPL (Provencio et al.,2002). Of these, only the outer tier, lying adjacent to theinner nuclear layer, matches the stratification of identi-

Fig. 8. X-gal-stained coronal sections illustrating inputs to theregion of the habenula and posterior limitans nucleus. A: Mid-habe-nular level; midline is at the left edge of the image. The main terminalfield lies at dorsolateral margin of the stria medullaris and lateralhabenula, with scattered fibers (arrowheads) within the fiber capsulebounding the habenular complex ventrolaterally. B: The caudal end ofthe terminal field, at the level of the habenular commissure. The

plexus of labeled fibers on the right lies at the dorsal surface near theboundary of the midbrain (represented by the anterior pretectal nu-cleus) and the thalamus (represented by the lateral posterior nu-cleus), in a region included within the posterior limitans nucleus inother rodents. The right eye was enucleated in this animal, leading tonearly complete loss of labeled axons on the left. For abbreviations,see list. Scale bar � 200 �m in A,B.



fied ipRGCs in rats (Berson et al., 2002; but see Warren etal., 2003). The second tier of melanopsin-immunopositivedendrites lies near the inner border of the IPL, close to theganglion cells. These dendrites appear to arise from asecond population of more weakly melanopsin-immunoreactive RGCs, with somewhat more radiate, or-derly dendritic arbors (A. Castrucci, I. Provencio, and D.Berson, unpublished observations). This second popula-tion of immunoreactive RGCs has been termed M2 cells todistinguish them from the heavily stained M1 cells, whichcorrespond to the ipRGCs. There are roughly equal num-bers of M1 and M2 cells. Nothing is currently known aboutwhether the M2 cells are intrinsically photosensitive orwhere they project in the brain. However, their immuno-reactivity appears to reflect real melanopsin expression,rather than cross-reactivity of the antibody with an unre-lated protein, because it disappears in melanopsin knock-out animals (A. Castrucci and I. Provencio, personal com-munication; X. Qiu, J. Tong, and D. Berson, unpublishedobservations). The primate retina appears to contain twobroadly similar subtypes of melanopsin-expressing gan-glion cells, both of which are intrinsically photosensitive(Dacey et al., 2005).

If mouse M2 cells synthesize melanopsin protein, oneshould expect them to express �-galactosidase in theknock-in mice studied here. However, we have found noevidence for labeling of M2 cells in the retinas of theseanimals with either X-gal or the anti-�-galactosidase an-tibody. Dendrites that were stained well enough to traceinvariably terminated in the outer IPL, like M1 but unlikeM2 cells. Because the tau signal sequence was fused to the�-galactosidase protein, the marker enzyme was preferen-tially routed to axons, thus reducing its abundance anddetectability in the somadendritic compartment. How-ever, it seems very unlikely that labeling marked theaxons but not the somata or dendrites of M2 cells becausethe number of labeled axons in the optic nerve closelymatched the number of labeled somas (approximately700–800). These counts, in turn, correspond well to esti-mates of the total number of M1 cells (A. Castrucci et al.,unpublished observations). Thus, the most parsimoniousinterpretation appears to be that M2 cells do not expressenough �-galactosidase to be detected by the methodsused. This may be because the expression level of themelanopsin gene (and hence of the tau-lacZ marker gene)is lower in M2 than in M1 cells, as implied by the weakermelanopsin immunostaining in M2 than in M1 cells inwild-type animals (A. Castrucci et al., unpublished obser-vations). Indeed, when anti-melanopsin immunostainingis suboptimal (e.g., by diluting primary antibody), only M1cells are visible (unpublished observations). In summary,although some caution is warranted, the weight of thepresent evidence suggests that most if not all of the fiberlabeling observed in the present study originates in M1cells and thus from RGCs known to be directly photosen-sitive.

Suprachiasmatic nucleus

The present data confirm the well-established projec-tion of photosensitive mRGCs to the SCN (Gooley et al.2001, 2003; Berson et al., 2002; Hattar et al., 2002; Han-nibal 2002, 2004; Berson, 2003; Sollars et al., 2003; Morinet al., 2004). The SCN has been subdivided into “core” and“shell” components on the basis of distinct patterns ofchemoarchitecture and connections (Moore et al., 2002).

The boundaries of these subregions are drawn differentlyby different investigators, in part because there is noconsensus on how they should be defined (e.g., Abraham-son and Moore, 2001; Yan and Silver, 2002). The core hasbeen reported to receive the bulk of the retinal input(Abrahamson and Moore, 2001; Moore et al., 2002) and, inturn, to relay photic information to the shell (Kriegsfeld etal., 2004), which may be the critical locus for light-inducedcircadian phase shifts (Yan and Silver, 2002). Our dataweigh against this sort of serial model in the mouse, atleast in its purest form, because they show that the axonsof mRGCs terminate throughout the SCN as defined cy-toarchitecturally or by the distribution of VIP-immunoreactive fibers, and certainly over a wider terri-tory than the core as defined by Yan and Silver (2002).This evidence for substantial direct retinal innervation ofthe shell is in agreement with several earlier reports (Cas-sone et al. 1988; Castel et al., 1993) and suggests that theshell may not be exclusively dependent on the core forphotic signals driving shifts in circadian phase.

The remarkable bilateral balance in input from mRGCsto the SCN parallels earlier observations on overall retinalinput to the SCN in C57BL/6J mice (Abrahamson andMoore, 2001). Some bias in favor of crossed input has beenreported in an albino strain of the house mouse (Miura etal., 1997) and in the white-footed mouse Peromyscus leu-copus (Youngstrom and Nunez, 1986) This stands inmarked contrast to the pronounced bias in favor of crossedprojections observed in all other targets of mRGCs andsuggests that most uncrossed mRGC axons end at theSCN and do not continue beyond it to the thalamus orbrainstem.

Electrophysiological and behavioral evidence stronglysuggests that conventional rod and cone photoreceptorscan drive SCN unit responses and circadian phase shifts(Aggelopoulos and Meissl, 2000; Panda et al, 2002, 2003;Ruby et al., 2002; Lucas et al., 2003; Hattar et al, 2003).One possible route for such influences is through intrareti-nal synaptic inputs to mRGCs, which have been detectedboth ultrastructurally (Belenky et al., 2003) and electro-physiologically (Dunn and Berson, 2002; Dacey et al.,2005). However, an alternative pathway suggested by ear-lier retrograde tracing studies is a minor contribution ofnon-mRGCs to the retinohypothalamic tract (Gooley et al.,2003; Morin et al., 2003; Sollars et al., 2003; Hannibal andFahrendrug, 2004). Although these earlier findings couldarguably have been due to a failure to detect melanopsinprotein or mRNA in some cells, the present data provideindependent confirmation by showing that some�-galactosidase-negative (non-mRGC) axons innervatethe SCN. Among the remaining challenges are to charac-terize the identity and functional properties of the non-mRGCs innervating the SCN; and to determine to whatdegree rod/cone influences reach the SCN directly frommRGCs, directly from non-mRGCs, or indirectly throughpolysynaptic circuits involving other brain nuclei.

Projections to other nuclei of thehypothalamus and limbic telencephalon

The mRGCs scatter a few afferent fibers through sev-eral preoptic nuclei. Among these is the VLPO, a keyplayer in sleep regulation (Sherin et al., 1996), confirmingearlier reports in the rat (Gooley et al., 2003; Hannibaland Fahrenkrug, 2004). Inputs from mRGCs to the pre-optic region may provide a basis for photic modulation not



Figure 9



only of sleep but also of circadian oscillations in core bodytemperature (Moore and Danchenko, 2002) or of neuroen-docrine processes related to reproductive function. Inagreement with the findings of Gooley et al. (2003) in therat (but see Hannibal and Fahrenkrug, 2004) and of Morinet al. (2003) in the hamster, we find that retinal inputs tothe vSPZ arise predominantly from mRGCs. This region,which also receives direct projections from the SCN, hasbeen implicated in circadian and photic modulation ofsleep and locomotor activity (Lu et al., 2001; Kramer et al.,2001; Moore and Danchenko, 2002).

The lateral hypothalamus, another destination of la-beled axons in this study, is a well-known target of theretinohypothalamic tract. Leak and Moore (1997) sug-gested, on retinotopic grounds, that ganglion cells inner-vating the rat lateral hypothalamus differed from thoseprojecting to the SCN. Our data, by contrast, indicate thatin the mouse both of these projections arise primarily frommRGCs. Another well-established target of the RHT is thesupraoptic nucleus and/or the overlying adjacent peri-SON (e.g., Johnson et al., 1988; Levine et al., 1991, 1994;Mikkelsen and Serviere, 1992; Nakagawa et al., 1992;Ling et al., 1998; Abrahamson and Moore, 2001). Thisprojection appears to influence the magnocellular neuronsin the SON proper (Cui et al., 1997a,b) and may thusmediate photic modulation of neuroendocrine output. Onepossible reflection of such influence is the acute disruptionby light of normal diurnal rhythms of plasma oxytocin andvasopressin (Windle et al., 1992; Kostoglou-Athanassiouet al., 1998). Our evidence indicates that some, althoughby no means all, of the direct retinal influence on the SONarises from intrinsically photosensitive RGCs.

Sparse retinal inputs to the medial amygdala have beenreported previously (Johnson et al., 1988; Cooper et al.,.1993; Elliott et al., 1995) and appear on the present evi-dence to arise in part from mRGCs. This region appears tobe a site of integration of retinal input with signals fromthe olfactory system and IGL and may be one site wherephotic and olfactory stimuli converge to modulate repro-ductive behavior (Cooper, et al., 1994; Elliott et al., 1995;Morin and Blanchard, 1999).

Lateral geniculate complex

The present data confirm earlier reports that rodentmRGCs provide very little input to the LGd, the main

thalamic relay of retinal information to the striate cortex(Hattar et al., 2002; Gooley et al.., 2004; Hannibal andFahrenkrug, 2004). This suggests that, at least in rodents,mRGCs may exert little influence on visual perceptualmechanisms, consistent with the view that these RGCsform the foundation of a specialized non-image-formingvisual network. However, a role for melanopsin RGCs ingeniculocortical function should not be entirely excluded.We observed sparse fiber labeling in the LGd within a thinshell bounding the nucleus ventromedially and rostrally.The ventromedial region may correspond to the beta sec-tor, a part of the murine LGd lying within the externalmedullary lamina (Godement, 1984), or to the alpha sub-division of the hamster LGd (Ling et al., 1997). The rostralzone may be related to the retinal input observed in thethalamic lateral posterior nucleus of some rodents (Ling etal., 1997). These thalamic targets are virtually certain toprovide a conduit for signals from ganglion-cell photore-ceptors to the visual cortex. In addition, mRGCs mayinfluence cortical visual areas of the neocortex by lessdirect paths, because two of their brainstem targets, theolivary pretectal nucleus and superior colliculus, emit as-cending projections to the visual thalamus. In monkeys,intrinsically photosensitive mRGCs have been reported toinnervate the LGd directly on the basis of retrogradetracing evidence (Dacey et al., 2005), suggesting a possiblerole for mRGCs in cortical vision. However, humans withadvanced outer retinal degeneration but preservation ofnon-image-forming visual responses presumably medi-ated by mRGCs (Provencio et al., 2000; Hannibal et al.,2004; Dacey et al., 2005) apparently lack any consciousappreciation of light (Czeisler et al. 1995; Klerman et al.,2002).

The IGL is clearly one of the major brain targets ofmRGCs. Although it is a part of the geniculate complex, itdoes not project to the cortex but is, instead, a well-recognized component of the non-image-forming visualsystem. The IGL is reciprocally interconnected with theSCN and OPN, both of which are also targets of mRGCs,and has been implicated in circadian photoentrainmentmechanisms (Harrington, 1997). Light responses of IGLneurons (Harrington and Rusak, 1989) are similar in sev-eral respects to those of mRGCs (Berson et al., 2002;Warren et al., 2003), including tonic ON responses that

Fig. 9. Distribution of afferents from melanopsin ganglion cellswithin the olivary pretectal nucleus (OPN), caudal IGL, and periaq-ueductal grey. A–E: Coronal series through the OPN stained withX-gal; section A is most rostral. F–J: Double-immunofluorescencemicrographs of coronal sections illustrating relationship of afferentsfrom melanopsin ganglion cells (red anti-�-galactosidase) with overallcrossed retinal input (green CTB). F, G, and H are at roughly thesame level as A, C, and D, respectively. I and J are higher powerviews of F and H, respectively. K,L: Confocal merged double-immunofluorescence images of the left OPN and adjacent pretectalnuclei illustrating the relationship between afferents from melanop-sin RGCs (green; anti-�-galactosidase) and contralateral retinal af-ferents (red; anti-CTB; note that assignment of fluorophores is re-versed from that in F–J). L is an enlarged, higher resolution scan ofthe boxed area in K. Note that the OPN, as defined by input frommelanopsin afferents, is a small part of a much larger field of crossedretinal input (K). In L, most melanopsin afferents in the OPN containanterograde label transported from the contralateral eye (green �red � yellow), but the terminal at the bottom right lacks red anti-CTB

labeling and presumably arises from the ipsilateral eye. M,N: Thesame as K and L, but from the other side of the brain (right), so thatanterograde CTB label (red) marks inputs from the ipsilateral eye. Nis enlarged from the boxed region in M. In M, note the modest overlapbetween the field of melanopsin afferents (green) and the patch ofuncrossed retinal input (red). Most melanopsin afferents in N lackanterograde label, presumably because they come from the contralat-eral eye. Within the OPN as defined by the melanopsin afferentterminal field, many retinal afferents, both crossed (K,L) and un-crossed (M,N), lack �-galactosidase (red �, green �) and thus pre-sumably arise from non-melanopsin ganglion cells. O: Coronal sectionthrough the OPN stained with X-gal after enucleation of the left eye.Note the pronounced reduction in the labeled terminal field contralat-eral to the enucleation (right). On the left, fibers descending from theOPN penetrate the posterior commissure (pc) to enter the periaque-ductal gray (PAG). For abbreviations, see list. Scale bar � 500 �m inA (applies to A–E), F (applies to F–H), and O; 200 �m in K (applies toK–M); 100 �m in I,J.



Fig. 10. Coronal sections illustrating X-gal staining of melanopsinafferents to the superior colliculus. The left eye was enucleated, leav-ing only right-eye afferents intact. A: Rostral superior colliculus.Fiber labeling is heaviest in the stratum opticum (SO), especiallymedially and laterally, but some fibers invade the stratum griseumsuperficiale (SGS) and stratum zonale (SZ). B: Higher magnification

view of left colliculus in a similar section from a different brain.C: Caudal pole of superior colliculus, from the same brain as shown inA. In A and C, a few melanopsin afferents from the ipsilateral eye areapparent in the right colliculus. For abbreviations, see list. Scale barin C � 500 �m in A,C and 200 �m in B.



encode light intensity over a dynamic range of 2–3 logunits.

The input from mRGCs to the IGL is much strongerfrom the contralateral than from the ipsilateral eye, asreported previously in the rodent for overall retinal input(Moore and Card, 1994; Muscat et al., 2003) and formRGCs input in particular (Morin et al., 2003; Hannibaland Fahrenkrug, 2004). Ipsilateral-eye influence, which ispredominantly inhibitory (Harrington, 1997; Tang et al.,2002), appears to come not only by way of this directpathway but also through an indirect pathway involvingthe contralateral IGL (Moore and Card, 1994; Harrington,1997). Our data indicate that although the bulk of retinalafferents to the IGL arise from mRGCs, at least a smallfraction of the input comes from non-mRGCs, in accordwith observations in the hamster (Morin et al., 2003).Inputs from non-mRGCs are a possible source for OFF-center or transient light responses in the IGL (Har-rington, 1997).

The boundaries of the IGL can be difficult to discern inconventional cell or fiber stains. It has been suggestedthat the nucleus be defined on the basis of its high con-centration of neurons expressing neuropeptide Y (NPY)and its projections to the SCN and contralateral IGL(Morin et al., 1992; Morin and Blanchard, 1995; Har-rington, 1997). The present results suggest that the pres-ence of dense input from mRGCs may be a useful supple-mentary defining feature of the IGL. The nucleus isunmistakable in X-gal-stained coronal sections throughthe middle of the geniculate complex, bounded by regionswhere fiber labeling is virtually absent (the LGd) or rela-tively sparse (the LGv). This zone of dense fiber labelingextends forward from both its caudoventral and rostralpoles into territory not traditionally included within theIGL. The caudoventral zone is traditionally includedwithin the LGv (Harrington, 1997) but has been suggestedto belong to the IGL in hamsters (Morin and Blanchard,1995). The extension of the labeled terminal field from therostral pole of the geniculate complex to the vicinity of thebed nucleus of the stria terminalis has been observed inseveral mammalian orders (Itaya et al., 1986; Johnson etal., 1988; Levine et al., 1991; Youngstrom et al., 1991;Cooper et al., 1993; Morin and Blanchard, 1995, 1999).The projection of mRGCs to this region was anticipated ina sense by the finding that this retinal terminal field isrelatively hypertrophic in the blind mole rat, along withother relatively enlarged components of the non-image-forming visual nuclei (Cooper et al., 1993). Functional,connectional, and neurochemical studies will be needed tocorroborate the provisional inclusion of these regionswithin the murine IGL.

As for the LGv, inputs from mRGCs clearly representonly a small fraction of its retinal afferents, in agreementwith observations in the rat (Hannibal and Fahrenkrug,2004). Few labeled fibers were present in the externaldivision of the nucleus, which represents the major reti-norecipient zone (Harrington, 1997). The densest labelingwithin the boundaries of the LGv as plotted by Paxinosand Franklin (2001) lies in two small regions that maywell be part of the IGL, judging from the pattern of fiberlabeling and its continuity with the IGL proper. The first,part of a possible rostral continuation of the IGL beyondthe geniculate complex (see above), lies just ventrolateralto the rostral pole of the LGd. In hamsters, this region hasbeen considered the anterior VLGNei by Harrington and

colleagues (Harrington and Rusak, 1989; Harrington,1997), but others have grouped it with the IGL on connec-tional and neurochemical grounds (Morin et al., 1992).The second, an apparent rostral extension of the cau-doventral pole of the IGL, appears to occupy the caudalpart of the internal division of the LGv.

Habenula and posterior limitans nucleus

The present findings demonstrate a substantial projec-tion from mRGCs to a small zone lying at the dorsolateralmargin of the lateral habenula. Retinal projections to thisregion were apparently first noted in the blind mole rat byCooper et al. (1993) and in the rat by Qu et al. (1996) andhave been observed subsequently in a variety of species(Morin and Blanchard, 1997; Reuss and Decker, 1997;Matteau et al., 2003). The functional role of the retinalprojection to this region is not understood. Generally, thehabenula is viewed as a relay from forebrain limbic cen-ters and the striatum to the midbrain (Herkenham andNauta, 1979). The lateral division of the lateral habenula,which lies closest to the retinal terminal zone, is impli-cated in polysynaptic pathways linking the entopeduncu-lar nucleus (internal segment of globus pallidus) to themidbrain and pontine reticular formation (Kim andChang, 2005). Light responses have been recordedthroughout the habenula (Zhao et al., 2005), but theseseem unlikely to be driven by direct retinal input becausemost habenular cells appear to have dendritic arbors toorestricted to reach the retinal terminal field (Kim andChang, 2005). Indirect pathways may instead be respon-sible (Zhao et al., 2005). In the cod, a teleost fish, melan-opsin is expressed in cells within the lateral part of thehabenula (Drivenes et al., 2003), and similar observationshave been made in the chick (Chaurasia et al., 2005). Thehabenula is embryologically associated with the pinealand appears to be connected to it synaptically (e.g., Ron-nekliev et al., 1980; Ronnekliev and Kelly, 1984; Mollerand Baeres, 2002). The mammalian pineal appears to bestrongly influenced by mRGCs, but this influence has beenthought to be very indirect, routed through a polysynapticcircuit including the SCN and sympathetic nervous sys-tem (for review, see Berson, 2003). There was nothing inour material to suggest the presence of melanopsin-expressing cells or mRGC axons in the pineal or its stalk.

In hamster, Morin and Blanchard (1997) consider theperi-habenular retinorecipient terminal field to be a ros-tral continuation of the posterior limitans nucleus, whichthey view as a component of what they have termed thesubcortical visual shell (Morin and Blanchard, 1998). Thisshell, comprising a constellation of functionally and con-nectionally interrelated retinorecipient nuclei, includesthe SCN, OPN, and IGL, all of which are targets ofmRGCs. The main body of the posterior limitans nucleuslies just lateral to the anterior pretectal nucleus, where itabuts the dorsal thalamus. In rats, mRGCs have beeninferred to project to this region (Hannibal and Fahrenk-rug, 2004; see also Pritchard et al., 2002), but a compara-ble pathway appears to be very weak in the mouse, judg-ing from our material.

OPN and other pretectal nuclei

Dense terminations of �-galactosidase-labeled fibers de-marcate a small portion of the retinorecipient pretectum.This field corresponds largely to the olivary pretectal nu-cleus as described in atlases and experimental studies in



rodents (Scalia, 1972; Scalia and Arango, 1979;Mikkelsen, 1992; Morin and Blanchard, 1997; Paxinosand Franklin, 2001). The OPN is a critical element in thepathway mediating the pupillary light reflex (Trejo andCicerone, 1984; Clarke and Ikeda, 1985; Young and Lund,1994), and the role of mRGCs in pupillary function is wellestablished (Lucas et al., 2001, 2003; Hattar et al, 2002,2003; Panda et al., 2003). Neurons in the OPN have tonic,irradiance-encoding light responses that resemble those ofthe intrinsically photosensitive ganglion cells that inner-vate them (Trejo and Cicerone, 1984; Clarke and Ikeda,1985). However, our data indicate that the OPN receivesconvergent input from non-mRGCs as well, as inferred forother rodents on the basis of retrograde tracing data(Gooley et al., 2003; Morin et al., 2003). Both mRGCs andnon-mRGCs are thus potential sources of the rod and/orcone signals known to reach the pupillomotor centers (Lu-cas et al., 2001; Hattar et al., 2003).

The OPN has been difficult to define in most prior stud-ies because it is indistinct in conventional cell and fiberstains and even in retinofugal fiber labeling studies, dueto its contiguity with other retinorecipient nuclei. Thespecificity of mRGC input suggests that this may be oneuseful defining characteristic of the nucleus. Other poten-tially specific markers for the OPN include parvalbuminimmunoreactivity, induction of the immediate early genec-fos by light, and selective labeling by axonally trans-ported adenoassociated virus injected into the eye(Okoyama and Moriizumi, 2001; Prichard et al., 2002;Gooley et al., 2004). All three of these markers, like theX-gal staining, reveal the hollow ovoid form of the rostralportion of the nucleus as viewed in coronal sections. Fur-ther study correlating these various markers would helpto define the boundaries of this nucleus. On present evi-dence, though, the OPN would appear to extend farthercaudally and laterally than commonly recognized, invad-ing territory often included in the posterior pretectal nu-cleus and nucleus of the optic tract and terminating justbelow the dorsal terminal nucleus of the accessory opticsystem.

The OPN may have functional roles beyond pupillarycontrol. For example, there are substantial ascending pro-jections from the OPN to the SCN, IGL, LGv, and lateralposterior-pulvinar complex of the thalamus (Weber et al.,1986; Mikkelsen and Vrang, 1994; Klooster et al., 1995;Moga and Moore, 1997; Morin and Blanchard, 1998). Theretinal projection to the OPN is robust in the blind molerat, despite the absence of any iris or ciliary body in its eye(Cooper et al., 1993).

Few mRGC axons extend beyond the confines of theOPN, as we have defined it, to other pretectal nuclei. Themost prominent target of such fibers is a region of the deeppretectum in and around the nucleus of the posteriorcommissure. This area exhibits light-dependent inductionof immediate early genes, as do many of the other targetsof mRGCs (Prichard et al., 2002). Some fibers continuebeyond this region to reach the rostral periaqueductalgray, a region that also receives input from the OPN andprojects to autonomic pupillary control centers (Kloosteret al., 1995; Klooster and Vrensen, 1998). In some mam-malian species, fibers continue along this trajectory toinnervate the dorsal raphe nucleus (Fite et al., 1999).However, in mouse this pathway is extremely weak, andexcept for a single fiber in one animal, we have not seen�-galactosidase-positive fibers in this nucleus.

Superior colliculus

Our data demonstrate an unequivocal, if relativelysparse, projection from mRGCs to the murine superiorcolliculus. Similar findings were reported by Morin et al.(2003) in the hamster. In the rat, several indirect lines ofevidence suggest a possible sparse collicular innervationby the axons of mRGCs (Gooley et al., 2003; Hannibal andFahrenkrug, 2004), but Gooley et al. (2003) were unable tolabel mRGCs by retrograde transport from the SC. Ourdata show that mRGC afferents terminate throughout thefull thickness of the superficial, retinorecipient collicularlaminae (Frost et al., 1986). The morphology of theseafferents did not match that of either of the two types ofretinotectal arbors so far described in mouse (Frost et al.,1986), presumably because they are a tiny minority ofretinotectal afferents and would have been unlikely toappear in a random sample of such fibers.

Although they are present throughout the collicularmap, terminations of mRGC axons are heaviest in themedial third of the colliculus (Fig. 10A), representing theupper visual field (Drager and Hubel, 1976). If mRGCsadhere to the retinotopic ordering characteristic of theretinocollicular projection, this would indicate a particu-larly heavy projection from mRGCs in the inferior retina.This seems to be at odds with reports indicating somewhathigher mRGC density in the superior than in the inferiorretina (Hannibal et al., 2002; Hattar et al., 2002; but seeMorin et al., 2003 and Sollars et al., 2003). However, theassumption of conventional topographic order may be un-warranted. Whereas the overall uncrossed retinotectalprojection is concentrated in a small collicular zone rep-resenting the binocular visual field (Godement et al.,1984), the uncrossed input from mRGCs is far more wide-spread. It clearly encroaches on the representation of thecontralateral temporal visual field, which cannot be seenby the eye from which these retinofugal fibers arise. Atleast one target of melanopsin RGCs, the IGL, lacks obvi-ous retinotopic order (Harrington, 1997), and it may bethat this unique ganglion cell population spurns retino-topy even when projecting to highly retinotopic targets.Neurons in the superficial collicular layers contribute toorienting movements toward visual targets by virtue oftheir descending influence on the deeper collicular layers(Isa, 2002). They may also participate in cortical visualmechanisms through ascending projections to thalamicvisual nuclei. Both of these functions call for considerablespatial fidelity, so a lack of retinotopy in the mRGC inputmay suggest that these ganglion cells play a modulatoryrole in the colliculus, rather than conveying informationabout target location or identity (see, e.g., Barash et al.,1998).

ACKNOWLEDGMENTS

We thank Motoharu Takao for technical help on thecholera toxin experiments, Mary Blue and Jonathan Brad-ley for assistance with histology and interpretation oflabeling distributions, Valerie Maine for technical help,and Yuqin Liang for assistance with genotyping.

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