Report. Melanopsin Regulates Visual Processing in the Mouse Retina. Results and Discussion

Transcription

1 Current Biology 16, , February 21, 2006 ª2006 Elsevier Ltd All rights reserved DOI /j.cub Melanopsin Regulates Visual Processing in the Mouse Retina Report Alun R. Barnard, 1 Samer Hattar, 2 Mark W. Hankins, 3, * and Robert J. Lucas 1, * 1 Faculty of Life Sciences Michael Smith Building University of Manchester Manchester M13 9PT United Kingdom 2 Department of Biology Johns Hopkins University Baltimore, Maryland Department of Visual Neuroscience Division of Neuroscience and Mental Health Faculty of Medicine Imperial College London St Dunstan s Road London W6 8RF United Kingdom Summary The discovery of melanopsin-dependent inner retinal photoreceptors in mammals [1, 2] has precipitated a fundamental reassessment of such non-image forming (NIF) light responses as circadian photoentrainment and the pupil light reflex. By contrast, it remains unclear whether these new photoreceptors also play a role in classical image-forming vision. The retinal ganglion cells that subserve inner retinal photoreception (iprgcs) project overwhelmingly to brain areas involved in NIF responses [3 6], indicating that, in terms of central signaling, their predominant function is non-image forming. However, iprgcs also exhibit intraretinal communication via gap junction coupling [7], which could allow them to modulate classical visual pathways within this tissue. Here, we explore this second possibility by using melanopsin knockout (Opn4 2/2 ) mice to examine the role of inner retinal photoreceptors in diurnal regulation of retinal function. By using electroretinography in wild-type mice, we describe diurnal rhythms in both the amplitude and speed of the retinal cone pathway that are a function of both prior light exposure and circadian phase. Unexpectedly, loss of the melanopsin gene abolishes circadian control of these parameters, causing significant attenuation of the diurnal variation in cone vision. Our results demonstrate for the first time a melanopsin-dependent regulation of visual processing within the retina, revealing an important function for inner retinal photoreceptors in optimizing classical visual pathways according to time of day. *Correspondence:m.hankins@imperial.ac.uk (M.W.H.); robert.lucas@ manchester.ac.uk (R.J.L.) Results and Discussion A Diurnal Variation in the Murine Cone ERG The day/night cycle in environmental illumination imposes a substantial variation in functional demands upon the visual system. The retinal response, recorded in a variety of vertebrate species, includes diurnal rhythms in several aspects of physiology and anatomy [8 25], which are thought to target regenerative events to appropriate times of day and optimize the twice daily transitions between scotopic (rod-based) and photopic (cone-based) vision. Despite their importance, the mechanisms underlying these events remain incompletely understood, especially in mammals. A potential new origin for the diurnal regulation of retinal physiology and anatomy comes from the recent discovery of inner retinal photoreceptors in mammals. A subset of retinal ganglion cells (RGCs) comprise a novel class of directly light-sensitive cells [26]. These intrinsically photosensitive RGCs (iprgcs) employ the photopigment melanopsin [27 29] and provide light information to brain areas responsible for the circadian and diurnal regulation of behavior and physiology [3 5]. In addition to such central functions, a local, intraretinal role for such nonrod noncone photoreceptors has been suggested in humans [30]. At present, direct evidence in support of such a function for melanopsin photoreceptors is lacking. However, iprgcs have been shown to maintain gap junction connections with other components of the ganglion cell layer (presumably amacrine and/or ganglion cells) in mice [7], providing a potential substrate for the intraretinal export of melanopsin signaling. The availability of Opn4 2/2 mice, in which iprgcs are no longer photoreceptive [2], provides a unique opportunity to test the hypothesis that inner retinal photoreceptors regulate retinal function. However, until now the adoption of such molecular genetic techniques to explore diurnal regulation of retinal function in mammals has been hampered by the lack of simple experimental protocols with which to study these events in mice. In order to overcome this problem, we first set out to explore time-of-day differences in the activity of visual pathways in mice by electroretinography. On the basis of preliminary investigations (data not shown), we chose to concentrate on the activity of events originating with cone photoreceptors. This was achieved by measuring responses to a series of bright white flashes (at 0.5 Hz) applied against a blue, rod-saturating background light. In all experiments, animals were exposed to at least 10 min of darkness prior to recording in order to ensure a standardized and essentially dark-adapted state for cone photoreceptor sensitivity. This approach elicited a characteristic electroretinogram (ERG) waveform from all subjects (Figure 1A), the major component of which was a large corneal positive deflection (b-wave). The generation of this b-wave has previously been attributed to the activity of ON bipolar cells [31, 32].

2 Current Biology 390 Figure 1. A Diurnal Variation in the Cone Pathway of Mice (A) Representative cone ERG traces from wild-type animals recorded at midday (upper trace) and midnight (lower trace) in response to a brief (10 ms) flash against a rod-saturating background following short-term (10 min) dark adaptation. A marked diurnal difference was apparent, quantifiable in terms of b-wave amplitude (baseline to peak) and implicit time (flash onset to peak) on a postfiltered (25 Hz low bandpass) trace (gray line). Scale bars equal 50 mv for y axis and 50 ms for time base. (B) Comparison of amplitude and implicit time (mean 6 SEM) between midday (n = 8) and midnight (n = 6) revealed that responses during the day were significantly larger (***p < 0.001, unpaired t test with Welch s correction) and faster (**p < 0.01) than those recorded at night. (C) After 10 min exposure to the rod-saturating light, ERG amplitude increased and implicit time decreased, indicative of light adaptation in the cone ERG. Nevertheless, diurnal differences in both parameters were retained (*p < 0.05, unpaired t tests with Welch s correction). We used this protocol to examine time-of-day differences in the activity of cone pathways by comparing the form of ERGs recorded from mice at midday and midnight. We found a large day/night difference in the cone ERG waveform (Figure 1), with both the amplitude and speed of the b-wave significantly enhanced during the day. As the bipolar cells, whose activity the b-wave reflects, are second order to the photoreceptors and an essential element in the transfer of information through the retina from photoreceptors to ganglion cells, these findings reveal an increase in the amplitude and speed of the inner retinal response to cone activation during the daytime. Consequently, these events would have the effect of optimizing photopic vision at a time of day when it is likely to be employed. To exclude the possibility that the strong diurnal variation in cone pathway activity that we observed was dependent on the specific characteristics of our ERG protocol, we continued to demonstrate similar time-ofday effects on the cone ERG under short-term (10 min) light adaptation (Figure 1C) and when a fast-flicker ERG was used to isolate cone pathways (see Figure S1 in the Supplemental Data available with this article online). The Cone Pathway Is Regulated According to Both Light History and Circadian Phase Animals sampled at midday differ from those at midnight in both their recent history of light exposure and the phase of their internal circadian clock. In order to determine the extent to which each of these features was responsible for the observed diurnal variation in cone ERG, we investigated the effects of extended dark exposure during the day and extended light exposure during the night (Figure 2A). Both treatments were able to partly, though not fully, reverse the diurnal variation in cone pathway function (Figures 2B and 2C). Thus, both recent light history and circadian phase contribute to the differences between midday and midnight. Interestingly, the precise nature of the interaction between these two drives differed for amplitude and implicit time (Figures 2B and 2C), implying a separation in the mechanisms regulating these two aspects of cone pathway function. Long-term exposure to light overrode any circadian influence to produce equally short implicit times at any time of day (Figure 2C). This suggests that light induces a strong positive drive for speed of response, perhaps through modulation of cellular excitability within the cones [33] and/or bipolar cells [34] or by altering the kinetics at the cone to bipolar cell synapse. The regulation of cone ERG amplitude was somewhat different. Darkness overcame circadian phase to produce equally suppressed responses at any time of day (Figure 2B). Thus, light appears to facilitate high amplitudes, suggesting the presence of a dominant dark-dependent inhibition that is interrupted by light. This may originate with an inhibitory effect of dark-adapted rods on cone pathway activity [35, 36]. Increasing evidence in mice indicates that rods can make direct synaptic connections with cone bipolar cells and that through gap junction coupling they may recruit the cone:bipolar cell synapse itself [37 41]. Long-term dark exposure may increase utilization of these recently revealed rod pathways in order to facilitate scotopic vision and, in the process, inhibit the activity of normal cone pathways. Under appropriate lighting regimes (extended darkness for implicit time and extended light for amplitude), an influence of circadian phase on both aspects of cone pathway activity was apparent. This indicates an important role for the circadian clock in regulating retinal function in mice. The origin of this circadian influence remains unclear. The mammalian retina contains an

3 Melanopsin Regulates Visual Processing 391 Figure 2. The Diurnal Variation in Cone ERG Is a Function of Both Light History and Circadian Phase (A) A graphical representation of experimental lighting conditions. Arrows indicate the approximate times (61 hr) of ERG recording under a 12 hr light:12 hr dark cycle (white and black bars indicate light and dark, respectively) and under extended exposure to darkness (day-dark) or light (night-light). In all cases, recordings were preceded by 10 min darkness to normalize short-term adaptation. (B) Light at night only partially restored b-wave amplitude (mean 6 SEM) to midday levels (one-way ANOVA p < ; post hoc Bonferroni s selected comparisons), indicating a role for both light history and circadian phase in determining this parameter. By contrast, darkness during the day fully suppressed amplitude to midnight levels. (C) Light at night fully restored daytime implicit time (mean 6 SEM; one-way ANOVA p < ; post hoc Bonferroni s selected comparisons). However, dark during the day did not fully suppress speed to midnight levels (p < 0.01 midnight versus day-dark; n = 6 and 5, respectively), indicating that both light history and circadian phase are important in determining this parameter. *p < 0.05, **p < 0.01, ***p < 0.001, and ns p > 0.05; n = 8 midday, 6 midnight, 7 night-light, and 5 day-dark. autonomous circadian oscillator [16, 42], which might drive these responses through purely intraretinal processes. Alternatively, both neural and humoral pathways have been described via which the primary circadian pacemaker in the suprachiasmatic nuclei could influence retinal rhythmicity [43, 44]. A Role for Melanopsin An important implication of our success in defining experimental protocols for measuring diurnal regulation of visual pathways at a functional level in mice is that it enables the power of genetic manipulation in this species to be harnessed in mechanistic analyses. In a first application of this approach, we used mice lacking melanopsin, the photopigment of iprgcs [27 29], to investigate the role of inner retinal photoreceptors in generating diurnal variations in retinal function. Opn4 2/2 mice retain normal numbers of iprgcs. However, while their morphology and central projections are unaffected by melanopsin loss, they are no longer photoreceptive [5]. Opn4 2/2 mice exhibited a clear diurnal variation in the cone ERG, with b-waves faster and larger during the day than at night (Figure 3A). However, the magnitude of these diurnal differences was reduced compared to wild-types. Thus, the difference in amplitude between midday and midnight was mv (mean 6 SEM) in wild-types but only mv (mean 6 SEM) in Opn4 2/2 (Figure 3A), while the difference in implicit time was reduced from ms in wildtype to ms in Opn4 2/2 (Figure 3A). We have previously presented evidence that a nonrod noncone photoreceptor transduces light history information for the diurnal regulation of the cone ERG in humans [30]. Consequently, we anticipated that the impaired diurnal variation in the cone ERG of Opn4 2/2 mice would be caused by a deficit in direct light regulation. To test this possibility, we examined the effects of long-term dark exposure during the day and long-term light exposure during the night in this genotype. To our surprise, light at night was able to completely restore midday values for amplitude and implicit time, while darkness during the day completely restored midnight values in both parameters (Figures 3B and 3C). Thus, while light history effects on cone pathway activity were retained in Opn4 2/2 mice, circadian control appeared to be absent. One implication of these data is that, in mice, the regulation of retinal function according to light history originates solely with rod and/or cone photoreceptors. This suggests that intrinsic cone events [33] and/or inhibitory influences from rods [35, 36] provide this aspect of retinal regulation. By contrast, iprgcs appear to fulfil an unexpected role in facilitating circadian regulation of the mouse retina. The mechanistic basis for this function will be an important area for future investigation. While it remains possible that melanopsin loss disrupts circadian control within the retina through effects on the central suprachiasmatic nucleus clock (which could in turn regulate retinal physiology via either endocrine or neuronal mechanisms), this seems unlikely. Opn4 2/2 mice show normal circadian rhythms in locomotor activity [45, 46], and although melanopsin loss does impair some aspects of circadian photoentrainment, these effects are not revealed under exposure to full light:dark cycles such as used in these experiments [45, 46] (and our

4 Current Biology 392 Figure 3. Opn4 2/2 Mice Show Reduced Diurnal Variation in Cone ERG (A) Representative cone ERG traces from Opn4 2/2 (dark lines) and littermate wild-type (gray lines) animals recorded at midday (upper panel) and midnight (lower panel) reveal an attenuated rhythm in the mutant genotype. Scale bars equal 50 mv for y axis and 50 ms for time base. (B and C) Both b-wave amplitude ([B]; mean 6 SEM) and implicit time ([C]; mean 6 SEM) were regulated according to light history, but not circadian phase in Opn4 2/2 mice, with significant differences (one-way ANOVA p < 0.001, post hoc Bonferroni s selected comparisons) between midday and midnight but not between midday and night-light or midnight and day-dark in either parameter. *p < 0.05, ***p < 0.001, and ns p > 0.05; n = 9 midday, 5 midnight, 9 night-light, and 6 day-dark. own data, not shown). Thus, substantial evidence confirms that central circadian rhythmicity is intact in Opn4 2/2 mice, suggesting that the effects of melanopsin loss observed here are a reflection of a local intraretinal interaction between iprgcs and circadian signaling. There are two ways in which inner retinal photoreception could be required for circadian regulation of the cone ERG. Perhaps the simplest explanation for our data is that iprgcs lie upstream of the retinal circadian clock and that a coherent circadian signal is possible only in the presence of melanopsin activity. Components of the circadian molecular clock are found in multiple cell types within the retina [42, 47 49], suggesting a relatively complex circadian organization in this tissue. The autonomy of individual cellular oscillators is largely unexplored, as are the nature and origin of entraining/ synchronizing signals. Should iprgcs alone provide photoentrainment of these heterogeneous retinal oscillators, the loss of melanopsin may result in desynchrony among this population [50] and the consequent abolition of coherent circadian signaling within the retina (Figure 4A). An alternative possibility is that a circadian clock gates the influence of melanopsin on the cone pathway by regulating the intraretinal output of iprgcs (Figure 4B), perhaps partly by modifying expression of melanopsin itself [51]. Such an arrangement would explain our findings only if light history modulates the cone ERG via at least two separate pathways: a direct route Figure 4. A Model for the Regulation of the Cone Pathway by Melanopsin and a Circadian Clock The effects of melanopsin loss on the circadian regulation of the cone pathway could be explained if melanopsin (shown in blue) lay either upstream (A) or downstream (B) of a circadian clock (shown in pink) in circadian control of the retina. Thus, iprgcs may facilitate circadian control by entraining local retinal clocks (A). In the absence of melanopsin, the clock cell population may become desynchronized, resulting in the loss of coherent circadian signaling. Alternatively, a clock may gate the effects of iprgcs on the cone pathway (B). In this case, the clock would have to facilitate both a light-dependent output at night and a dark-dependent output during the day in order to explain the timeof-day dependence of light and dark treatments and predict the loss of circadian control in Opn4 2/2 mice. In all cases, arrowheads represent stimulatory and bar-ends inhibitory relationships.

5 Melanopsin Regulates Visual Processing 393 originating in rod/cone photoreceptors that underlies the light history effects retained in Opn4 2/2 mice (Figures 3C and 3D), and a melanopsin-dependent pathway whose activity is regulated by a circadian clock. In this case, loss of melanopsin would result in the abolition of circadian but not light history control of the cone pathway. To date, the only described route by which melanopsin activity could either entrain local clocks or more directly influence elements of the cone pathway is via gap junction coupling within the ganglion cell layer [7]. The target cells for this coupling are unknown; however, they may include clock neurons and/or cells with the ability to modify inner retinal function, either inherently or perhaps through further coupling/signaling to other cell types. Dopaminergic amacrine (DA) cells represent a prime candidate for such targets as they are known to regulate retinal function through paracrine and synaptic action and to express elements of the molecular circadian clock [42, 52]. Conclusions We have described a robust diurnal modification of retinal function in mice in which the processing of visual information arising in cone photoreceptors is enhanced in terms of both speed and magnitude at midday compared to midnight. These modifications are determined by two complementary factors: assessments of longterm light history and the influence of a circadian clock. Genetic ablation of melanopsin blunts the rhythm in both amplitude and speed through the loss of circadian regulation of these parameters. Our results represent the first direct demonstration that melanopsin modulates the processes of image-forming vision and reveal the importance of inner retinal photoreceptors in optimizing retinal physiology according to time of day. Experimental Procedures Animals Adult (w80 days) melanopsin knockout (Opn4 2/2 [5]) and wild-type littermate mice (mixed strain, 129/sv and C57/bl6) were used for all experiments. Animals were bred and housed in the University of Manchester under a 12 hr light:12 hr dark cycle. We found that the magnitude and reliability of diurnal variations in cone ERG were dependent on standardizing the entraining light (fluorescent source, 160 mw/cm 2 at cage floor). Electroretinography Anesthesia was induced and maintained with ketamine (70 mg/kg) and xylazine (7 mg/kg). Mydriatics (tropicamide 1% and phenylephrine 2.5%) and hypromellose solution (0.3%) were used to dilate the pupil and retain corneal moisture. The recording electrode, comprising a silver chloride loop with integral saline-soaked cotton wick was placed concentrically centered on the corneal-scleral margin, by means of a micromanipulator. A similar silver chloride/saline wick electrode was attached to the ear as a reference, and a surfacecontacting, adhesive gel electrode was attached to the tail to act as ground. Electrode installation was conducted under dim red illumination (>650 nm, irradiance at the cornea <10 mw/cm 2 ) where necessary. Electrode leads were connected via a signal conditioner (Model 1902 Mark III, Cambridge Electronic Design, Ltd., CED, UK), in which the signal was differentially amplified (33000) and filtered (band-pass filter cut-off 0.5 to 200 Hz), and digitizer (Model 1401, CED) to a Windows PC that displayed and recorded the information (sampling rate 10 khz) using the Signal 2.15 Software (CED). Core body temperature was maintained (37ºC, by rectal probe) by a warm water reservoir heated by an electrical mat. Mice in all experimental groups were left in the dark for 10 min after electrode placement in order for cone photoreceptors of animals previously exposed to light to regain a high degree of dark-adapted sensitivity. Hence, cone photoreceptors in all groups attained a standard and essentially dark-adapted sensitivity state, irrespective of time of day or prior light exposure. This time also allowed electrodes to stabilize and preparations that displayed persistent baseline instability during this period to be rejected. Isolating the Cone Pathway Rod-Saturating Background Our primary method for isolating cone-driven activity was to measure responses to a bright white flash applied against a background light of sufficient intensity to saturate rods. Cone ERGs were recorded via a bright white flash (10 ms duration, peak irradiance 615 mw/cm 2 at the level of the cornea, Grass Model PS33 Photic Stimulator, Astro-Med, Inc., RI) presented at a 0.5 Hz stimulus frequency against a broad blue filtered (Grass blue filter, Astro-Med) rod-saturating background light (272 mw/cm 2 at the cornea; PM203 Optical Power Meter, Macam Photometrics Ltd, Livingston, UK), rendered uniform and full field by reflection within the light stimulator dome positioned close to the test eye. Flash ERGs were collected immediately after the commencement of background exposure. Twenty 2 s sweep repetitions were averaged for analysis, giving a total recording time of 40 s at 0.5 Hz. We also recorded ERGs after 10 min of exposure to the rodsaturating background light. In all groups, b-wave amplitude became larger and implicit time smaller with increasing time spent under the rod-saturating background illumination (Figures 1Band1C). These classical signs of light adaptation within the cone ERG [53, 54] occurred in all experimental groups and genotypes tested. Overall, light adaptation acted to reduce differences in speed and amplitude between experimental groups. Nonetheless, analysis of these lightadapted recordings supported all of the conclusions reported in this manuscript (Figure 1C and data not shown). Amplitude and implicit time of the b-wave were measured offline, by Signal 2.15 Software (CED) on records filtered (low bandpass 25 Hz) to exclude oscillatory potentials. All such analysis was undertaken blind, and recordings exhibiting large signal interference due to excessive noise or flash artifact were excluded. Implicit time of the b-wave was measured from the flash onset to time of peak. Because of the presence of flash artifacts and the sporadic appearance of a small a-wave, we chose to measure b-wave peak amplitude from the stable baseline immediately preceding the flash. Flicker ERG To confirm the results obtained with the rod-saturating background, we used a fast flickering stimulus under otherwise scotopic conditions to isolate the cone pathway in a different cohort of individuals. After short-term (10 min) dark adaptation, flicker ERGs were recorded in response to a 30 Hz stimulus presented against a dark background. Forty 500 ms epochs were averaged for analysis, giving a total recording time of 20 s at 30 Hz. The amplitude of the evoked ERG was measured manually and by Signal 2.15 (CED) power spectrum analysis. Experimental Light Conditions To investigate diurnal variation of the cone pathway, flash and flicker ERGs were recorded under four experimental conditions (Figure 2A): (1) midday, tested in middle of the light phase of a 12:12 hr LD cycle; (2) midnight, tested in middle of the dark phase; (3) day-dark, tested at midday but lights not turned on at the previous dawn, i.e., subjects maintained in darkness for the previous 18 hr; and (4) night-light, tested at midnight but lights not turned off at the previous dusk, i.e., subjects held in the light for the previous 18 hr. Supplemental Data The supplemental figure can be found with this article online at Acknowledgments This work was supported by the BBSRC. We would like to thank Dr. K.-W. Yau for generously providing our original Opn4 2/2 colony.

6 Current Biology 394 Received: October 17, 2005 Revised: December 20, 2005 Accepted: December 21, 2005 Published: February 21, 2006 References 1. Berson, D., Dunn, F., and Takao, M. (2001). Phototransduction by ganglion cells innervating the circadian pacemaker. Invest. Ophthalmol. Vis. Sci. 42, S Lucas, R.J., Hattar, S., Takao, M., Berson, D.M., Foster, R.G., and Yau, K.W. (2003). Diminished pupillary light reflex at high irradiances in melanopsin-knockout mice. Science 299, Gooley, J.J., Lu, J., Fischer, D., and Saper, C.B. (2003). A broad role for melanopsin in nonvisual photoreception. J. Neurosci. 23, Hannibal, J., Hindersson, P., Knudsen, S.M., Georg, B., and Fahrenkrug, J. (2002). The photopigment melanopsin is exclusively present in pituitary adenylate cyclase-activating polypeptidecontaining retinal ganglion cells of the retinohypothalamic tract. J. Neurosci. 22, RC Hattar, S., Liao, H.W., Takao, M., Berson, D.M., and Yau, K.W. (2002). Melanopsin-containing retinal ganglion cells: architecture, projections, and intrinsic photosensitivity. Science 295, Dacey, D.M., Liao, H.W., Peterson, B.B., Robinson, F.R., Smith, V.C., Pokorny, J., Yau, K.W., and Gamlin, P.D. (2005). Melanopsin-expressing ganglion cells in primate retina signal colour and irradiance and project to the LGN. Nature 433, Sekaran, S., Foster, R.G., Lucas, R.J., and Hankins, M.W. (2003). Calcium imaging reveals a network of intrinsically light-sensitive inner-retinal neurons. Curr. Biol. 13, Green, C.B., and Besharse, J.C. (2004). Retinal circadian clocks and control of retinal physiology. J. Biol. Rhythms 19, Anderson, F.E., and Green, C.B. (2000). Symphony of rhythms in the Xenopus laevis retina. Microsc. Res. Tech. 50, Li, L., and Dowling, J.E. (1998). Zebrafish visual sensitivity is regulated by a circadian clock. Vis. Neurosci. 15, Douglas, R.H., and Wagner, H.J. (1982). Endogenous patterns of photomechanical movements in teleosts and their relation to activity rhythms. Cell Tissue Res. 226, Chiu, J.F., Mack, A.F., and Fernald, R.D. (1995). Daily rhythm of cell proliferation in the teleost retina. Brain Res. 673, LaVail, M.M. (1976). Rod outer segment disk shedding in rat retina: relationship to cyclic lighting. Science 194, Goldman, A.I., Teirstein, P.S., and O Brien, P.J. (1980). The role of ambient lighting in circadian disc shedding in the rod outer segment of the rat retina. Invest. Ophthalmol. Vis. Sci. 19, Nir, I., Haque, R., and Iuvone, P.M. (2000). Diurnal metabolism of dopamine in the mouse retina. Brain Res. 870, Tosini, G., and Menaker, M. (1996). Circadian rhythms in cultured mammalian retina. Science 272, Bassi, C.J., and Powers, M.K. (1986). Daily fluctuations in the detectability of dim lights by humans. Physiol. Behav. 38, O Keefe, L.P., and Baker, H.D. (1987). Diurnal changes in human psychophysical luminance sensitivity. Physiol. Behav. 41, Roenneberg, T., Lotze, M., and von Steinbuchel, N. (1992). Diurnal variation in human retinal sensitivity determined by incremental thresholds. Clin. Vis. Sci. 7, Brandenburg, J., Bobbert, A.C., and Eggelmeyer, F. (1983). Circadian changes in the response of the rabbits retina to flashes. Behav. Brain Res. 7, Rosenwasser, A.M., Raibert, M., Terman, J.S., and Terman, M. (1979). Circadian rhythm of luminance detectability in the rat. Physiol. Behav. 23, Birch, D.G., Berson, E.L., and Sandberg, M.A. (1984). Diurnal rhythm in the human rod ERG. Invest. Ophthalmol. Vis. Sci. 25, Sandberg, M.A., Pawlyk, B.S., and Berson, E.L. (1986). Electroretinogram (ERG) sensitivity and phagosome frequency in the normal pigmented rat. Exp. Eye Res. 43, Hankins, M.W., Jones, R.J., and Ruddock, K.H. (1998). Diurnal variation in the b-wave implicit time of the human electroretinogram. Vis. Neurosci. 15, Hankins, M.W., Jones, S.R., Jenkins, A., and Morland, A.B. (2001). Diurnal daylight phase affects the temporal properties of both the b-wave and d-wave of the human electroretinogram. Brain Res. 889, Berson, D.M., Dunn, F.A., and Takao, M. (2002). Phototransduction by retinal ganglion cells that set the circadian clock. Science 295, Melyan, Z., Tarttelin, E.E., Bellingham, J., Lucas, R.J., and Hankins, M.W. (2005). Addition of human melanopsin renders mammalian cells photoresponsive. Nature 433, Qiu, X., Kumbalasiri, T., Carlson, S.M., Wong, K.Y., Krishna, V., Provencio, I., and Berson, D.M. (2005). Induction of photosensitivity by heterologous expression of melanopsin. Nature 433, Panda, S., Nayak, S.K., Campo, B., Walker, J.R., Hogenesch, J.B., and Jegla, T. (2005). Illumination of the melanopsin signaling pathway. Science 307, Hankins, M.W., and Lucas, R.J. (2002). The primary visual pathway in humans is regulated according to long-term light exposure through the action of a nonclassical photopigment. Curr. Biol. 12, Hood, D.C., and Birch, D.G. (1996). Beta wave of the scotopic (rod) electroretinogram as a measure of the activity of human on-bipolar cells. J. Opt. Soc. Am. A Opt. Image Sci. Vis. 13, Masu, M., Iwakabe, H., Tagawa, Y., Miyoshi, T., Yamashita, M., Fukuda, Y., Sasaki, H., Hiroi, K., Nakamura, Y., Shigemoto, R., et al. (1995). Specific deficit of the ON response in visual transmission by targeted disruption of the mglur6 gene. Cell 80, Elias, R.V., Sezate, S.S., Cao, W., and McGinnis, J.F. (2004). Temporal kinetics of the light/dark translocation and compartmentation of arrestin and alpha-transducin in mouse photoreceptor cells. Mol. Vis. 10, Shiells, R.A., and Falk, G. (2002). Potentiation of on bipolar cell flash responses by dim background light and cgmp in dogfish retinal slices. J. Physiol. 542, Goldberg, S.H., Frumkes, T.E., and Nygaard, R.W. (1983). Inhibitory influence of unstimulated rods in the human retina: evidence provided by examining cone flicker. Science 221, Arden, G.B., and Frumkes, T.E. (1986). Stimulation of rods can increase cone flicker ERGs in man. Vision Res. 26, Soucy, E., Wang, Y., Nirenberg, S., Nathans, J., and Meister, M. (1998). A novel signaling pathway from rod photoreceptors to ganglion cells in mammalian retina. Neuron 21, Hack, I., Peichl, L., and Brandstatter, J.H. (1999). An alternative pathway for rod signals in the rodent retina: rod photoreceptors, cone bipolar cells, and the localization of glutamate receptors. Proc. Natl. Acad. Sci. USA 96, Tsukamoto, Y., Morigiwa, K., Ueda, M., and Sterling, P. (2001). Microcircuits for night vision in mouse retina. J. Neurosci. 21, Deans, M.R., Volgyi, B., Goodenough, D.A., Bloomfield, S.A., and Paul, D.L. (2002). Connexin36 is essential for transmission of rod-mediated visual signals in the mammalian retina. Neuron 36, Protti, D.A., Flores-Herr, N., Li, W., Massey, S.C., and Wassle, H. (2005). Light signaling in scotopic conditions in the rabbit, mouse and rat retina: a physiological and anatomical study. J. Neurophysiol. 93, Witkovsky, P., Veisenberger, E., LeSauter, J., Yan, L., Johnson, M., Zhang, D.Q., McMahon, D., and Silver, R. (2003). Cellular location and circadian rhythm of expression of the biological clock gene Period 1 in the mouse retina. J. Neurosci. 23, Brandenburg, J., Bobbert, A.C., and Eggelmeyer, F. (1981). Evidence for the existence of a retino-hypothalamo-retinal loop in rabbits. Int. J. Chronobiol. 8, Doyle, S.E., Grace, M.S., McIvor, W., and Menaker, M. (2002). Circadian rhythms of dopamine in mouse retina: the role of melatonin. Vis. Neurosci. 19,

7 Melanopsin Regulates Visual Processing Ruby, N.F., Brennan, T.J., Xie, X., Cao, V., Franken, P., Heller, H.C., and O Hara, B.F. (2002). Role of melanopsin in circadian responses to light. Science 298, Panda, S., Sato, T.K., Castrucci, A.M., Rollag, M.D., DeGrip, W.J., Hogenesch, J.B., Provencio, I., and Kay, S.A. (2002). Melanopsin (Opn4) requirement for normal light-induced circadian phase shifting. Science 298, Gekakis, N., Staknis, D., Nguyen, H.B., Davis, F.C., Wilsbacher, L.D., King, D.P., Takahashi, J.S., and Weitz, C.J. (1998). Role of the CLOCK protein in the mammalian circadian mechanism. Science 280, Miyamoto, Y., and Sancar, A. (1998). Vitamin B2-based bluelight photoreceptors in the retinohypothalamic tract as the photoactive pigments for setting the circadian clock in mammals. Proc. Natl. Acad. Sci. USA 95, Namihira, M., Honma, S., Abe, H., Masubuchi, S., Ikeda, M., and Honmaca, K. (2001). Circadian pattern, light responsiveness and localization of rper1 and rper2 gene expression in the rat retina. Neuroreport 12, Carr, A.J., and Whitmore, D. (2005). Imaging of single lightresponsive clock cells reveals fluctuating free-running periods. Nat. Cell Biol. 7, Sakamoto, K., Liu, C., and Tosini, G. (2004). Classical photoreceptors regulate melanopsin mrna levels in the rat retina. J. Neurosci. 24, Gustincich, S., Contini, M., Gariboldi, M., Puopolo, M., Kadota, K., Bono, H., LeMieux, J., Walsh, P., Carninci, P., Hayashizaki, Y., et al. (2004). Gene discovery in genetically labeled single dopaminergic neurons of the retina. Proc. Natl. Acad. Sci. USA 101, Ekesten, B., Gouras, P., and Moschos, M. (1998). Cone properties of the light-adapted murine ERG. Doc. Ophthalmol. 97, Peachey, N.S., Goto, Y., al-ubaidi, M.R., and Naash, M.I. (1993). Properties of the mouse cone-mediated electroretinogram during light adaptation. Neurosci. Lett. 162, 9 11.