Brief light stimulation during the mouse nocturnal activity phase simultaneously induces a decline in core temperature and locomotor activity followed by EEG-determined sleep.

Light exerts a variety of effects on mammals. Unexpectedly, one of these effects is the cessation of nocturnal locomotion and the induction of behavioral sleep (photosomnolence). Here, we extend the initial observations in several ways, including the fundamental demonstration that core body temperature (T(c)) drops substantially (about 1.5°C) in response to the light stimulation at CT15 or CT18 in a manner suggesting that the change is a direct response to light rather than simply a result of the locomotor suppression. The results show that 1) the decline of locomotion and T(c) begin soon after nocturnal light stimulation; 2) the variability in the magnitude and onset of light-induced locomotor suppression is very large, whereas the variability in T(c) is very small; 3) T(c) recovers from the light-induced decline in advance of the recovery of locomotion; 4) under entrained and freerunning conditions, the daily late afternoon T(c) increase occurs in advance of the corresponding increase in wheel running; and 5) toward the end of the subjective night, the nocturnally elevated T(c) persists longer than does locomotor activity. Finally, EEG measurements confirm light-induced sleep and, when T(c) or locomotion was measured, show their temporal association with sleep onset. Both EEG- and immobility-based sleep detection methods confirm rapid induction of light-induced sleep. The similarities between light-induced loss of locomotion and drop in T(c) suggest a common cause for parallel responses. The photosomnolence response may be contingent upon both the absence of locomotion and a simultaneous low T(c).

Absence of normal photic integration in the circadian visual system: response to millisecond light flashes.

Light is the most prominent synchronizing stimulus for circadian rhythms. The circadian visual system responds in accordance with the energy content of photic stimuli longer than a few seconds. Here, as few as three flashes (2 ms each delivered to hamsters over 5 or 60 min at circadian time 19) elicited large phase advances. Ten or more flashes were required to induce FOS protein in the suprachiasmatic nucleus (SCN), and such induction occurred throughout the entire SCN, as well as outside the nucleus. High-density flash stimulation (0.5 s interflash interval) was ineffective, but response increased as the interval increased up to 4 s. In an irradiance response test, phase shifts appeared to be all-or-none with threshold irradiance between 140 and 1070 microW/cm2, implying lack of stimulus energy summation. Nevertheless, an irradiance ineffective when delivered as 10 flashes induced phase shifts when given as 100 flashes, but the response was substantially smaller than elicited by 10 flashes, each with approximately 1 log unit more irradiance. The results also show reduced sensitivity of flash-induced FOS response in the intergeniculate leaflet compared with the SCN, contrary to studies using longer light stimuli. Masking was robust and prolonged in response to 10 flashes. The data demonstrate that the circadian visual system responds markedly to brief, intense light stimuli without normal photic integration. This may involve a second input pathway different from that mediating the effects of longer, dimmer photic stimuli.

SCN organization reconsidered.

The SCN has long had organizational schemas imposed on it. In most, the SCN is dichotomized, with one region typically associated with the presence of vasopressin cells and the other associated with cells containing vasoactive intestinal polypeptide and certain afferent terminal fields. If assumed to be accurate, the schemas that have been intended to simplify and conceptually organize the known anatomy may actually interfere with the understanding of how various cell types and input pathways contribute to circadian rhythm regulation. This review describes inadequacies of existing schemas and notes several practical difficulties that undermine their usefulness. These include "static" versus "dynamic" anatomy, generalizations about SCN organization in relation to the plane or level of section, and the concept of differential density, all of which contribute to a view in which the SCN is substantially more complex than typically depicted in oversimplified line drawings. The need for accurate topographical description is emphasized.

Binocular contributions to the responsiveness and integrative capacity of the circadian rhythm system to light.

The retinohypothalamic tract (RHT), a monosynaptic retinal projection to the SCN, is the major path by which light entrains the circadian system to the external photoperiod. The circadian system of rodents effectively integrates or counts photons, and the magnitude of the rhythm phase response is proportional to the total energy of the photic stimulus. In the present studies, responsiveness to light and integrative capacity of the circadian system were tested in hamsters after reduction of retinal photoreceptor input by 50%. At CT 19, animals in constant darkness with or without unilateral retinal occlusion were exposed to 1 of 6 irradiances of 5-min white-light pulses ranging from 0.0011 to 70 microW/cm(2) or 5 white-light pulses of 0.6 microW/cm(2) with durations ranging from 0.25 to 150.0 min. Assessment of light-induced circadian rhythm phase response and Fos expression in the SCN by these animals revealed that a 50% reduction in input from photoreceptors stimulated directly with light caused a decrease in responsiveness to the longest duration and highest irradiance pulses presented. Despite this effect, both the magnitude of Fos induction in the SCN and phase-shift response remained directly proportional to the total energy in the photic stimuli. The results support the view that a reciprocal relationship between stimulus irradiance and duration persists despite the 50% reduction in retinal photoreceptor input. The mechanism of integration neither resides in the retina nor in the RHT.

Descending projections of the hamster intergeniculate leaflet: relationship to the sleep/arousal and visuomotor systems.

The intergeniculate leaflet (IGL), homolog of the primate pregeniculate nucleus, modulates circadian rhythms. However, its extensive anatomical connections suggest that it may regulate other systems, particularly those for visuomotor function and sleep/arousal. Here, descending IGL-efferent pathways are identified with the anterograde tracer, Phaseolus vulgaris leucoagglutinin, with projections to over 50 brain stem nuclei. Projections of the ventral lateral geniculate are similar, but more limited. Many of the nuclei with IGL afferents contribute to circuitry governing visuomotor function. These include the oculomotor, trochlear, anterior pretectal, Edinger-Westphal, and the terminal nuclei; all layers of the superior colliculus, interstitial nucleus of the medial longitudinal fasciculus, supraoculomotor periaqueductal gray, nucleus of the optic tract, the inferior olive, and raphe interpositus. Other target nuclei are known to be involved in the regulation of sleep, including the lateral dorsal and pedunculopontine tegmentum. The dorsal raphe also receives projections from the IGL and may contribute to both sleep/arousal and visuomotor function. However, the locus coeruleus and medial vestibular nucleus, which contribute to sleep and eye movement regulation and which send projections to the IGL, do not receive reciprocal projections from it. The potential involvement of the IGL with the sleep/arousal system is further buttressed by existing evidence showing IGL-efferent projections to the ventrolateral preoptic area, dorsomedial, and medial tuberal hypothalamus. In addition, the great majority of all regions receiving IGL projections also receive input from the orexin/hypocretin system, suggesting that this system contributes not only to the regulation of sleep, but to eye movement control as well.

Medial vestibular connections with the hypocretin (orexin) system.

The mammalian medial vestibular nucleus (MVe) receives input from all vestibular endorgans and provides extensive projections to the central nervous system. Recent studies have demonstrated projections from the MVe to the circadian rhythm system. In addition, there are known projections from the MVe to regions considered to be involved in sleep and arousal. In this study, afferent and efferent subcortical connectivity of the medial vestibular nucleus of the golden hamster (Mesocricetus auratus) was evaluated using cholera toxin subunit-B (retrograde), Phaseolus vulgaris leucoagglutinin (anterograde), and pseudorabies virus (transneuronal retrograde) tract-tracing techniques. The results demonstrate MVe connections with regions mediating visuomotor and postural control, as previously observed in other mammals. The data also identify extensive projections from the MVe to regions mediating arousal and sleep-related functions, most of which receive immunohistochemically identified projections from the lateral hypothalamic hypocretin (orexin) neurons. These include the locus coeruleus, dorsal and pedunculopontine tegmental nuclei, dorsal raphe, and lateral preoptic area. The MVe itself receives a projection from hypocretin cells. CTB tracing demonstrated reciprocal connections between the MVe and most brain areas receiving MVe efferents. Virus tracing confirmed and extended the MVe afferent connections identified with CTB and additionally demonstrated transneuronal connectivity with the suprachiasmatic nucleus and the medial habenular nucleus. These anatomical data indicate that the vestibular system has access to a broad array of neural functions not typically associated with visuomotor, balance, or equilibrium, and that the MVe is likely to receive information from many of the same regions to which it projects.

Functional analysis of the role of the median raphe as a regulator of hamster circadian system sensitivity to light.

The retinohypothalamic tract, a monosynaptic retinal projection to the suprachiasmatic nucleus (SCN), is the path by which light entrains the circadian system to the external photoperiod. Serotonergic neurons in the mesencephalic median raphe nucleus (MnR) also give rise to a major SCN afferent projection. The present study was designed to determine the extent to which MnR serotonergic projections regulate sensitivity of the circadian rhythm system to light. Serotonergic neurons in the MnR were destroyed by the direct application of the neurotoxin, 5,7-dihydroxytryptamine. Animals in constant darkness were given 5-min white light pulses at circadian time 19. Light intensity varied from 0.0011 to 70 microW/cm2. Assessment of rhythm phase response to light by lesioned and control animals revealed that animals lacking the MnR serotonergic projection are considerably more sensitive to light at high irradiances. The results are consistent with behavioral and physiological evidence implicating serotonin as an inhibitory modulator of the effects of light on circadian rhythmicity.

A Path to Sleep Is through the Eye

Light has long been known to modulate sleep, but recent discoveries support its use as an effective nocturnal stimulus for eliciting sleep in certain rodents. "Photosomnolence" is mediated by classical and ganglion cell photoreceptors and occurs despite the ongoing high levels of locomotion at the time of stimulus onset. Brief photic stimuli trigger rapid locomotor suppression, sleep, and a large drop in core body temperature (Tc; Phase 1), followed by a relatively fixed duration interval of sleep (Phase 2) and recovery (Phase 3) to pre-sleep activity levels. Additional light can lengthen Phase 2. Potential retinal pathways through which the sleep system might be light-activated are described and the potential roles of orexin (hypocretin) and melanin-concentrating hormone are discussed. The visual input route is a practical avenue to follow in pursuit of the neural circuitry and mechanisms governing sleep and arousal in small nocturnal mammals and the organizational principles may be similar in diurnal humans. Photosomnolence studies are likely to be particularly advantageous because the timing of sleep is largely under experimenter control. Sleep can now be effectively studied using uncomplicated, nonintrusive methods with behavior evaluation software tools; surgery for EEG electrode placement is avoidable. The research protocol for light-induced sleep is easily implemented and useful for assessing the effects of experimental manipulations on the sleep induction pathway. Moreover, the experimental designs and associated results benefit from a substantial amount of existing neuroanatomical and pharmacological literature that provides a solid framework guiding the conduct and interpretation of future investigations.

Retinal ganglion cell projections to the hamster suprachiasmatic nucleus, intergeniculate leaflet, and visual midbrain: bifurcation and melanopsin immunoreactivity.

The circadian clock in the suprachiasmatic nucleus (SCN) receives direct retinal input via the retinohypothalamic tract (RHT), and the retinal ganglion cells contributing to this projection may be specialized with respect to direct regulation of the circadian clock. However, some ganglion cells forming the RHT bifurcate, sending axon collaterals to the intergeniculate leaflet (IGL) through which light has secondary access to the circadian clock. The present studies provide a more extensive examination of ganglion cell bifurcation and evaluate whether ganglion cells projecting to several subcortical visual nuclei contain melanopsin, a putative ganglion cell photopigment. The results showed that retinal ganglion cells projecting to the SCN send collaterals to the IGL, olivary pretectal nucleus, and superior colliculus, among other places. Melanopsin-immunoreactive (IR) ganglion cells are present in the hamster retina, and some of these cells project to the SCN, IGL, olivary pretectal nucleus, or superior colliculus. Triple-label analysis showed that melanopsin-IR cells bifurcate and project bilaterally to each SCN, but not to the other visual nuclei evaluated. The melanopsin-IR cells have photoreceptive characteristics optimal for circadian rhythm regulation. However, the presence of moderately widespread bifurcation among ganglion cells projecting to the SCN, and projection by melanopsin-IR cells to locations distinct from the SCN and without known rhythm function, suggest that this ganglion cell type is generalized, rather than specialized, with respect to the conveyance of photic information to the brain.

Intergeniculate leaflet and ventral lateral geniculate nucleus afferent connections: An anatomical substrate for functional input from the vestibulo-visuomotor system.

The intergeniculate leaflet (IGL) has widespread projections to the basal forebrain and visual midbrain, including the suprachiasmatic nucleus (SCN). Here we describe IGL-afferent connections with cells in the ventral midbrain and hindbrain. Cholera toxin B subunit (CTB) injected into the IGL retrogradely labels neurons in a set of brain nuclei most of which are known to influence visuomotor function. These include the retinorecipient medial, lateral and dorsal terminal nuclei, the nucleus of Darkschewitsch, the oculomotor central gray, the cuneiform, and the lateral dorsal, pedunculopontine, and subpeduncular pontine tegmental nuclei. Intraocular CTB labeled a retinal terminal field in the medial terminal nucleus that extends dorsally into the pararubral nucleus, a location also containing cells projecting to the IGL. Distinct clusters of IGL-afferent neurons are also located in the medial vestibular nucleus. Vestibular projections to the IGL were confirmed by using anterograde tracer injection into the medial vestibular nucleus. Other IGL-afferent neurons are evident in Barrington's nucleus, the dorsal raphe, locus coeruleus, and retrorubral nucleus. Injection of a retrograde, trans-synaptic, viral tracer into the SCN demonstrated transport to cells as far caudal as the vestibular system and, when combined with IGL injection of CTB, confirmed that some in the medial vestibular nucleus polysynaptically project to the SCN and monosynaptically to the IGL, as do cells in other brain regions. The results suggest that the IGL may be part of the circuitry governing visuomotor activity and further indicate that circadian rhythmicity might be influenced by head motion or visual stimuli that affect the vestibular system.

Crossed and uncrossed retinal projections to the hamster circadian system.

The hamster suprachiasmatic nucleus (SCN), site of the circadian clock, has been thought to be equally and completely innervated by each retina. This issue was studied in animals that had received an injection of the tracer cholera toxin subunit B (CTb) conjugated to Alexa 488 into the vitreous of one eye, with CTb-Alexa 594 injected into the other. Retinal projections to the SCN and other nuclei of the circadian system were simultaneously evaluated by using confocal laser microscopy. Each retina provides completely overlapping terminal fields throughout each SCN. Although SCN innervation by the contralateral retina is slightly denser than that from the ipsilateral retina, there are distinct SCN regions where input from one side is predominant, but not exclusive. A dense terminal field from the contralateral retina encompasses, and extends dorsally beyond, the central SCN subnucleus identified by calbindin-immunoreactive neurons. Surrounding the dense terminal field, innervation is largely derived from the ipsilateral retina. The densest terminal field in the intergeniculate leaflet is from the contralateral retina, which completely overlaps the ipsilateral projection. Most nuclei of the pretectum receive innervation largely, but not solely, from the contralateral retina, although the olivary pretectal nucleus has very dense patches of innervation derived exclusively from one retina or the other. Retina-dependent variation in terminal field density within the three closely examined nuclei may indicate areas of specialized function not previously appreciated. This issue is discussed in the context of the melanopsin-containing retinal ganglion cell projections to several nuclei in the circadian visual system.

Light pulse duration differentially regulates mouse locomotor suppression and phase shifts.

Brief exposure of mice to nocturnal light causes circadian rhythm phase shifts, simultaneously inducing locomotor suppression, a drop in body temperature, and associated sleep. The exact nature of the relationship between these light-induced responses is uncertain, although locomotor suppression and phase shift magnitudes are related to stimulus irradiance. Whether stimulus duration has similar effects is less clear. Here, the relationship between stimulus duration and response magnitude was evaluated further using 100 µW/cm(2) white light-emitting diode pulses administered for 30, 300, 1200, or 3000 sec. The results show that, in general, shorter pulses yielded smaller responses and larger pulses yielded larger responses. However, the 300-sec pulse failed to augment locomotor suppression compared with the effect of a 30-sec pulse (44.7 ± 4.8 vs 40.6 ± 2.0 min) but simultaneously induced much larger phase shifts (1.28 ± 0.20 vs 0.52 ± 0.11 h). The larger phase shifts induced by the 300-sec stimulus did not differ from those induced by either the 1200- or 3000-sec pulses (1.43 ± 0.10 and 1.30 ± 0.17 h, respectively). The results demonstrate differential photic regulation of the two response types. Pulses ranging from 300 to 3000 sec produce equal phase shifts (present data); pulses ranging from 30 to 600 sec produce equal locomotor suppression levels. Greater suppression can occur additively in response to pulses of 1200 sec or more (present data), but this is not true for phase shifts. Nocturnal light appears to trigger a fixed duration event, locomotor suppression, or phase shift, with the latter followed by a light-refractory interval during which locomotor suppression can additively increase. The results also provide further support for the view that temporal integration of photic energy applies, at best, across a limited set of stimulus durations for both light-induced locomotor suppression/sleep and phase shift regulation.

Retinofugal projections in the mouse.

The laboratory mouse is increasingly a subject for visual system investigation, but there has been no comprehensive evaluation of this species' visual projections. Here, projections were visualized and mapped following intraocular injection of cholera toxin B subunit. Tissue was processed using standard procedures applied to 30 µm free-floating sections with diaminobenzidine as the chromogen. The mouse retina projects to ~46 brain regions, including 14 not previously described in this species. These include two amygdaloid nuclei, the horizontal limb of the diagonal band, the paraventricular hypothalamic nucleus, several visual thalamic nuclei, the paranigral nucleus, several pretectal nuclei, and the dorsal cortex of the inferior colliculus. Dense retinal patches were also observed in a narrow portion of the ipsilateral intermediate layer of the superior colliculus. The superior fasciculus of the accessory optic tract, which innervates the medial terminal nucleus, was also determined to be a terminal zone throughout its length. The results are compared with previous descriptions of projections from mouse intrinsically photoreceptive retinal ganglion cells, and with data from the hamster, Nile grass rat, and laboratory rat. The retinal projection patterns are similar in all four species, although there are many differences with respect to the details. The specific visual functions of most retinorecipient areas are unknown, but there is substantial convergence of retinal projections onto regions concerned with olfaction and audition.

Neuroanatomy of the extended circadian rhythm system.

The suprachiasmatic nucleus (SCN), site of the primary clock in the circadian rhythm system, has three major afferent connections. The most important consists of a retinohypothalamic projection through which photic information, received by classical rod/cone photoreceptors and intrinsically photoreceptive retinal ganglion cells, gains access to the clock. This information influences phase and period of circadian rhythms. The two other robust afferent projections are the median raphe serotonergic pathway and the geniculohypothalamic (GHT), NPY-containing pathway from the thalamic intergeniculate leaflet (IGL). Beyond this simple framework, the number of anatomical routes that could theoretically be involved in rhythm regulation is enormous, with the SCN projecting to 15 regions and being directly innervated by about 35. If multisynaptic afferents to the SCN are included, the number expands to approximately brain 85 areas providing input to the SCN. The IGL, a known contributor to circadian rhythm regulation, has a still greater level of complexity. This nucleus connects abundantly throughout the brain (to approximately 100 regions) by pathways that are largely bilateral and reciprocal. Few of these sites have been evaluated for their contributions to circadian rhythm regulation, although most have a theoretical possibility of doing so via the GHT. The anatomy of IGL connections suggests that one of its functions may be regulation of eye movements during sleep. Together, neural circuits of the SCN and IGL are complex and interconnected. As yet, few have been tested with respect to their involvement in rhythm regulation.

Nocturnal light and nocturnal rodents: similar regulation of disparate functions?

Investigators typically study one function of the circadian visual system at a time, be it photoreception, transmission of photic information to the suprachiasmatic nucleus (SCN), light control of rhythm phase, locomotor activity, or gene expression. There are good reasons for such a focused approach, but sometimes it is advantageous to look at the broader picture, asking how all the parts and functions complete the whole. Here, several seemingly disparate functions of the circadian visual system are examined. They share common characteristics with respect to regulation by light and, to the extent known, share a common input neuroanatomy. The argument presented is that the 3 hypothalamically mediated effects of light for which there are the most data, circadian clock phase shifts, suppression of nocturnal locomotion ("negative masking"), and suppression of nocturnal pineal function, are regulated by a common photic input pathway terminating in the SCN. For each, light triggers a relatively fixed interval response that is irradiance-dependent, the effective stimulus can be very brief light exposure, and the response continues to completion in the absence of additional light. The presence of a triggered, fixed-length response interval is of particular importance to the understanding of the circuitry and mechanisms regulating circadian rhythm phase shifts because it implies that the SCN clock response to light is not instantaneous. It also may explain why certain stimuli (neuropeptide Y or novel wheel running) administered many minutes after light exposure are able to block light-induced phase shifts. The understanding of negative masking is complicated by the fact that it can be represented as a positive change, that is, light-induced sleep, not just as a reduction in locomotion. Acute nocturnal light exposure also induces adrenal hormone secretion and a rapid drop in body temperature, physiological responses that appear to be regulated similarly to the other light effects. The likelihood of a common regulatory basis for the several responses suggests that additional light-induced responses will be forthcoming and raises questions about the relationships between light, SCN cellular anatomy, the molecular clockworks of SCN neurons, and SCN throughput mechanisms for regulating disparate downstream activities.

Neurons identified by NeuN/Fox-3 immunoreactivity have a novel distribution in the hamster and mouse suprachiasmatic nucleus.

The suprachiasmatic nucleus (SCN) has several structural characteristics and cell phenotypes shared across species. Here, we describe a novel feature of SCN anatomy that is seen in both hamster and mouse. Frozen sections through the SCN were obtained from fixed brains and stained for the presence of immunoreactivity to neuronal nuclear protein (NeuN-IR) using a mouse monoclonal antibody which is known to exclusively identify neurons. NeuN-IR did not identify all SCN neurons as medial NeuN-IR neurons were generally not present. In the hamster, NeuN-IR cells are present rostrally, scattered in the dorsal half of the nucleus. More caudally, the NeuN-IR cells are largely, but not exclusively, scattered inside the lateral and dorsolateral border. At mid- to mid-caudal SCN levels, a dense group of NeuN-IR cells extends from the dorsolateral border ventromedially to encompass the central subnucleus of the SCN (SCNce). The pattern is similar in the mouse SCN. NeuN-IR does not co-localize with either cholecystokinin- or vasoactive intestinal polypeptide, but does with vasopressin-IR in the caudal SCN. In the hamster SCNce, numerous cells contain both calbindin- and NeuN-IR. The distribution of NeuN-IR cells in the SCN is unique, especially with regard to its generally lateral location through the length of the nucleus. The distribution of NeuN-IR cells is not consistent with most schemas representing SCN organization or with terminology referring to its widely accepted subdivisions. NeuN has recently been identified as Fox-3 protein. Its function in the SCN is not known, nor is it known why a large proportion of SCN cells do not contain NeuN-IR.

Two components of nocturnal locomotor suppression by light.

In nocturnal rodents, millisecond light ("flash") stimuli can induce both a large circadian rhythm phase shift and an associated state change from highly active to quiescence followed by behavioral sleep. Suppression of locomotion ("negative masking") is an easily measured correlate of the state change. The present mouse studies used both flashes and longer light stimuli ("pulses") to distinguish initiation from maintenance effects of light on locomotor suppression and to determine whether the locomotor suppression exhibits temporal integration as is thought to be characteristic of phase shift responses to pulse, but not flash, stimuli. In experiment 1, locomotor suppression increased with irradiance (0.01-100 microW/cm( 2)), in accordance with previous reports. It also increased with stimulus duration (3-3000 sec), but interpretation of this result is complicated by the ability of light to both initiate and maintain locomotor suppression. In experiment 2, an irradiance response curve was determined using a stimulus series of 10 flashes, 2 msec each, with total flash energy varying from 0.0025 to 110.0 J/m(2). This included a test for temporal integration in which the effects of two equal energy series of flashes that differed in the number of flashes per series (10 vs 100), were compared. The 10 flash series more effectively elicited locomotor suppression than the 100 flash series, a result consistent with prior observations involving flash-induced phase shifts. In experiment 3, exposure of mice to an 11-h light stimulus yielded irradiance-dependent locomotor suppression that was maintained for the entire stimulus duration by a 100-microW/cm(2) stimulus. Light has the ability to initiate a time-limited (30-40 min) interval of locomotor suppression (initiation effect) that can be extended by additional light (maintenance effect). Temporal integration resembling that seen in phase-shifting responses to light does not exist for either phase shift or locomotor suppression responses to flashes or for locomotor suppression responses to light pulses. The authors present an alternative interpretation of data thought to demonstrate temporal integration in the regulation of phase shift responses to light pulses.

Targeted destruction of photosensitive retinal ganglion cells with a saporin conjugate alters the effects of light on mouse circadian rhythms.

Non-image related responses to light, such as the synchronization of circadian rhythms to the day/night cycle, are mediated by classical rod/cone photoreceptors and by a small subset of retinal ganglion cells that are intrinsically photosensitive, expressing the photopigment, melanopsin. This raises the possibility that the melanopsin cells may be serving as a conduit for photic information detected by the rods and/or cones. To test this idea, we developed a specific immunotoxin consisting of an anti-melanopsin antibody conjugated to the ribosome-inactivating protein, saporin. Intravitreal injection of this immunotoxin results in targeted destruction of melanopsin cells. We find that the specific loss of these cells in the adult mouse retina alters the effects of light on circadian rhythms. In particular, the photosensitivity of the circadian system is significantly attenuated. A subset of animals becomes non-responsive to the light/dark cycle, a characteristic previously observed in mice lacking rods, cones, and functional melanopsin cells. Mice lacking melanopsin cells are also unable to show light induced negative masking, a phenomenon known to be mediated by such cells, but both visual cliff and light/dark preference responses are normal. These data suggest that cells containing melanopsin do indeed function as a conduit for rod and/or cone information for certain non-image forming visual responses. Furthermore, we have developed a technique to specifically ablate melanopsin cells in the fully developed adult retina. This approach can be applied to any species subject to the existence of appropriate anti-melanopsin antibodies.