EXTRAOCULAR PHOTOTRANSDUCTION AND CIRCADIAN TIMING SYSTEMS IN VERTEBRATES

CHRONOBIOLOGY INTERNATIONAL, 18(2), 137–172 (2001)

REVIEW

EXTRAOCULAR PHOTOTRANSDUCTIONAND CIRCADIAN TIMING SYSTEMS

IN VERTEBRATES

Scott S. Campbell,* Patricia J. Murphy, and Andrea G. Suhner

Laboratory of Human Chronobiology, Department of Psychiatry,Weill Medical College of Cornell University, White Plains, New York

ABSTRACT

It is widely accepted that, for organisms with eyes, the daily regulationof circadian rhythms is made possible by light transduction through thoseorgans. Yet, it has been demonstrated repeatedly in recent years that ocularlight receptors that mediate vision, at least in mammals, are not the samephotoreceptors involved in circadian regulation. Moreover, it has been recog-nized for many years that circadian regulation can occur in organisms withouteyes. In fact, extraocular circadian phototransduction (EOCP) appears to bea phylogenetic rule for the vast majority of species. EOCP has been reportedin every nonmammalian species studied to date. In mammals, however, thestory is very different. This paper presents findings from studies that haveexamined specifically the capacity for EOCP in vertebrate species. In addi-tion, the literature addressing noncircadian aspects of extraocular phototrans-duction is briefly discussed. Finally, possible mechanisms underlying EOCPare discussed, as are some of the implications of the presence, or absence,of EOCP across phylogeny. (Chronobiology International, 18(2), 137–172,2001)

Key Words: Circadian rhythms; Extraocular; Extraretinal; Phototransduc-tion; Phylogeny.

*Corresponding author. Scott Campbell, Ph.D., Laboratory of Human Chronobiology, New YorkPresbyterian Hospital, 21 Bloomingdale Road, White Plains, NY 10605. E-mail: [email protected]

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Copyright 2001 by Marcel Dekker, Inc. www.dekker.com

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138 CAMPBELL, MURPHY, AND SUHNER

INTRODUCTION

The vertebrate form is remarkably transparent. Light passing through furand feather makes polar bears white and cardinals red; light passing throughthe skin enables an organism to synthesize vitamin D. And, experiments havedemonstrated that the transparency is not only skin deep. Light passing throughthe skulls of many nonmammalian vertebrates may be instrumental in the regula-tion of seasonal reproductive cycles (1–4). How much light can pass through theskull to illuminate deep brain structures? A light source of moderate intensity,directed at the human scalp, provides illumination at the level of the hypothala-mus adequate for reading (5). Recent evidence also suggests that, at least in somevertebrate species, light that reaches organs such as the heart and lungs may beinvolved in the daily synchronization of the endogenous clock to the 24h day(6). Of course, light that passes through the vitreous (from the Latin vitriummeaning glass) fluid of the eyes makes sight possible.

It is widely accepted that, for organisms with eyes, the daily regulation ofcircadian rhythms is also made possible by light transduction through those or-gans. Yet, it has been demonstrated repeatedly in recent years that ocular lightreceptors that mediate vision, at least in mammals, are not the same photorecep-tors involved in circadian regulation (7–10). Moreover, it has been recognizedfor many years that circadian regulation can occur in organisms without eyes.This is the case both for species that have not evolved complex ocular systems,such as the green flagellate Euglena and the jellyfish, and for laboratory animalsblinded experimentally. In fact, extraocular circadian phototransduction (EOCP)appears to be a phylogenetic rule for the vast majority of species. EOCP hasbeen reported in every nonmammalian species studied to date. In mammals,however, the story is very different. Only two studies in humans, and three morein laboratory rats, have reported a circadian response to nonretinal photic stimu-lation, while several have reported no such capacity.

In this article, we present findings from studies that have examined specifi-cally the capacity for EOCP in vertebrate species. In addition, we discuss brieflythe literature that addresses noncircadian aspects of extraocular phototransduc-tion. We examine what is known about the possible mechanisms underlyingEOCP, and we discuss some of the implications of the presence, or absence, ofEOCP across phylogeny. First, though, we examine some of the methodologicalissues that need to be considered when interpreting the results.

METHODOLOGICAL CONSIDERATIONS

Organ Extirpation

Although several general approaches have been used to examine the issueof EOCP, the majority of studies have used the traditional functional approach

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EOCP IN VERTEBRATES 139

in which the organ or system of interest is removed, and one determines whethera given physiological or behavioral response is changed or eliminated. With re-spect to the study of circadian phototransduction, it is not surprising that the firstorgan to draw the attention of researchers was the eye. However, the early dis-covery, in several species, that enucleation alone was not sufficient to eliminatecircadian responses to photic cues led investigators to excise a variety of organs,both singly and in various combinations, that have been hypothesized to mediatecircadian photoentrainment (e.g., Ref. 11). Based on their putative locations,most of the targets can be grouped in the category of “deep brain” photorecep-tors, although non–central nervous system structures have also been examined(12).

A major challenge and important limitation of the approach involves thedifficulty in keeping experimental animals alive and relatively healthy followingenucleation. Underwood and Menaker’s pioneering series of studies to examineEOCP in a variety of lizard species, for example, was hampered by the fact thatthe iguanid lizards survived surgery for an average of only 2 months (13). Assuch, animals died before experiments could be completed. By the same token,Menaker cited the difficulty in keeping blinded birds alive for the dearth of stud-ies following Benoit’s landmark demonstration of extraretinal light sensitivity inblinded ducks (reviewed in Ref. 1), and he selected sparrows for study specifi-cally because that species survived blinding reasonably well (14).

For animals that do survive, there is little question that experimentally in-duced blindness, pinealectomy, or both have a significant impact on behaviorand physiology well beyond the direct effects on circadian regulation. For exam-ple, the circadian rhythm of food intake is not only altered by enucleation, butalso feeding behavior frequently disappears entirely (see, for example, Ref. 15).Likewise, it has been shown that pinealectomy in fish and lizards can disruptcircadian rhythms and possibly photoneuroendocrine organization, confoundingthe examination of other putative extraocular receptors (16).

Less consequential noncircadian effects on physiology and behavior mayalso feed back on the “blinded” circadian timing system, further altering its usualfunctioning and/or the integrity of output measures. For example, in Syrian ham-sters, surgical blinding results in altered functioning of the hypothalamic-pitu-itary-adrenal and hypothalamic-pituitary-gonadal axes (17). Circulating levels ofthyroid, gonadal, and adrenal hormone levels are reduced compared to sightedanimals of the same species. In addition, blinding is associated with a reductionin serotonin production in hypothalamic extracts (18). Such widespread and sub-stantial neuroendocrine effects of blinding might also have an impact on thefunctioning of the circadian timing system and its response to photic input. It isalso quite likely that lesioning, enucleation, and transection of neural pathwaysmay result in scarring, as well as retrograde and anterograde degeneration. This,in turn, may have an impact on other systems and functions that may have adirect or secondary influence on circadian organization.

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Mutant Strains

The study of mutant strains can be viewed as the “organ extirpation” ofmolecular biology. Through naturally occurring or engineered genetic alteration,animal strains are bred for use in studies to examine the effects on physiologyand behavior of a particular mutation or gene knockout. The approach has beenhighly productive in the discovery and investigation of nonvisual ocular circadianphotoreception. In a series of studies using a strain of mice characterized by avirtual absence of rods and cones, Foster and coworkers showed convincinglythe existence of circadian photoreceptors distinct from those responsible for vi-sion (8–10). More recently, rats with a genetic mutation that blocks developmentof the ocular system ventral to the optic chiasm have been used to examine theexistence of extraocular circadian photoreception (19,20).

Yet, there is some evidence to suggest that such mutant strains may sufferfrom deficits in the circadian system well beyond blindness. For example, a ge-netically anophthalmic mouse strain (ZRDCT-An) is characterized by abnormali-ties in several brain areas, including the suprachiasmatic nucleus (SCN) (21).Moreover, heterozygous mice of this anophthalmic strain, with normal visualperception, are nevertheless unable to entrain to a light-dark cycle (22). Thus, aswith organ excision, genetic mutation may potentially have an impact on noncir-cadian physiology and behavior that may feed back directly or indirectly on thecircadian timing system. For example, Scheuch and coworkers found wide inter-individual variation in the circadian activity cycles of their anophthalmic micethat could not always be traced to neuroanatomical abnormalities (21).

Natural Blindness

Distinctive to the study of EOCP in humans is the study of individuals withcongenital blindness or blindness from accidents or disease. It is unclear, how-ever, to what extent findings in such individuals can be generalized to the circa-dian systems of sighted populations. By way of analogy, it has been demon-strated that many congenitally blind individuals can effectively utilizeecolocation to orient themselves in space. In one study, for example, it was foundthat one blind man could detect 4-cm and 5-cm disks almost perfectly fromalmost a meter away (23). Similarly, anophthalmic mice have a more sensitiveauditory system than normal mice, presumably as a measure to compensate fortheir blindness (22).

That such cross-modal plasticity in function is associated with structuralchanges in neuronal pathways has been demonstrated by electrophysiologicaland imaging studies (24–27). In both early- and late-onset blindness, a reorgani-zation, or rewiring, of cortical pathways occurs (24). In degenerative diseasessuch as nondecussating retinal-fugal fiber syndrome, in which profound “miswir-ing” of visual pathways occurs, nonvisual retinal projections might be abnormal

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as well. In short, the structure and function of the central nervous system differsin blind and sighted humans, making generalization of any effects of photic inputin these disparate populations problematic.

It seems likely that other physiological systems of congenitally blind indi-viduals may undergo similar functional modifications. Indeed, the generally con-sistent finding that the free-running circadian period of a totally blind individualaverages close to 25h (e.g., 28), whereas sighted subjects average very close to24h, suggests that there may be fundamental differences in the manner in whichthe endogenous clock functions under the “constant conditions” of total blind-ness.

Environmental Factors/Stimulus Characteristics

The intensity and duration of a phase-shifting stimulus, or the spectral com-position of a putative entraining agent or zeitgeber, may have a significant impacton the outcome of a given investigation. It is conceivable, for example, that aphotic stimulus with an intensity sufficient to induce phase shifts in sighted ani-mals may be too weak to achieve the same result in blinded animals. Underwoodreported this effect in some, but not all, species of lizards studied and calculatedthat the threshold of intact animals was approximately 20 times lower than thethreshold for blinded lizards. For investigations using poikilotherms, it is impor-tant to eliminate the possibility that entrainment or phase shifting is not inducedby temperature cycles associated with the experimental setup.

EXTRAOCULAR CIRCADIANPHOTOTRANSDUCTION: THE DATA

The following section is divided into the five classes of vertebrate species.In addition to the body of literature reviewed here, there is substantial literaturethat addresses the issue of EOCP in nonvertebrate species. While a review ofthat literature is beyond the scope of this article, there are several reviews of thatspecific topic available (29,30).

Fish

In a series of experiments using the lake chub (Couesius plumbeus), Kava-liers (31,32) examined the role of extraretinal photoreceptors in mediating en-trainment of the circadian activity rhythm, as well as acute response to pulses oflight (32). As with intact fish, blinded fish showed initiation of activity, or in-creases in the amplitude of ongoing locomotor activity, in response to a series of30-second light pulses. However, blinded fish were sensitive only to light be-

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tween 568 and 742 nm, whereas intact fish showed responsiveness across theentire visible spectrum, with maximum responsiveness at 538–568 nm. In addi-tion, light of significantly higher intensity was required to induce the responsein blinded fish. Pinealectomies in the blinded fish did not alter the spectral sensi-tivity (relative to blinding alone), but the light intensity required to induce activ-ity was significantly greater.

Both pinealectomized fish and blinded fish also showed entrainment to a12h:12h light-dark cycle (LD 12:12), qualitatively similar to that exhibited byintact fish. However, when compared to intact animals, the blinded fish requiredup to 1 week longer to entrain to the light-dark cycle. Blinded pinealectomizedfish also showed entrainment to the light-dark cycle, although these animals re-quired even longer to establish stable phase relationships with the light-dark cy-cle. Relative to that required for the elicitation of an acute behavioral response,all animals required a higher light intensity to achieve entrainment. For a givenexperimental group (i.e., intact, blinded, pinealectomized, blinded pinealectom-ized), there was no difference in action spectra required for acute response versusentrainment.

Using the circadian rhythm of skin pigment changes as a dependent mea-sure, Reed examined the effects of blinding in another teleost, the pencil fish(Nannostomus beckfordi anomalus) (33). He reported no differences between in-tact and blinded fish when exposed to a light-dark cycle. Moreover, when thelight period was extended into the evening hours, both normal and blinded fishcontinued to exhibit daytime coloring until the room was darkened.

Similar results were reported for the nocturnally active European eel (An-guilla anguilla) (15). Neither bilateral enucleation nor bilateral enucleation com-bined with pinealectomy had any impact on the capacity of the animals to reen-train to a reversed light-dark cycle (LD 12:12, 680:0 lux). However, an opaquecover over the skull caused the animals to become arrhythmic. As in lake chub,blinded eels maintained in constant darkness (DD) were also responsive to acutelight stimuli by exhibiting characteristic backward swimming behavior, but onlywhen the light was directed toward the head. The response was qualitativelysimilar to that observed in intact animals, although response latency was 3 to 10times longer in the blinded eels.

Amphibians

Studying activity rhythms of a nocturnal amphibian, the slimy salamander(Plethodon glutinosus), Adler reported no differences in the phase-shifting ca-pacity of light between blinded and intact animals (34). Moreover, both groupsshifted activity onset to coincide with successive 1h delays of an artificial light-dark cycle (LD 9:15), indicating that the animals were not simply free runningwith a period of exactly 24h. A subsequent experiment sought to localize thepossible site(s) responsible for EOCP in these animals. Various portions of the

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head were covered with opaque paint, and reaction times were recorded follow-ing activation of a fluorescent light source. Although behavioral reactions wereobserved in all conditions, including when the head was completely painted, themost rapid reaction times were recorded when the anterior part of the brain wasexposed. This led the authors to conclude that the putative extraretinal photore-ceptors were concentrated in, but not unique to, that portion of the brain.

Demian and Taylor also reported apparent EOCP in a diurnal newt (Not-ophthalmus viridescens) (35). An enucleated animal exhibited statistically differ-ent activity patterns in continuous light (LL) than in light-dark conditions, al-though the activity record differed in several respects from that observed in anintact animal: The intact animal showed transients when introduced into a light-dark cycle, but the blinded animal did not; the activity periods of the blindedanimals were less concentrated, but of higher amplitude, compared to the intactanimal. Because individual animals were studied, any generalizations must bemade with caution.

Reptiles

In one of the most thorough examinations of EOCP, Underwood and Men-aker assessed the entrainability of locomotor activity rhythms in seven speciesof lizard, representing three lizard families, under several lighting conditions andbefore and after blinding (13). Both diurnal and nocturnal species were repre-sented. Removal of the lateral eyes did not prevent entrainment of activityrhythms to a light-dark cycle (LD 12:12, 30–50:0 lux). This was the case forevery animal studied (n = 44). As with lake chub, the activity cycles of allblinded animals followed a shift in the onset and offset times of light. Moreover,control experiments showed that the low-amplitude temperature changes associ-ated with the artificial light-dark cycle were not responsible for the entrainment.

In an effort to identify the possible locus of the observed EOCP, Under-wood (13) excised the parietal eye and the pineal organ, two structures that hadbeen identified previously as having well-organized photoreceptors. Yet, com-plete removal of those organs did not prevent entrainment to the light-dark cyclein two of the species tested. Removal of the pineal in a blinded third species thatlacks a parietal eye (the Western banded gekko) produced the same result. Thus,the mechanism underlying EOCP in these lizard species remains unknown.Based on earlier studies using juvenile rats (see below), the author next targetedthe Harderian gland as a possible locus of EOCP. Yet, blinded, pinealectomized,and parietalectomized Texas spiny lizards exhibited entrainment to the imposed24h light-dark cycle subsequent to having the Harderian glands removed.

The author (13) then attempted to localize the brain areas responsible forEOCP by shielding areas of the brain with black lacquer applied to the surfaceof the head or carbon black injected under the skin of the head. Three of the 4blinded iguanid lizards tested remained entrained after their heads were painted.

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In contrast, blinded Western banded gekkos subjected to carbon black injectionexhibited free-running activity rhythms. Despite these mixed results, the authorconcluded that “the [extraretinal photoreceptor] in lizards, as in amphibians andbirds, is located in the brain” (13, p. 203).

Underwood (13) went on to show that the mechanism responsible forEOCP is capable of discriminating variations in light intensity similar to thatcharacterizing ocular mechanisms. Blinded lizards were able to entrain to a pho-toperiod with a light intensity of 0.6 lux. Under these conditions, they exhibiteda characteristic phase angle between activity and lights off of just under 5h. Withan identical photoperiod, but an increase in light intensity to 50 lux, the activityrhythms assumed a phase angle of 0 relative to lights off. In addition, the lizardsshowed more variability in activity onset under 0.6 lux when compared to the50-lux condition, providing further evidence of intensity discrimination by theextraretinal photoreceptors.

Kavaliers and Ralph found similar results in an eighth reptilian specieslacking a pineal organ, the American alligator (Alligator mississippensis) (16).Rest-activity rhythms were measured in young animals fitted with opaque, ace-tate caps placed over the lateral eyes. When the animals were also fitted withopaque caps over the entire dorsal surface of the head, they showed no entrain-ment response to a light-dark cycle. However, with only the eyes shielded, ani-mals showed entrainment to an LD 12:12 cycle. The exact time required forstable entrainment was dependent on the ratio of light to dark and the phase atwhich the entraining stimulus was introduced, but did not differ from that ofsighted animals. The author proposed several sites for the location of photorecep-tors responsible for entrainment (diencephalon, paraphysis, third ventricle) andspeculated that they may be part of an “integrated photoendocrine system” (36).

Birds

The first study to examine the possible existence of EOCP in birds waspublished by Menaker (14). The perching behavior of bilaterally enucleated spar-rows (Passer domesticus) was studied continuously, in several different photope-riods, across periods ranging from several weeks to several months. Figure 1shows the perching record of one such animal and demonstrates dramatically thecapacity for EOCP in this species. Following a 6-day period in constant darkness,during which the bird free ran at a period of slightly longer than 24h, a lightcycle (LD 12:12, 500:0 lux) was presented. After 3 to 4 days of transient activitypatterns, the animal became entrained to the 24h light cycle, with activity onsetoccurring shortly before daily lights on. On day 40, the light portion of the light-dark cycle was extended by 6h (LD 18:6); in response, the blinded bird extendedits daily activity bouts to correspond to the lengthened light interval. Whenpower outages in the laboratory caused the timing of the light cycle to be delayedby 9.5h, the sparrow shifted its activity to follow the newly phased light cycle.

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Figure 1. Continuous perching activity record (155 days, double plotted) of a bilaterally enucle-ated sparrow. The various light regimens (500 lux) employed throughout the study (see text) areshown schematically in the upper portion of the figure. Note that when a light cycle is present, itcontrols the phase and period of the activity, as well as the duration of activity per day (compareLD 12:12 with LD 18:6). Hour 0 = midnight Central Standard Time. Blank spaces are due tooccasional pen failure.

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On day 117, the animal was returned to constant darkness, in which it exhibiteda free-running activity rhythm with a characteristic period.

Each of the 53 birds studied under similar conditions showed entrainmentto the light-dark cycles imposed, and subsequent experiments to rule out alterna-tive zeitgebers, such as cyclic changes in ambient temperature and even second-ary entrainment by entrained external parasites on the birds, confirmed that en-trainment was achieved by EOCP. One effort to reduce the possible effects ofambient temperature involved replacing the 500-lux fluorescent light source withan electroluminescent panel that produced green light with an intensity of only0.1 lux. Whereas all sighted birds entrained to this light cycle (LD 12:12, 0.1green:0), only half of the blinded animals showed entrainment.

As with the lizard studies by Underwood (11,13,37) and Kavaliers’ studiesof lake chub (31,32), then, more intense light appears to be required for EOCP,at least when the eyes are not present. (That is, it is unclear from these datawhether more intense light is required for EOCP per se, or whether removal ofthe eyes increases the threshold for photic entrainment of the circadian clock.)Also, as with lizards and fish, enucleated sparrows required longer to entrain toa light-dark cycle than did sighted birds, and under some lighting conditions, thephase angle of entrainment differed between intact and blinded birds. Finally, aswith lizards, intact birds became arrhythmic in constant light (LL) of 500 lux orhigher, whereas blinded birds remained rhythmic even in constant light of 2000lux.

Mammals

One of the earliest studies to address EOCP in mammals, at least indirectly,was conducted by Hunt and Schlosberg (38). Since at that time the very role oflight in the synchronization of circadian rhythms was still unclear, the focus ofthe study was a more general examination of the influence of illumination onactivity rhythms of albino rats. After establishing that normal rats responded toa reversal of artificial light-dark cycles by shifting their activity rhythms to con-form to the new cycle, the authors blinded 5 of the animals “as a final check” toensure that it was, indeed, the light-dark cycle, and not temperature or socialfactors, that induced the reversal in activity rhythms. Their assumption, then, wasthat light could affect activity rhythms only via the eyes. That assumption ap-peared to be confirmed since the blinded animals were unaffected by a reversalof the light-dark cycle.

Klein and Weller also concluded that extraocular photoreception was notinvolved in circadian regulation in adult rats. This was based on the findings that(1) blinded rats showed no suppression of serotonin N-acetyltransferase in re-sponse to a light pulse, and (2) they continued to show circadian rhythmicity inN-acetyltransferase output when exposed to continuous lighting (39).

A study of EOCP in newborn rats produced opposite results (40). The focus

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of the study was to examine the development of circadian rhythms in pinealgland serotonin content as a function of ontogeny. Assaying serotonin content in6- and 12-day-old rats at two times during the circadian day (8h into the lightperiod and 4h into the dark period), the authors established that pineal serotoninconcentrations showed clear day-night changes consistent with those observed inadult rats. Moreover, exposure to 4 additional hours of light prevented the noc-turnal decline in serotonin in the same manner observed in adult animals.

In an effort to clarify whether the newborn rats were receiving the light cuethrough their still-closed eyelids, through communication with the mother, or byan extraretinal pathway, a group of 11-day-old rats were bilaterally enucleated,exposed to one light-dark cycle, and subsequently killed (40). As with sightedyoung rats, these animals showed a “circadian rhythm in pineal serotonin” identi-cal to normal controls. A group of blinded rats exposed to 4 additional hours oflighting showed significantly higher serotonin levels than the blinded rats left indarkness. Yet, placing an opaque hood over the heads of the blinded animalscompletely abolished the effect of additional lighting, indicating that lightreached the pineal by a direct route through the skull.

Using a similar experimental design, Wetterberg and coworkers essentiallyconfirmed these findings (41). However, instead of hooding their animals, theseinvestigators removed the Harderian glands of the blinded rats and achieved thesame result, suggesting that the glands were the site of the extraretinal receptorresponsible for entrainment by light in juvenile rats.

Employing food intake rhythms as a measure of the circadian timing sys-tem, Groos and van der Kooy reported a failure to entrain to a LD 12:12 cycle of300–400 lux in 20 male rats blinded by binocular enucleation (42). In a secondexperiment, designed to increase the likelihood that light of sufficient intensitywould reach putative photoreceptive brain sites, the authors performed craniot-omies on 5 rats, exposing over 28 mm2 of brain surface, which was sealed withtransparent plastic. The animals were then exposed to 1h pulses of white light(1600 µW/cm2) at various phases of their circadian cycles. Such light pulsesfailed to generate a coherent phase-response curve.

While the results of the two experiments are quite convincing, there werecritical limitations in the experimental design. For example, the rats in the phase-shifting study were anesthetized prior to receiving the 1h light pulses. There issubstantial evidence that anesthesia alters physiological responses to a wide vari-ety of manipulations (e.g., Refs. 43 and 44). Although the authors acknowledgethat anesthesia could have reduced the sensitivity of the putative photoreceptors,an appropriate control condition was not included. Rather, they simply cite anearlier finding that showed an absence of electrical activity in the hypothalamusof blinded rats in response to a light stimulus.

Nelson and Zucker used a more naturalistic approach to examine EOCP intwo rodent species, one nocturnal and the other diurnal (45). Their hypothesiswas similar to that of Groos and van der Kooy (42), that light of an intensitylikely to be encountered in nature may impinge on deep brain photoreceptors,

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whereas light typically used in laboratory settings may not. Following enucle-ation, 13 night-active grasshopper mice (Onychomys leucogaster) and 8 day-active golden mantled ground squirrels (Spermophilus lateralis) were exposed tonatural daylight for 3h to 7h each day over a period of 3 months, during whichwheel-running activity was monitored. In contrast to control animals, the blindedanimals did not entrain to the natural light-dark cycle. They continued to freerun with a period characteristic of the species. Interestingly, the investigatorswere unable to entrain the blinded animals to nonphotic cues, although at leastone other species of diurnal rodent entrains to such signals (46).

Perhaps because these early studies reported consistently negative results,few investigators have examined EOCP in mammals in the past 15 years. In1999, several laboratories examined the issue with conflicting results. Yamazakiand coworkers found no evidence of EOCP in Syrian hamsters studied in eitherof two similar experimental conditions (47). Following entrainment to a reversedlight-dark cycle, animals were enucleated, their backs were shaved, and theywere exposed to natural sunlight at times when either a phase delay or a phaseadvance would be expected. Six animals received 1h exposures (60,000–80,000lux) at times when either a phase delay or a phase advance would be expected,and 6 received a 3h exposure during the presumed phase-delay portion of thecircadian cycle.

Based on comparisons of running wheel activity measured for several daysbefore and after light exposure, the authors reported no phase change in responseto the 1h exposures and “no (or very slight) phase changes” following the 3hexposure (47). In addition, they reported no evidence of melatonin suppressionin response to sunlight exposure (40,000 lux) in a separate group of 4 enucleatedanimals, whereas intact hamsters exhibited strong suppression.

The authors noted that, in the study in which animals were exposed to a 3hlight pulse (47), both enucleated animals and controls exhibited robust and exten-sive bouts of activity immediately following the light exposure interval. Thesmall phase shifts observed in the blind hamsters were attributed to this nonpho-tic stimulus. Whether the light exposure was instrumental in the increased motoractivity in either the blinded or the control animals was not addressed, but anumber of studies using nonmammalian species have reported similar results(see sections above).

Using a similar experimental design, but an artificial light source (the sameone used on humans in the study in Ref. 48), Meijer and colleagues also reportedno effect of extraocular light on the circadian systems of hamsters (49). Afterrelease into constant darkness for 7 days, intact and blinded animals were ex-posed to a 15-minute light pulse of 100 lux (sighted hamsters), or they wereplaced on a light-emitting pad for either 30 minutes or 3h (blinded hamsters) attimes in the circadian cycle when either phase advances or delays would beexpected. Compared to intact animals, which exhibited characteristic phase-shift-ing responses to light, the blinded animals showed no such evidence of a circa-dian response.

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Using a different approach and a different nocturnal rodent, Jagota and co-workers arrived at the opposite conclusion (20). The investigators studied con-genitally anophthalmic mutant rats under various light conditions to assess theeffect of the light conditions on pineal rhythms. First identified among the labo-ratory’s breeding stock, these animals lack a complete visual system; they haveventricular dilation and a total absence of eyeballs and the corresponding opticnerve up to the optic chiasm. The mutation is not associated with any othercongenital abnormalities, and preliminary analyses by the authors revealed a nor-mal circadian rhythm of food intake and other behaviors.

Circadian photoreception was assessed by measuring across the circadianday serum melatonin concentrations and the frequency and ultrastructure of syn-aptic ribbon complexes in pinealocytes in normal and mutant rats maintained inboth constant conditions and under a light-dark cycle (20). Both blind and con-trol animals showed normal circadian rhythms in both measures when main-tained in constant darkness and disappearance of the rhythms in constant light.Moreover, both sighted and anophthalmic rats exhibited entrainment to a 12:12LD cycle, as well as reentrainment to reversed LD conditions. In a separate studyby the same authors, activity rhythms of the mutant rats were also found toentrain to a light-dark cycle (50).

After ruling out the possibility that ambient temperature or some other envi-ronmental cue was responsible for the observed effect, the authors (20) con-cluded that such endocrine and behavioral entrainment was due to

the existence of a light perception pathway involving a nonvisual system,i.e., the photosensitive capacity of pineal either directly via skull . . . or thephotoreceptive capacity of the harderian gland . . . or [an] unknown systemsuch as photosensitive skin. (p. 101)

This was not the conclusion reached by Ibuka (19), who studied both unilat-erally and bilaterally anophthalmic mutant rats. Using polygraphically recordedsleep-wake measures as a measure of circadian rhythmicity, rats were challengedwith a protocol in which the light-dark cycle was delayed by 6h. Unilaterallyanophthalmic rats reentrained at the same rate and to the same degree as sightedrats, but bilaterally anophthalmic animals failed to reentrain after 6 days. In addi-tion, the 10 bilaterally blind animals in which wheel-running activity was re-corded showed no evidence of reentrainment in that measure following the 6hshift in the light-dark cycle.

Interestingly, neither unilaterally-, bilaterally anophthalmic mice (“eyeless”ZRDCT/An strain) nor heterozygous mice of the same strain with normal visualperception exhibited entrainment of sleep cycles to a light-dark schedule (22).The authors attributed this result to an absence of anatomical or functional con-nections between the retina and the endogenous biological clock in the unilater-ally blinded and sighted mutant animals rather than to a nonfunctional or alteredclock per se. As such, and based on the lack of entrainment in the bilaterallyanophthalmic animals, the authors excluded the possibility of EOCP in these

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mutant mice. Subsequent experiments by Scheuch and coworkers did show, how-ever, that anophthalmic mice often exhibit varying degrees of hypogenesis of theSCN (21). Animals with substantial SCN hypogenesis showed abnormal circa-dian rhythmicity.

Several studies have examined the issue of EOCP in humans, again withmixed results. Inspired by Oren’s theoretical paper (51) that introduced the con-cept of humoral phototransduction (see section on mechanisms), we examinedEOCP in a group of healthy, sighted adults between the ages of 18 and 62 years(48). Using a fiber-optic light source applied to the popliteal fossa of each leg(the area directly behind the kneecap), we administered light (approximately13,000 lux, 450–540 nm) to subjects on one occasion for a period of 3h. Thecircadian phase of body core temperature and melatonin onset were determinedat baseline and following light exposure, and the degree of phase shift was deter-mined by comparing baseline and postlight measures.

Figure 2 shows that there was a systematic relationship between the timingof light exposure and the magnitude and direction of phase shifts, resulting inthe generation of a phase-response curve (48) remarkably similar to that for ocu-lar light exposure. In contrast, a sham light exposure condition, in which the

Figure 2. Response of the circadian pacemaker, as measured by core body temperature, to asingle 3h presentation of bright light to the popliteal region. Each point represents the phase shiftobserved (advances are designated by positive numbers and delays by negative numbers on the yaxis) in response to bright light presented at a given time relative to the phase of body coretemperature at baseline. “Timing of light relative to Tmin” (x axis) refers to the interval betweenthe midpoint of light presentation and the fitted temperature minimum. The magnitude of theobserved phase shifts varied systematically as a function of this relation.

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apparatus was attached to the legs, but no light exposure occurred, did not inducephase shifts.

Based on these findings in healthy sighted people, Erman and coworkers,sought to alleviate sleep disturbance reported by a 36-year-old woman with bilat-eral enucleation by resetting her circadian timing system using a similar approach(52). Broadband white light (approximately 10,000 lux) was applied to the popli-teal region for several consecutive days at a time calculated to induce a phasedelay. Comparisons of phase of body temperature and melatonin onset betweenbaseline and postlight treatment revealed a 4h delay in the course of body tem-perature and a 3h–4h delay in melatonin onset. Self-selected bedtime was de-layed by approximately 2.5h, and sleep became more consolidated.

Lindblom and coworkers also employed broad-spectrum white light (53),directed at the chests and abdomens of five healthy sighted subjects, to examinethe effects of extraocular light on several hormonal rhythms. In two differentlaboratory sessions, subjects were exposed to either bright light (�13,000 lux)or to no light on one occasion during each lab session, between the hours of22:00 and 01:00. Circadian profiles of melatonin, cortisol, and thyrotropin weremeasured at baseline and compared to those measured on the day following light(or sham light) exposure. In both the active and control conditions, all threerhythms showed a tendency to delay relative to baseline, but the average phasedelay measured in the two conditions did not differ significantly from one an-other. The authors interpreted the findings as providing no evidence of EOCP.

A similar conclusion was reached by Eastman et al. (54) based on resultsfrom a study using light exposure devices identical to those employed by Camp-bell and Murphy (48). Healthy young adults, the popliteal regions of whom wereexposed for 3h to either about 13,000 lux or to no light, showed no systematicdifferences in the degree to which rhythms of salivary melatonin and body coretemperature were shifted relative to baseline measures. The majority of subjectsin a third experimental group who received ocular light exposure (1000 lux) atthe same time of day (03:00–06:00) also showed no significant difference incircadian phase following light administration. Interestingly, although light expo-sure in all groups was timed to induce phase delays (based on the human phase-response curve to ocular light), half of the subjects exposed to extraocular lightexhibited phase advances in their temperature rhythms.

NONCIRCADIAN EXTRAOCULAR PHOTOTRANSDUCTION

There is a substantial body of literature that addresses the issue of extraocu-lar phototransduction not with respect to the circadian timing system specifically,but within the framework of seasonal/reproductive influences and orientation.Such studies are germane to the current review inasmuch as the endogenoustiming system is clearly involved in those components of physiology and behav-ior. The following sections provide a brief overview of that literature.

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Photoperiodism

In many vertebrate species, seasonal changes in day length are the primarysource of information for controlling such important events as molting, changeof fur color, fattening, thermoregulation, migration, and reproduction (55).

The extraocular control of photoperiodically induced gonadal growth inbirds has been extensively documented. Benoit was the first one to show that theeyes are not essential for the photosexual reflex in birds. From a series of experi-ments in ducks in the 1930s (56,57; summarized in Ref. 1), he concluded thatthe “eye acts as a superficial photoreceptor,” but that a “second, deep photore-ceptor apart from the eye” is involved in the mechanism. He showed that stimu-lation of the testes occurred when male ducks were exposed to light, even whenthe optic nerves had been severed or when the eyes had been removed.

Other studies followed, demonstrating that blinding did not prevent longdays from stimulating testicular growth in chicken (58,59), quail (60–62), spar-row (13,63), canary (64), and white-crowned sparrow (65,66) as reviewed inRef. 67. Thus, there is strong evidence that birds detect the photoperiodicity notwith the retina, but by brain photoreceptors, which probably lie in the hypothal-amus.

A more recent study in bilaterally enucleated American tree sparrowsshowed that an extraocular mechanism not only mediates photoinduced gonadalgrowth, but also mediates the transition from photosensitivity to photorefractori-ness, which terminates reproduction (68). In white-crowned sparrows, the basalhypothalamus was directly illuminated via implanted optic fibers for 20h perday, superimposed on the 8h daily ambient photophase. In addition to testiculargrowth, a migratory reflex (Zugunruhe) was triggered, as indicated by increasedperch-hopping activity (4). This experiment demonstrated that both photoperiodi-cally induced migratory behavior and gonadal development are mediated by ex-traocular receptors located in the hypothalamus.

The involvement of extraretinal photoreceptors in photoperiodic responseshas also been demonstrated in fish and reptiles. Underwood (37), for example,demonstrated that long daily photoperiods can induce testicular growth inblinded, as well as sighted, lizards (Anolis carolinensis). However, no testiculardevelopment occurred when the heads of sighted lizards were painted withopaque color. In the Gekkonid lizard Hemidactylus flaviridis, tail regeneration isinfluenced by photoperiod (69). Following autotomy of the tail, the lizards weresubjected to different lighting schedules. It has been shown that long-day photo-periods stimulate the regeneration process of the tail, whereas short-day photope-riods depress tail regeneration. This phenomenon was observed in both intact andblinded lizards, suggesting that the eyes do not participate in photoperiodicallysignificant photoreception. Pinealectomy or painting of the head with India inksignificantly retarded tail regeneration. The authors concluded that extraocularphotoreceptors, most probably located in the pineal gland, are responsible formediating photic information necessary for tail regeneration.

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Light sensitivity of blinded fish was studied extensively by de la Motte(44), who demonstrated that illumination of the head elicited reactions (alteredswimming behavior) in all 18 species of freshwater fish tested. Effects of extra-ocular photoreception on fish reproduction have been shown in several species,including the medaka (70) and the three-spined stickleback (71). Urasaki (70)studied ovarian development in blinded, blinded-pinealectomized, and intact fe-males of the medaka subjected to different photoperiods. The gonasomatic index(GSI; the percentage of the gonadal weight to the body weight) of both intactand blinded medaka increased during long photoperiods and decreased duringshort photoperiods. However, seasonal changes in GSI were less profound inblinded than in intact animals, and pinealectomy completely abolished the photo-periodic response.

From these and earlier experiments (72), it was concluded that the pinealis an endocrine organ that transmits photic information from the eyes and at thesame time is a photoreceptive organ that directly responds to light. Extraretinalphotoreception has also been observed in three-spined sticklebacks (71). Sexualdevelopment of intact male three-spined sticklebacks during the breeding seasonwas manifested by breeding colors, kidney hypertrophy, and spermatogenesis.These photoperiodic responses also occurred in blinded fish subjected to longphotoperiods, although it did not always occur to the same extent as in the intactfish.

Studies in the blind mole rat Spalax Ehrenbergi suggest that mammals alsopossess extraretinal receptors for mediating photoperiodic responses (23). Themole rat is a blind fossorial mammal with atrophic, subcutaneous eyes. Despitetheir highly degenerated visual system, these animals are capable of detectingchanges in day length, as evidenced by photoperiodic-dependent alteration intheir thermoregulatory capacities: Mole rats increase their resistance to coldwhen days get shorter. It has been suggested that a retinal pathway is involvedin mediating photoperiodic responses in Spalax (78). However, intact and enucle-ated mole rats increased their thermoregulatory capacity when experimentallytransferred from a long to a short photoperiod (73). Based on experiments usingvarious surgical manipulations, the authors concluded that the Harderian glandand possibly other extraocular photoreceptors are implicated in the detection ofshortened photoperiod.

Orientation

Extraretinal photoreception has also been implicated in various kinds oforiented movements, ranging from simple phototaxis to the more complex sun-compass and magnetic orientations. Phototactic behavior (i.e., orienting move-ments toward or away from a light source) has been described in many species ofamphibians, fish, and reptiles. Early studies in eyeless frogs and toads providedpreliminary evidence that phototactic behavior may be mediated by dermal pho-

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toreceptors (75,76). Studying nine different species of amphibians, Pearse re-ported blinded animals showed photic responses similar to intact individuals.Additional examples of phototactic behavior in vertebrates thought to be medi-ated through the skin (e.g., in Epinephelus and Proteus) have been described inan extensive review on the “dermal light sense” by Steven (30).

Brain photoreceptors appear to be responsible for photonegative (i.e., re-treat) behavior in eels (15,44). Illumination of the head with a focused light beamresulted in characteristic backward swimming in intact fish, as well as in blindedand blinded-pinealectomized fish, although response latency was 3 to 10 timeslonger in blinded eels. However, blinded animals with aluminum foil coveringthe skull showed an altered motor activity pattern compared to intact or blindedeels bearing a transparent plastic cover. In addition, de la Motte found that illu-mination of the eel’s tail induced forward swimming, while illumination of thetrunk elicited no significant response (44). Interestingly, the eels reacted fasterto stimulation of the tail than to stimulation of the head.

Phototactic behavior in hatchling alligators is dependent on ambient tem-perature, but only when extraretinal photoreceptors are stimulated (77). Whenthe eyes were covered, limiting photoreception to extraocular sites, the light in-tensities required to induce a positive phototactic response was greater whenambient temperature was warmer. In contrast, when the heads of sighted alliga-tors were covered with an opaque cap, a phototactic response was elicited regard-less of ambient temperature. The author interpreted such findings as reflectingthe adaptive functional significance (i.e., for thermoregulation) of extraocularphotoreceptors in the presence of a fully developed ocular photoreceptor system.

A more sophisticated type of orientation is the sun-compass orientation ob-served in many species of amphibians (78–81). Such orientation allows the ani-mal to move in a given direction without the use of landmarks by using celestialcues in combination with a biological clock mechanism synchronized to localtime to compensate for the rotation of the earth (34). Studies in frogs (78,79,81)and salamanders (80) showed that a laboratory-induced shift of the 24h light-dark cycle by 6h results in a 90° rotation in orientation. The orientational capa-bilities of these animals have been tested after various surgical manipulationsand demonstrate that they can utilize either the eyes or extraocular photorecep-tors located in the brain for compass orientation. However, simultaneous blindingand covering of the head with black plastic results in a complete loss of orienta-tion.

MECHANISMS OF EXTRAOCULAR CIRCADIANPHOTOTRANSDUCTION

The preceding sections have reviewed the literature concerning behavioraland physiological responses to extraocular light exposure in vertebrates. In con-trast to that relatively abundant literature, knowledge about the processes in-

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volved in EOCP is much more limited. Indeed, the specific location(s) of extra-ocular photoreceptor(s) are still unknown, and very little is understood abouthow extraocular light may be transduced into a signal that mediates circadian orphotoperiodic responses.

Due to the lack of data specifically addressing the mechanisms of EOCP,here we have broadened the discussion to include extraocular photoreception forphotoperiodic responses as well. Moreover, although we have attempted to limitthis discussion to mechanisms of EOCP in vertebrates, models of phototransduc-tion in nonvertebrates are, in many cases, more well developed. Therefore, wehave included some of the literature concerning nonvertebrates if it may be gen-eralizable to vertebrate species.

One difficulty in determining the location of extraocular circadian photore-ceptors in vertebrates may be that there are too many choices. For example, thereis recent evidence that cryptochromes, which have been found, among other ar-eas, in the skin, hypothalamus, and retina of mammalian vertebrates, can trans-duce circadian photic signals (82,83). Even cells within individual organs, suchas the liver and heart, exhibit circadian responses to light-dark cycles (6,84). Itis now clear that many tissues, cells, and even cell components in vertebratesare capable of producing a rhythmic response to photic stimulation. While muchhas been learned about transduction of photic information, a fundamental chal-lenge remaining for chronobiologists is to determine which of these mechanismsof extraocular phototransduction are involved in rhythmic processes at the behav-ioral level.

Deep Brain Photoreceptors

Evidence that light that impinges directly on neural tissue can alter photo-periodic responses in vertebrates was reported by Benoit in a series of studies(1,56,57). In blinded ducks, light directed through the excavated eye socket viaquartz rods to the pituitary, hypothalamus, or rhinencephalon resulted in gonadalcrudescence (i.e., a 5- to 10-fold increase in gonadal weight in anticipation ofbreeding). While Benoit’s data suggested that the eye played a role in modulatinggonadal stimulation by light, removal of the eye did not eliminate testiculargrowth (1). Yet, a black cloth placed over the heads of the ducks prevented theresponse, indicating that the extraocular photoreceptors were probably localizedto the brain.

Benoit’s landmark work was not pursued by other researchers until theearly 1960s, following a study by Ganong and colleagues (5), which demon-strated that visible light penetrates deep into the brain of mammals. Using alight-sensitive photovoltaic cell mounted on a 14-gauge needle, they quantifiedlight that reached the temporal lobe and hypothalamus of sheep, dogs, rabbit,and rat. While alive, the animals were placed outside on sunny or cloudy daysin the afternoon hours; after being decapitated, their heads were placed outside

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on days with similar weather conditions. Detectable light was measured in allanimals in both brain sites under all weather conditions and were identical inliving and dead animals. Although the amount of irradiation falling on the sur-face of the heads of the animals was attenuated by the skull bone, blood vessels,and dura mater covering the brain tissue, a significant amount of light, particu-larly longer wavelengths, did penetrate to neural tissue. Indeed, the amount oflight detected in the hypothalamus of the rat outside on a partly cloudy dayexceeded the sensitivity of the photovoltaic gauge.

Based on these findings, Lisk and Kannwischer investigated the sensitivityof hypothalamic neurons to direct light stimulation (85). These workers demon-strated that female rats would enter a persistent estrouslike state if exposed toconstant light via glass rods implanted in the suprachiasmatic region of the hypo-thalamus. If the arcuate region was exposed instead, ovarian weight increased ina manner similar to that observed with constant exposure of the eyes to photicstimulation. These authors concluded that, although rats’ eyes are primary for thefunctioning of the estrous cycle, the central nervous system of these mammalsnevertheless retained the ability to respond to direct visible radiation.

Other studies in birds focused light on smaller brain regions using smallglass rods or by implanting light-emitting diodes (4,86,87). These studies haveimplicated the basal hypothalamus, ventromedial hypothalamus, and paraventric-ular nucleus as photoreceptive sites for inducing testicular regression or growth.

During embryonic development, the pineal complex is formed as an out-growth from the diencephalic roof, which also gives rise to the retina (e.g., Ref.34). Its direct photosensitivity has been demonstrated in several species, andphotosensitive cells in the fish and rat pineal share morphological and functionalfeatures with ocular photoreceptors (e.g., Ref. 88). Dodt and Meissl (89) re-ported that light received by pineal photoreceptors is transduced into nervousimpulses. The pineal is also situated advantageously for light reception. In frogs,only a translucent layer of skin covers the pineal and frontal organs, which arelocated on the dorsal surface of the head. Similarly, in birds, the skull bone isthinner directly over the pineal gland than any other area (3). These characteris-tics render the pineal complex a natural choice in the search for extraocular pho-toreceptors.

In fish, the pineal complex has been shown to play a role in modulatingcircadian rhythm responses to light. For example, in a study measuring the circa-dian rhythm of skin coloration in pencil fish, the principle product of the pineal,melatonin, was shown to be responsible for the day-night change in color pat-terns (33). Although the author was unable to pinealectomize these fish withoutcausing death, he concluded, nevertheless, that the presence of the pineal wasrequired for expression of the coloration rhythm. Yet, other studies in fish havedemonstrated that removal of the pineal organ does not abolish entrainment ofthe daily pattern of swimming in lake chub (32) or reentrainment of nocturnalmotor activity patterns to a reversal of the light-dark cycle in eels (15). The

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possibility remains that the pineal organ is required in the phototransductionpathway and may be involved in the control of circadian period length and ex-pression of ultradian rhythmicities (31,72), but it is unlikely that the pineal actsas the photoreceptive site for entrainment of rhythmic processes.

A thorough examination of extraocular photoreception in amphibians ledresearchers to the conclusion that light reception for perceiving photic cyclicityin the environment most likely occurred at the level of the pineal complex (12).Evidence cited for this inference included the finding that blinded frogs couldnot be phase shifted by light if the frontal organ was removed (12). The authorinterpreted this result as due not to the loss of photoreception by the frontalorgan per se, but to removal of connections between the frontal organ and pineal.Also, in newts (which lack a frontal organ, but have a pineal body), blinding didnot prevent entraining effects of a light-dark cycle, but pinealectomy of blindedanimals resulted in similar activity patterns in a light-dark cycle and constantconditions, suggesting that the pineal is involved the perception of alternatinglight conditions (35).

Extraocular circadian photoreception in reptilian species has also beenshown to be mediated by a deep brain photoreceptor. Although the parietal eyeand pineal organ in lizards had been identified as having well-organized photore-ceptors, excision of either or both of these structures did not prevent entrainmentin two of seven lizard species tested (13,90). Removal of the pineal in a blindedthird species (the Western banded gekko) produced the same result.

Underwood further attempted to localize the brain areas responsible forEOCP in lizards by painting the head with black lacquer, decreasing or eliminat-ing light transmission through the head (13). Of the 4 blinded iguanid lizards, 3tested remained entrained after their heads were painted. In contrast, blindedWestern banded gekkos with carbon black injected under the skin (thereby leav-ing possible dermal photoreceptors unshielded) exhibited free-running activityrhythms. Despite these mixed results, the author concluded that “the [extraretinalreceptor] for entrainment, in lizards, as in amphibians and birds, is located in thebrain” (p. 203).

Additional evidence for extraocular, extrapineal circadian photoreceptors inreptiles has been reported (16). These authors studied the locomotor activityrhythm in American alligators, a species that lacks a pineal organ, and found thatanimals with opaque patches placed over their eyes could still entrain to a light-dark cycle. In contrast, when their eyes were shielded and opaque skull capscovered their heads, the alligators exhibited free-running locomotor rhythms inlight-dark cycles. The capacity of the deep brain photoreceptor in alligators todiscriminate light intensities was demonstrated by showing that animals withtheir lateral eyes covered obeyed Aschoff’s rule: When maintained in constantlight, lower light intensities consistently increased the locomotor activity periodlength.

Among avian species, the house sparrow has been most frequently studied

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with regard to EOCP. An elegant series of studies by Menaker and colleagues(2,3,14,91–93) compared the contribution of the eyes, the pineal complex, andextraocular, extrapineal receptors to light mediation of both circadian and photo-periodic responses. Following pinealectomy and hooding experiments, the authorconcluded that (1) the pineal was not the sole deep brain photoreceptor in birds,(2) the eyes did not play a role in the photoperiodic response of testicular recru-descence in this species, and (3) the photoreceptor for both circadian entrainmentand seasonal reproductive rhythmicity was in the brain, although a more preciseanatomical location could not be determined.

In addition to the house sparrow, another bird species that thrives followingvisual blinding, and therefore has been used for studies of photic entrainment, isthe Japanese quail. Results from a recent investigation of photic control of theoviposition rhythm in this bird suggest that ocular light receptors and pacemakersthat exist in the eyes of Japanese quail may not be functional in terms of photicentrainment (94). Blinding by eye removal or patching intact eyes with black-painted adhesive bandages did not significantly alter the circadian rhythm ofoviposition. One interpretation of these findings suggested by the authors wasthat, even if the eyes play a role in entraining the reproductive rhythm of Japa-nese quail, extraretinal mechanisms alone are sufficient for this purpose.

At least one possible location of extraocular circadian photoreceptors inJapanese quail has been identified by a study that demonstrated the direct photo-sensitivity of neural tissue (87). In that study, low-intensity light emitted fromradioluminescent disks inserted into the rhinencephalon and an area adjacent tothe hypothalamus elicited the photosexual response.

As described above, the capacity for EOCP in mammals has been testedrarely and observed only in neonatal rats (40); some species of anophthalmicmutant rats (20), but not others (19); normally sighted adult humans (48); and ina case study of one blind adult human female (53). As detecting a circadianresponse to extraocular light exposure in mammals has thus been difficult, in-vestigating mechanisms by which EOCP might occur in mammals has not beena research focus. Nonetheless, the finding that light affected the pineal N-acetyl-transferase rhythm in neonatal rat pups before their visual systems were devel-oped (40) prompted the examination of possible mediators of this response. Wet-terberg et al. suggested that the Harderian gland, which contains a red porphyrinphotopigment and is also capable of synthesizing melatonin, was the site of ex-traocular photoreception in neonatal rats (41).

The Harderian gland has also been implicated in the perception of photope-riod for thermoregulatory responses in the blind mole rat (73). Removal of theseverely atrophied eyes in this species did not prevent the photoperiodic response(i.e., systematic changes in thermoregulation), but animals without Harderianglands exhibited impaired thermoregulatory responses. Yet, it is unlikely that theHarderian gland is the only site for perception of the photoperiod since adapta-tion to the light cycle changes was observed in Harderianectomized rats after 5weeks in the new photic environment.

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Blood-Borne Photoreceptors/Humoral Phototransduction

A light-mediated time-measuring system not involving deep brain photore-ceptors was proposed by McDonagh (95), who hypothesized a novel functionfor the blue-light-absorbing chromophore bilirubin, typically thought to be awaste product in healthy adult humans. The proposed role of bilirubin was basedon the findings that a stable photoisomer of bilirubin was produced in an adultmale runner during daylight, but not produced when he ran at night, and that thesame photoisomer was produced in two male sunbathers. The author speculatedthat bilirubin might be a vestigial photoreceptor, which “in former times” mea-sured day length by the proportion of the photoisomer in plasma. The authoralso compared the photoisomer to light-absorbing molecules in plants and ob-served that “phytochrome, which has a very similar chemical makeup as biliru-bin, functions as the photoreceptor in plants in exactly this way” (p. 121).

The remarkable similarities in chemical structure between phytochromes inplants and chromophores such as bilirubin in humans also formed the basis of a“humoral phototransduction” hypothesis proposed by Oren (51). This hypothesisposits that light of sufficient intensity, falling on a vascular surface, initiates acascade of events that eventually stimulate the neural pathways that entrain bio-logical rhythms. Although the author initially proposed that this process occursprimarily via irradiation of retinal vasculature, the mechanism could apply toextraocular, peripherally mediated circadian phototransduction as well. In sup-port of the model is the fact that there is extensive vascularization of componentsof the circadian timing system, including the SCN. In addition, bright light candissociate neuroactive gases, including carbon monoxide and nitric oxide (NO),from heme moieties (51,96), and bright light can further increase NO in circula-tion by directly stimulating the production of NO synthase (97). Moreover, thedirect role of NO in phase shifting the biological clock has now been established(e.g., Refs. 98 and 99). Combined with the vasodilating properties of oxidativemolecules such as NO (100), this model constitutes a mechanism by which pho-tic cues from virtually any site in the body could be conveyed via blood to thecircadian timing system. Although it may be unlikely that peripherally producedNO is in circulation for a sufficient duration to be transported to and act locallyin the central nervous system, a recent study reported that peripheral blood flowvelocity is increased by extraocular light exposure in rats, and that this phenome-non was reversed by a NO synthase inhibitor (101). Thus, peripheral NO stimula-tion could increase the speed by which an (as yet undefined) humoral signal istransported to the brain.

Photoreceptivity at the Molecular Level

In recent years, the inner workings of the biological clock have been eluci-dated using molecular and biochemical techniques. The current model explaining

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the mechanisms of photic entrainment and phase-response curves to light includeat their core a transcription-translation feedback loop of a series of gene prod-ucts. Lakin-Thomas has noted that nearly all so-called canonical clock genes invertebrates, including mice and humans, are affected by light and has suggestedthat many genes thought to be core components of the clock may also functionas photoreceptors for circadian entrainment (102). For example, cry-1 and cry-2genes, which code for the blue-light-absorbing cryptochrome proteins that modu-late light-induced phase shifts, could be viewed as part of the mechanism ofentrainment by light rather than (or in addition to) part of the clock itself.

The role of cryptochromes in vertebrate circadian photoresponses is sup-ported by recent data from invertebrates. Emery et al. have determined thatcryptochromes are functional circadian photoreceptors in the fruit fly Drosophilamelanogaster (103). Thresher and colleagues demonstrated that cry-1 and cry-2knockout mice, although able to be entrained by light-dark cycles, did so at anabnormal phase angle (83). Such observations in circadian responses to light donot establish cryptochromes as a circadian photoreceptor in vertebrates, but theyimplicate these proteins in the phototransduction process by which the SCN re-ceives information concerning the photic environment.

Evidence for light-responsive molecular clocks in vertebrate peripheral tis-sues has recently been reported. Liver and heart cells of zebrafish have beenshown to exhibit a strong circadian oscillation in the expression of the Clockgene in vivo, as well as in culture (6). The rhythmic output from these cells canbe phase shifted by light in vitro after removal from the influence of centrallylocated pacemaker structures (6). Moreover, immortalized zebrafish-derived celllines continued to express rhythmicity in response to light (104). Unknown asyet are the photopigment molecules in these cells that are responsible for absorb-ing light, or whether other components of the putative phototransduction cascadein zebrafish cell cultures respond to light cycles as well. The current findingsstrongly support the possibility that the capacity for EOCP exists in peripheralstructures of some vertebrates and suggest that organization of the circadian sys-tem and its response to light in this vertebrate species may be similar to inverte-brates such as Drosophila (105).

Alternative Phototransduction Mechanisms

Dermal Photoreception

In addition to mechanisms by which extraocular light influences the circa-dian timing system, there are several alternative mechanisms by which centralnervous system processes are altered by extraocular light exposure. For example,it has long been known that there is a “dermal light sense,” and it appears that,in some species, the entire body surface may be light sensitive (106). Dermalstimulation was implicated in Parker’s studies of phototropic behavior in the frogRana pipiens (75), which was later confirmed in nine other terrestrial and aquatic

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amphibians (76). The studies by Pearse determined further that skin-mediatedphototropism in blinded amphibians required an intact spinal cord and mesen-cephalon for the signal to be transduced and result in motor behavior, but thatbrain regions anterior to the mesencephalon were not required (76).

Light stimulation of dermal receptors can evoke additional behavioral re-sponses that are not necessarily rhythmic in nature, but that rely on photic cues,such as basking in lizards (108) or phototaxis in eels (15,44). De la Motte re-ported that eels showed the fastest negative phototactic responses when their tailsand trunk were exposed to light (44). Attempts to localize the photoreceptivesites within the skin were made by excising skin from the tails of the eels or bycutting the skin away from the tails and then placing it back over the wounds(thereby eliminating neuronal connections between the skin and rest of the body).Both preparations agitated the eels to such an extent that assessing changes inmotor activity patterns was not feasible. A later study, however, could not findevidence of the dermal sensitivity in eels and found instead that the photorecep-tor for entraining motor activity must be located in the brain (15).

Peripheral Nerve Stimulation

Despite attempts of some investigators to locate dermal photoreceptors andto describe possible sequelae of dermal photoreception that would result in trans-duction of the light signal into a behavioral response (e.g., see Refs. 107–109),we could not identify any report of a photic response that was unequivocallyskin mediated. But, as suggested by Steven (30) and others (e.g., in Refs. 75 and76), “dermal” responses to light might actually be due to photic sensitivity ofperipheral nerves that innervate the skin. Photic stimulation of peripheral nervesites has been shown to affect centrally mediated physiological responses suchas suppression of muscle spasms (clonus) on the side of the body contralateralto light administration (110).

Direct Neural Stimulation

Direct photic stimulation of the caudate nucleus in rats has been shown tofacilitate learning of a complex behavior (conditioned avoidance) and to increasetransmission of serotonin and norepinephrine acutely (111,112). Exposure of ratcortical tissue to light alters GABA transmission (113). Such results suggest thatthe investigation of the location, identification, and functioning of extraocularphotoreceptors may be far from complete.

Photoreception in Vascular and Smooth Muscle Tissues

The stimulation of endothelium by light results in release of an endothe-lium-derived relaxation factor (EDRF), which has been identified as either NO

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or a closely related compound (100). The capacity of smooth muscle tissue torelease this compound has also been demonstrated (97,114). In addition, lighthas been shown to induce acetylcholine release in excised pig ileum (115,116),most likely as the result of an NO-mediated increase in production of the neuro-transmitter.

The sequelae of NO production include an increase in membrane perme-ability and increased production and release of neurotransmitters from synapticvesicles, and there is substantial evidence that NO itself acts as a neurotransmit-ter (117). Moreover, as mentioned above, direct application of NO to the SCNof rats mimics light-induced c-fos induction and behavioral phase shifts (98),and a role of NO in the photic entrainment pathway has now been established(98,99).

From Photoreception to Phototransduction

While the search for a photoreceptor is paramount in the investigation ofmechanisms of EOCP, identification of the “cascade of events” following recep-tion of a light signal is required to characterize the entire phototransduction pro-cess accurately. To this end, some researchers have identified extraocular photo-pigments by characterizing their action and absorption spectra. For example, thepioneering studies of blinded ducks by Benoit showed that the putative hypothal-amic receptor was sensitive to the entire visible spectrum (1), but was moresensitive at long wavelengths. In a smaller vertebrate species, the European yel-low eel, only exposure to shorter (but not longer) wavelengths resulted in en-trainment of locomotor activity rhythms via an extraocular, extrapineal receptor(118). These authors suggested that, although shorter wavelengths do not pene-trate neural tissue as well as longer wavelengths (119), the size of the speciesmay influence the biologically effective wavelengths for entrainment by extraoc-ular light exposure.

Oksche and Hartwig identified a photopigment in the encephalon of theEuropean minnow Phonixus phonixus that was sensitive to longer wavelengthsof light (120). More recently, Blackshaw and Snyder have described an opsin inthe mammalian brain that they have termed encephalopsin (121). This photopig-ment is more highly expressed in encephalic regions of adult mammals than inneonates. Foster and colleagues have described novel photopigments that areneither rodlike nor conelike photopigments. These circadian photoreceptor candi-dates are found not only in the retina, but also, at least in nonmammalian species,throughout the central nervous system (10,122–125). In addition to these opsin-based photopigments, the cryptochromes, as described above, may transducephotic information for circadian rhythmicity (82,83).

With regard to subsequent components of the phototransduction cascade,there have been virtually no investigations in vertebrates of the means by whicha deep brain photoreceptor might transmit a signal absorbed by a photopigment

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to the primary pacemaker/SCN. This, despite the fact that the putative deep brainphotoreceptors are thought to be “at some remove from the organs whose activi-ties they entrain” (45, p. 148). Nor are there cohesive theories that define a puta-tive pathway from peripherally detected light to the central nervous system ingeneral or the SCN in particular. As described above, Oren has suggested ahumoral phototransduction process, which permits the possibility that a signal oflight detected at any vascular site could be transported in the form of a humoralsubstance in blood to the circadian timing system (51,126). Others have sug-gested that the position of the SCN—adjacent to the third ventricle—might beadvantageous for detecting signals carried in the cerebrospinal fluid (8,118). In-deed, there are dendritic and axonal projections of SCN neurons into the thirdventricle; these have functions that are yet to be determined (123,125).

COMMENTS

There is a vast body of literature that leaves little question that extraocularphototransduction occurs in every vertebrate species studied to date. Moreover,results of the studies reviewed here provide compelling evidence that such photo-transduction can, and does, have an impact on the circadian timing systems ofall nonmammalian vertebrates thus far studied. In the case of mammals, how-ever, the data are far less definitive. Of the 14 studies reviewed here, less thanhalf have found evidence of EOCP in mammals. Based on such negative results,it is generally acknowledged throughout the literature that mammals do not pos-sess the capacity for EOCP.

One obvious conclusion to be drawn from such findings is that the abilityof an organism to employ extraocular light to provide the endogenous clock withtemporal cues has been lost in evolution, that the adaptive advantage of EOCPdoes not extend to a mammal’s survival. Yet, overwhelming evidence suggeststhat, if this is the case, it would be a rare exception to the phylogenetic rule ofwhat may be called time-sense conservation. Duplicates or analogues of basicmechanisms that are hypothesized to underlie EOCP in nonmammalian species,as well as invertebrates and even plants, are present in mammals, both at themolecular and cellular levels. Likewise, the ability to respond to ocular light isconserved across phylogeny and the nature of the resulting physiological andbehavioral output exhibited across species are governed by a remarkably consis-tent set of biological guidelines. In addition, there is good evidence to suggestthat noncircadian, yet temporally mediated, behaviors, such as seasonal repro-ductive cycles and more sophisticated forms of orientation, are strongly influ-enced by extraocular phototransduction even in mammals.

Why, then, would the ability to use extraocular cues for circadian temporalstructuring be selectively lost in mammals? Elaborating on speculation originallyoffered by Jerison (127), Menaker and Underwood (93) have hypothesized that,in the course of early mammalian evolution, the brain circuitry that underlies

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EOCP in nonmammalian species was perhaps converted for use in the detectionand utilization of stimuli more salient for nocturnal organisms (i.e., olfactoryand auditory cues), which early mammals primarily were. There is suggestiveanatomical support for such a notion inasmuch as photosensitive cells have beenreported to exist in olfactory areas of the brains of quail and ducks.

It is certainly conceivable that, for this and/or for other reasons, the mam-malian brain, indeed, has lost the capacity to detect and transduce extraocularlight for use in the circadian timing system. But, if this is the case, how can themixed findings in the few mammalian studies thus far conducted be reconciled?The previous argument concerning conversion of function might lead one tospeculate that the diurnality of a species may be a factor in its ability to employextraocular light as a temporal cue. And, indeed, with the exception of recenthuman studies, most investigators have examined EOCP in nocturnal rodents.Yet, of the studies that have reported EOCP in mammals, both nocturnal (rats)and diurnal (humans) species have been studied.

Perhaps mammals have retained the capacity for EOCP, but for some reasonthat capacity is more difficult to elicit and/or measure. For example, most non-mammalian vertebrates are hairless and have relatively thin skulls. Yet, studiesthat have attempted to account for such differences (e.g., see Refs. 42, 47, and49) nevertheless have reported negative findings. The vast majority of studies toexamine EOCP in both mammals and nonmammals have employed enucleation.There is no question that such an experimental manipulation has a widespreadimpact on anatomy, physiology, and behavior. It is unlikely, yet conceivable, thatenucleation has a differential impact on the mammalian brain such that the eyesmust be present, if not functional, for EOCP to be exhibited in these species.Yet, anophthalmic rats reported to exhibit EOCP have no eyeballs (20) and Er-man’s blind patient who responded to extraocular light had undergone enucle-ation (52).

In conclusion, it may be impossible to reconcile these inconsistent and puz-zling findings until a great deal more data become available and until a widervariety of species is studied. As Rusak and Zucker emphasized in a 1979 reviewof the neural regulation of circadian rhythms,

Until one can document the absence of photic entrainment in a numberof blind diurnal and nocturnal mammals exposed to light intensities approxi-mating those of direct sunlight, the conclusion that the eyes of adult mam-mals contain the only photoreceptors for entrainment must remain tentative.(128, p. 457)

Likewise, it is critical that we learn more about the mechanisms underlyingEOCP across all phylogeny. In this regard, Menaker’s statement in a 1971 paperseems apropos today:

It seems worthwhile to take stock of many intriguing questions, as yetunanswered. . . . No vertebrate extraretinal photoreceptor has been specifi-cally localized and as yet nothing is known of the neural or hormonal inputs

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to or outputs from any ERR [extraretinal photoreceptor]. We do not knowhow these brain structures interact with the eyes, the pineal gland, or thehypothalamus although the available evidence suggest that they do interactwith all three. . . . It may be that the whole vertebrate brain is photoreceptive,that a single structure is present, or that there are multiple, discrete ERRs.(91, p. 305)

While some progress has been made with regard to the mechanisms of EOCP invertebrates since that time, the most basic questions remain: Where are extraocu-lar photoreceptors mediating rhythmic processes located, and how do they trans-mit a signal to the circadian timing system?

Finally, as reviewed above, the general focus on the brain as the site forextraocular photorecptors seems well justified. Results from virtually all studiesthat have specifically examined extraocular photoreceptivity in terms of circadianor photoperiodic responses indicate that preventing light transmission to the braineliminates the capacity of the organism to entrain or phase shift in response tosuch light. Yet, given recent reports of light-sensitive peripheral clocks in speciesranging from Drosophila (105) to zebrafish (6), there is ample reason to continueand perhaps expand the search for extraocular photoreceptors and phototransduc-tion mechanisms in vertebrates.

ACKNOWLEDGMENT

This work was supported by NIH grants R01 MH45067, R01 AG12112,R01 MH54617, R01 AG15370, R01 HL64581, K02 MH01099, P20 MH49762,and M01 RR00047.

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EXTRAOCULAR PHOTOTRANSDUCTION AND CIRCADIAN TIMING SYSTEMS IN VERTEBRATES

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