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The role of retinal photoreceptors in the regulation of circadian
rhythms
Ketema N. Paul, Talib B. Saafir, and Gianluca Tosini
Circadian Rhythms and Sleep Disorders Program, Neuroscience Institute, Morehouse School of
Medicine, Atlanta, GA, USA
Abstract
The circadian clock is an evolutionarily, highly conserved feature of most organisms. This internal
timing mechanism coordinates biochemical, physiological and behavioral processes to maintain
synchrony with the environmental cycles of light, temperature and nutrients. Several studies have
shown that light is the most potent cue used by most organisms (humans included) to synchronize
daily activities. In mammals, light perception occurs only in the retina; three different types of
photoreceptors are present within this tissue: cones, rods and the newly discovered intrinsically
photosensitive retinal ganglion cells (ipRGCs). Researchers believe that the classical photoreceptors
(e.g., the rods and the cones) are responsible for the image-forming vision, whereas the ipRGCs play
a key role in the non-image forming vision. This non-image-forming photoreceptive system
communicates not only with the master circadian pacemaker located in the suprachiasmatic nuclei
of the hypothalamus, but also with many other brain areas that are known to be involved in the
regulation of several functions; thus, this non-image forming system may also affect several aspects
of mammalian health independently from the circadian system.
Keywords
Circadian; Melanopsin; Retina
1 Introduction
The circadian clock is an evolutionarily, highly conserved feature of bacteria, plants and
animals that allows organisms to adapt their physiological processes to the time of day in an
anticipatory fashion [1,2]. This internal timing mechanism coordinates biochemical,
physiological and behavioral processes to maintain synchrony with the environmental cycles
of light, temperature and nutrients. Circadian rhythms reflect extensive programming of
biological activities that meet and exploit the challenges and opportunities offered by the
periodic nature of the environment [2].
In mammals, circadian rhythms are driven by a timing system comprised of a master pacemaker
in the suprachiasmatic nuclei (SCN) of the hypothalamus, and peripheral oscillators located
throughout the organism. Independent circadian oscillators exist within each cell of almost
every tissue and/or organ investigated, including the liver and heart [3,4]. The mammalian
circadian system appears to be arranged in a hierarchical manner, with the SCN acting as the
© Springer Science + Business Media, LLC 2009
Neuroscience Institute, Morehouse School of Medicine, 720 Westview Dr, Atlanta, GA 30310, USA gtosini@msm.edu.
NIH Public Access
Author Manuscript
Rev Endocr Metab Disord. Author manuscript; available in PMC 2010 December 1.
Published in final edited form as:
Rev Endocr Metab Disord. 2009 December ; 10(4): 271–278. doi:10.1007/s11154-009-9120-x.
NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript
“master pacemaker.” The SCN drives and coordinates peripheral clocks through as yet poorly
defined humoral and neural signals, as well as indirectly by modulating activity and feeding.
Several studies have shown that light is the most potent Zeitgeber to entrain the mammalian
circadian system. In mammals, light perception occurs only in the retina [5], where three
different types of photoreceptors are present: cones, rods and the newly discovered intrinsically
photosensitive retinal ganglion cells (ipRGCs). Researchers believe that the classical
photoreceptors (e.g., the rods and the cones) are responsible for the image-forming vision,
whereas the ipRGCs play a key role in the non-image-forming vision.
In the present review, we will summarize the experimental evidence collected thus far on the
role that the different photoreceptors play on the photic entrainment of the mammalian
circadian rhythms and investigate how the ipRGC may contribute to the regulation of other
behavior.
2 Circadian photoreception
The study of the photoreceptors that mediate non-image-forming vision in mammals is
emerging as a new and promising aspect of retinal neurobiology. The current view is that, while
all effects of light on the circadian visual system are accounted for by the three types of
photoreceptors, no single photoreceptor is necessary for entrainment. For example, mice that
lack rod photoreceptors (rd) or rods and cones (rdcl) have a normal phase response to light
[6,7], and the action spectrum for phase shifts peaks at 480 nm [8]. These findings indicate that
an undiscovered photoreceptor/photopigment in the mammalian retina is responsible for the
photo-entrainment of circadian rhythms.
Data from many laboratories have provided compelling evidence that a mammalian homologue
of Xenopus melanopsin (also known as Opn4) is a new photo-pigment responsible for a key
role in the photic entrainment of the circadian system [9]. Studies have also shown that most
vertebrates express two melanopsin genes, termed Opn4m (mammal) and Opn4x (Xenopus).
In non-mammalian vertebrates, Opn4m and Opn4x are expressed in many tissue types, e.g.,
retina, brain, and skin [10–12], whereas mammals express only Opn4m [13].
In mammals, melanopsin mRNA and protein are present in only a small population (about 2%)
of the retinal ganglion cells (RGCs) [14]. These RGCs express pituitary adenylate cyclase-
activating polypeptide (PACAP) [15] and form the retinohypothalamic tract [16,17].
A series of elegant studies has demonstrated that these cells, named “intrinsically
photosensitive RGCs” (ipRGCs), are directly photosensitive and have an absorption peak
around 470–480 nm [18,19]. These ipRGCs were no longer intrinsically photosensitive in
melanopsin knock-out (KO) mice, although their number, morphology, and projections were
unchanged [20].
Studies have also reported that, since light response from these melanopsin cells can be
obtained before the rods and cones become functional, ipRGCs are the first photoreceptive
system to develop in mammalian retina [21,22]. The role played by these photoreceptors at
this early stage is not yet known. Some investigations have also reported that the regulation of
melanopsin levels (at least in the rat) is dependent on the light and the length of the photoperiod
[23–25].
Although these experimental data provided compelling evidence to suggest that melanopsin is
a functional photopigment, a direct demonstration was obtained in 2005 when three groups
published concurrent and mutually supportive evidence of the full functionality of expressed
melanopsin in human, mouse and Xenopus cell lines [26–28].
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Several studies using behavioral assay have also reported that removal of the melanopsin gene
in the mouse (melanopsin knock-out, KO) does not prevent the entrainment of the circadian
system and does not affect the free-running period in constant dark conditions. However, phase
response to light is attenuated in the KO animals since the magnitude of the phase-shift is about
half (40%) that of wild-type mice at each of three non-saturating irradiance levels [29]. A
saturating white light pulse also produced a diminished phase shift in the KO animals [30].
The length of the free-running period that follows exposure to constant light is reduced to about
55–65% of that of controls in melanopsin KO [29,30].
Melanospin levels in the Royal College of Surgeons (RCS) rat homozygous for retinal
dystrophy (rdy) are dramatically down-regulated once the classical photoreceptors have
degenerated [31]; however, despite this massive reduction in the photoreceptive apparatus,
their capability to phase shift in response to a pulse of light is not affected [32].
Melanopsin has also been implicated in the regulation of pupillary light reflex (PLR).
Transgenic mice that lack both rod and cone photoreceptors (rdcl) retain a PLR, a response
driven by a photopigment with peak sensitivity around 479 nm [33]. Melanopsin KO animals
showed a PLR indistinguishable from that of the wild-type mice at low irradiances, but at high
irradiances the reflex was incomplete. This result suggests that the melanopsin-associated
system and the classical rod/cone system are complementary in function [34]. Melanopsin KO
mice and mice that carry the rd (or rdcl) mutation, show normal light-induced suppression of
pineal melatonin [35], whereas melatonin in mice lacking rods, cones and melanopsin cannot
be suppressed by light [36], and the circadian rhythms of locomotor activity cannot be entrained
by light [20,36].
Accumulated experimental data indicate that melanopsin also plays an important role in
mediating the photic entrainment of human circadian rhythms. Brainard et al. [37] reported
that, in humans, the action spectra for melatonin suppression has a lambda max around 460
nm, suggesting that melanopsin is a key player in the photic regulation of melatonin levels and
of the circadian rhythms and sleep in humans. Studies have also shown that blue light in the
range of 440–480 nm is highly effective in phase-shifting the human circadian clock [38].
A few studies have reported that light can modulate sleep. In the rat, light exposure during the
night (the normal active period for a nocturnal animal) induced sleep, and exposure to darkness
promoted wakefulness [39], while neuroanatomical studies have described a retinal pathway
in the mouse that originates in ipRGCs and innervates the ventrolateral preoptic nucleus
(VLPO) [20,40]. Two recent studies investigated the contribution of the image-forming and
the non-image-forming system to the regulation of sleep. In the first study, Lupi et al., [41]
investigated whether the removal of melanopsin or of the rods and cones affected light-induced
sleep. As was the case in rats, light exposure during the night induced sleep in normal mice
and in mice that lacked the classical photoreceptor, whereas the photic regulation of sleep was
almost completely abolished in melanopsin KO mice, light exposure did not induce c-fos
expression in the brain areas that are believed to mediate this response (VLPO and superior
colliculus).
In the second study, Altimus et al. [42] reported the effects that light exposure during night, or
dark exposure during the day, have on sleep in mutant mice that lack the ipRGCs or lack
functional rods and cones, or melanopsin. The data presented in the study indicated that the
acute regulation of sleep and wake by light and dark requires both rod-cone and melanopsin-
signaling through ipRGCs and that such regulation is independent of image formation. The
differences between these two studies are probably due to the different experimental designs
used by the two research teams. Another study reported that only classical photoreceptors are
involved in the modulation of the cardiovascular system by acute exposure to light [43].
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Studies have also shown that blue light in the range of 440–480 nm is highly effective in phase-
shifting the human circadian clock, can increase alertness, and may be used to treat seasonal
affective disorders [44–46].
Because of these widespread effects on many different regulatory systems, several studies have
focused their attention on identifying the central projections of these new photoreceptors. Most
of these projections were initially identified by retrograde labeling [16–18] and then by using
a transgenic mouse line in which the melanopsin gene was replaced with tau-lacZ gene [17,
40]. These studies identified the SCN as one of the main recipients of the ipRGCs [16,18,40].
Another brain area that receives projections from these cells has been identified in the
intergeniculate leaflet, a brain area known to be involved in the regulation of the photic
entrainment of the circadian system [40]. In addition, the ipRGC cells project to the olivary
pretectal nucleus (a key center in the control of PLR) [17,47], the VLPO (a well established
center in the regulation of sleep), the lateral habenula, and the superior colliculus [40–47].
Although the number of ipRGCs in the mouse that project to the visual center seems to be
negligible or even absent, in primates it has been reported that an anatomically distinct
population of “giant” melanopsin-expressing ganglion cells projects to the lateral geniculate
nucleus, which is the thalamic relay to the primary visual cortex [48]. Hence, at least in the
primate, the non-image-forming and the image-forming pathways may merge, so the
melanopsin-based photoreception may contribute to conscious visual perception. A similar
situation may be present in humans, since new experimental evidence has suggested that visual
awareness may be present in humans with no conscious light perception [49].
Finally, newly obtained data have shown that there are at least two types of ipRGCs: the
melanopsin ipRGC type 1 (M1) cells, which mostly project to the SCN, and the melanopsin
ipRGC type 2 (M2) cells, which mostly project to the olivary pretectal nucleus (OPN)
responsible for the pupillary light reflex ([40,50]; see Fig. 1 for more details).
3 Circadian photoreception within the retina
Previous work has demonstrated that the mammalian retina contains its own autonomous
circadian clocks that control several retinal functions [51–53]. One of the most intensely
investigated of these functions is melatonin biosynthesis, which displays a clear circadian
rhythm in vivo and in vitro [51–53].
Cultured mammalian retinas show a clear circadian rhythm in melatonin release [54], and
Arylalkylamine N-acetyltransferase (an important regulatory component in the synthesis of
melatonin) mRNA is rhythmic in animals in which the suprachiasmatic nuclei of the
hypothalamus have been lesioned [55]. These findings indicate that the retinal clock can
generate circadian rhythmicity independent of the circadian clock located in the SCN.
Melatonin and dopamine (DA) play opposing roles in the regulation of retinal adaptive
physiology (reviewed in: [51–53]). Dopamine functions as a humoral signal for light and
produces light-adaptive physiology, while melatonin has dark-adaptive effects. In many
species, the synthesis and release of both melatonin and dopamine are under circadian control,
with melatonin released at night and dopamine during the daytime. Melatonin inhibits the
release of dopamine through an action on melatonin receptors, and dopamine inhibits the
synthesis of melatonin from photoreceptor cells by acting on dopamine receptors [51–53].
Thus, the melatonin-secreting photoreceptors and dopamine-secreting amacrine/IP cells form
a cellular feedback loop that functions to regulate circadian retinal physiology.
More recently, it has been reported that, in the RCS rat (rdy), light-dependent regulation of the
dopaminergic system within the retina is not affected by the loss in the photoreceptive system
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[56], and it has been proposed that dopamine and its metabolites may provide an important
signal for entrainment of retinal circadian rhythms [57]. A recent study using long-term
monitoring of PER2::LUC mice retina in vitro has also shown that, among the major retinal
neurotransmitters, dopamine, acting through D1 receptors, is responsible for the resetting
(entraining) of the phase of the retinal circadian clock [58].
Studies have also investigated whether melanopsin-based photoreception is involved in the
modulation of the retinal circadian rhythms. Hartwick et al. [59] investigated the light responses
evoked in DA neurons in the presence of L-AP4, a blocker of retinal ON-bipolar cells, and
found that the light responses observed were similar to those observed in ipRGCs. Moreover,
these light responses persisted in mice with degenerated retinas, suggesting the presence of
centrifugal outflow signals within the retina.
A second study that measured DA release reported an increase in the DOPAC:DA ratio in wild-
type mice and in mice lacking melanopsin or lacking rod phototransduction, whereas the light
response was not observed in mice that lacked both rods and cones. These data suggest that
light regulation of DA depends on the presence of rods and cones and that melanopsin is not
involved in the regulation of DA release [60]. The reasons for the discrepancy between the
results obtained in these two studies are unclear, and further studies will be needed to address
the role of melanopsin-based photoreception in the regulation of the retinal circadian clock.
Finally, the loss of the melanopsin gene abolishes circadian control in some parameters of cone
electroretinogram, causing significant attenuation of the diurnal variation in cone vision [61].
These new data suggest a melanopsin-dependent regulation of visual processing within the
retina and reveal an important function for inner retinal photoreceptors in optimizing classical
visual pathways according to time of day. In humans, it has also been reported that this new
photoreceptive system may actually modulate the entire visual system [45].
4 Role of the photoreceptors in regulation of circadian rhythms
Retinal photoreceptors play a key role in the circadian organization of the whole organism
since they are the only source of photic input to the SCN and, hence, to the rest of the body.
Some investigations have reported changes in several parameters of the circadian rhythm in
locomotor activity after photoreceptor degeneration or bilateral enucleation [32,62,63].
Bilaterally enucleated hamsters showed a wider range of free-running periods than did intact
hamsters held for the same length of time in constant darkness; this effect seems to be
independent from the age at which the animals were enucleated (postnatal days 1, 7, or 28);
on the other hand the average free-running period of intact animals maintained in DD from
days 7 or 28 was longer than that of intact animals kept in DD from day 1 or that of any of the
enucleated groups, suggesting that the exposure to light-dark cycles in the early days of
postnatal life affect the free-running period [63].
Lupi et al. [62] showed that, in rdta mice (a transgenic mouse in which the rod photoreceptors
degenerate very rapidly), the free-running period is significantly shorter the magnitude of light-
induced phase shift is significantly increased, and the irradiance required to produce a
saturating phase shift is also significantly increased with respect to the wild-type. In RCS rats,
the free-running period of the rat with dystrophic retina was significantly shorter, and the
magnitude of the light-induced phase shifts tended to be larger in rats with dystrophic retinas
than in the control group [32]. However, in rd mice and in mice lacking of all the photopigments
(i.e., rhodopsin, cone opsin, and melanopsin), the free-running period was not different from
that of the wild-type [6,36].
A few authors have proposed that the changes in the free-running period observed after
photoreceptor degeneration or enucleation are due to alteration in the relationships between
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the retinal and the SCN clocks [62–64]. However, previous studies have shown that, in RCS
rats with retinal dystrophy, the retinal circadian clock still functions [56,65], so it is unlikely
that the changes we have observed in the free-running period of rdy rats are a consequence of
the presence or absence of the retinal circadian clock. The same explanation is probably valid
also for the mouse since the loss of photoreceptors in the mouse also does not necessarily
destroy the retinal clock [66].
We believe that the changes in the circadian parameters observed in animals with retinal
degeneration are likely to be due to the inappropriate signals transmitted from the retina to the
SCN, either via the retinal hypothalamic tract or via accessory pathways, or that the changes
in the free-running periods observed are the result of a secondary effect of photoreceptor
degeneration on the developing SCN.
Recent studies have reported that retinal degeneration in mice may change the phase at which
the animals are active (nocturnal vs. diurnal). Under normal conditions, mice are nocturnal;
however, mice that lack rods and functional ipRGCs [67] and mice that lack melanopsin and
RPE65 (a key protein used in retinal chromophore recycling) became diurnal [68], suggesting
that changes in retinal input can affect the temporal niche at which the animals are active.
Overall, these data indicate that the influence of the retinal photoreceptors on the regulation of
circadian rhythm of locomotor activity may be difficult to predict (Table 1). It is not clear
whether these changes are the result of interactions between the two clock systems or to other
unknown factors that may be altered by photoreceptor degeneration or removal of the eyes.
5 The SCN plays a role in light transduction
Finally, there is evidence that light has separate and discrete effects on the circadian system.
When light is presented at night, glutamate release will phase-shift the circadian pacemaker
located in the SCN through its actions on NMDA and non-NMDA excitatory amino acid (EAA)
receptors [69–71]. In addition to this effect on the circadian clock, light exposure at night
induces a rapid decrease in the melatonin levels (within 5 min), which is due to the action of
proteasomal proteolysis on the arylalkylamine N- acetyltransferase protein [72]. Since
melatonin is not stored within the pineal but freely diffuses as soon as it has been synthesized,
the rapid destruction of the AA-NAT protein has, as a consequence, an almost immediate
decrease in pineal, and then plasma, melatonin levels.
A series of recent studies has investigated the converging mechanisms that underlie these two
different responses to light. First, it was shown that injections of either NMDA or non-NMDA
excitatory amino acid (EAA) receptor antagonists into the SCN inhibit the ability of light to
increase Per1 mRNA levels but do not disrupt the ability of light to suppress pineal melatonin
levels [73]. Then it was demonstrated that sodium-dependent action potentials in the SCN
region are necessary for the retina to signal the pineal gland that light is present [74]. Although
tetrodotoxin (TTX) was unable to block the effects of NMDA on Per1 and Per2 mRNA levels
and phase-shifting, TTX did block the ability of NMDA to suppress pineal melatonin,
suggesting that the SCN cells that mediate the suppression of pineal melatonin in response to
light are separate from and independent of those that phase-shift circadian rhythms. The
simplest explanation for these observations is that TTX blocks the electrochemical activity of
SCN cells that are essential for light to suppress melatonin but that action potentials in these
cells are not involved in regulating Per1 and Per2 mRNA levels or phase-shifting [74]. Another
study has also shown that NPY-receptor activation can inhibit the light-induced increase in
Per1 mRNA but not the light-induced suppression of melatonin [75]. Therefore, it is possible
to modulate pineal melatonin levels via the SCN without modulating Per1 mRNA levels in
the SCN, and vice versa. Taken together, these results suggest that the photic transduction
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pathway within the SCN that results in suppression of pineal melatonin during the night is
independent of the photic transduction pathway that upregulates Per1 mRNA.
An important implication of these studies is that SCN cells have a discrete phototransductory
role in which they function as a neural relay between retinal ganglion cells and the sympathetic
nervous system. It may not seem economical for SCN cells to develop a separate mechanism
to communicate light signals to extra-circadian processes, but the SCN exert control over the
pineal through a sympathetic pathway that includes the paraventricular nucleus, the intermedio-
lateral cell column of the spinal cord, and the superior cervical ganglion [76-78]. In rodents
whose SCN are intact, light acutely influences an array of autonomic functions that include
stimulatory and inhibitory effects (depending on the time of day) on heart rate [43,79,80] and
blood pressure [80]. Light also acutely reduces core body temperature [81] and acutely
increases glucocorticoid secretion from the adrenal glands [82]. These studies further
demonstrated that, in addition to abolishing circadian rhythms, SCN ablation eliminates the
acute effects of light on each of these processes. If the SCN serve as both the primary circadian
pacemaker and a relay point for sympathetic structures downstream of the SCN, and both
functions have to be responsive to light, then it makes sense for SCN cells to have developed
separate mechanisms to communicate light information. An extension of this view is that, under
certain circumstances, SCN cells can discriminate between the two roles and convey light
signals to one pathway at the expense of the other. Such a mechanism would be effective for
enhancing sympathetic activity without disturbing the pacemaker or, conversely, for entraining
the pacemaker without influencing sympathetic activity.
6 Conclusions
Studies in the last 10 years have demonstrated that a new type of photoreceptor is present in
the mammalian retina and that this new photoreceptive system plays a key role in the
entrainment of the circadian rhythms. This non-image-forming photoreceptive system projects
not only to the SCN, but also to several brain areas that are involved in the regulation of several
functions (e.g., sleep). Therefore, this non-image-forming system may also affect several
aspects of the mammalian health independently from the circadian system.
Acknowledgments
Supported by NIH grants NS 43459 to G.T., NS060659 to K.N.P., and T32MH65740 T.B.S.
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Fig. 1.
Two types of melanopsin containing retinal ganglion cells (mRGCs) M1 and M2 are present
in the retina. Earlier research highlighted type III mRGC'scells and type II mRGC'smore recent
studies refer to these as M1 cells (type III) and M2 cells (type II) based upon morphologic and
physiologic comparisons. The width of the projection from the M1 cell to the suprachiasmatic
nucleus (SCN) of the hypothalamus represents the proportion of innervation, 80%, versus 20%
from the M2 cells. The innervation to the olivary pretectal nucleus (OPN) from M1 cells is
55% versus 45% from M2 cells. A recent study shows that the dendrites of M1 mRGC'sare
localized to sublamina A of the innerplexiformlayer (off), while M2 dendrites localize to
sublamina B (on) as depicted by the difference in stratification. The dendrites of the M2
mRGC'sare considerably more complex and span a larger diameter than the M1 mRGC's. The
M1 cells are considerably smaller but respond with significantly larger depolarizations and
light-induced currents than do the M2 cells. The red and blue arrows represent the exclusive
dendrodentdriticplexus between M1 mRGC'sand the dopaminergicamacrine cells of the inner
nuclear layer. The other neural targets of mRGCs not shown in the figure include the pre-optic
area, sub paraventricular zone, anterior hypothalamic nucleus, lateral hypothalamus, medial
amygdaloid-nucleus, lateral habenula, lateral geniculate nucleus (dorsal division), bed nucleus
of the stria terminalis, periaqueductal gray, and superior colliculus. (OS outer segments; IS
inner segments; ONL outer nuclear layer; OPL outer plexiform layer; INL inner nuclear layer;
IPL innerplexiform layer; GCL ganglion cell layer)
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Paul et al. Page 13
Table 1
Summary of the effects that retinal photoreceptor removal and enucleation produces on the period length and
phase in nocturnal rodents (KO=knock out)
Species Phenotype Effect References
Mouse rodless (rd) no change [6,36,62]
Mouse rodless/coneless (rdcl) no change [7]
Mouse rodless (rdta) shorter (0.3 h) [62]
Mouse Opn4 KO no change [30,31,36]
Mouse Opn4-Rpe65 KO phase-change [68]
Mouse Only cones phase-change [67]
Mouse Opn/rd KO no change [36]
Mouse Opn4/Gnat1/Cnga3 KO no change [20]
Mouse enucleation no change [64]
Hamster enucleation no change [64]
Hamster enucleation broader free-running [63]
Rat enucleation no change [32]
Rat rdy (retinal degeneration) shorter (1 h) [32]
Rev Endocr Metab Disord. Author manuscript; available in PMC 2010 December 1.
