A mammalian neural tissue opsin (Opsin 5) is a deep

brain photoreceptor in birds

Yusuke Nakanea, Keisuke Ikegamia, Hiroko Onoa, Naoyuki Yamamotob, Shosei Yoshidac, Kanjun Hirunagid,
Shizufumi Ebiharae, Yoshihiro Kubof, and Takashi Yoshimuraa,g,h,1
a
Laboratory of Animal Physiology, bLaboratory of Fish Biology, eDivision of Biomodeling, gAvian Bioscience Research Center, Graduate School of
Bioagricultural Sciences, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8601, Japan, hGlobal COE Program Advanced Systems-Biology, Nagoya
University, Furo-cho, Chikusa-ku, Nagoya 464-8601, Japan; dNagoya University Museum, Furo-cho, Chikusa-ku, Nagoya 464-8601, Japan; cDivision of Germ Cell
Biology, National Institute for Basic Biology, Myodaiji, Okazaki, Aichi 444-8787, Japan; and fDivision of Biophysics and Neurobiology, Department of
Molecular Physiology, National Institute for Physiological Sciences, Myodaiji, Okazaki, Aichi 444-8585, Japan

Edited by Joseph S. Takahashi, Howard Hughes Medical Institute, University of Texas Southwestern Medical Center, Dallas, TX, and approved July 9, 2010
(received for review May 7, 2010)

It has been known for many decades that nonmammalian vertebrates detect light by deep brain photoreceptors that lie outside the
retina and pineal organ to regulate seasonal cycle of reproduction.
However, the identity of these photoreceptors has so far remained
unclear. Here we report that Opsin 5 is a deep brain photoreceptive
molecule in the quail brain. Expression analysis of members of
the opsin superfamily identied as Opsin 5 (OPN5; also known as
Gpr136, Neuropsin, PGR12, and TMEM13) mRNA in the paraventricular organ (PVO), an area long believed to be capable of phototransduction. Immunohistochemistry identied Opsin 5 in neurons
that contact the cerebrospinal uid in the PVO, as well as bers
extending to the external zone of the median eminence adjacent
to the pars tuberalis of the pituitary gland, which translates photoperiodic information into neuroendocrine responses. Heterologous
expression of Opsin 5 in Xenopus oocytes resulted in light-dependent activation of membrane currents, the action spectrum of which
showed peak sensitivity (max) at 420 nm. We also found that
short-wavelength light, i.e., between UV-B and blue light, induced
photoperiodic responses in eye-patched, pinealectomized quail.
Thus, Opsin 5 appears to be one of the deep brain photoreceptive
molecules that regulates seasonal reproduction in birds.

circadian rhythms Japanese quail photoperiodism

organ cerebrospinal uid-contacting neuron

| paraventricular

nimals living outside the tropics use changes in the photoperiod to adapt to seasonal changes in the environment. In
addition to the photoreceptive retina and pineal organ, nonmammalian vertebrates are known to have additional photoreceptors that mediate seasonal changes in physiology and
behavior (1). In 1911, Karl von Frisch rst reported evidence for
the existence of deep brain photoreceptors that mediate skin
color changes in minnows (Phoxinus laevis) (2). Subsequently,
Benoit demonstrated that blind ducks showed normal testicular
growth in response to a long-day stimulus, whereas a black cap
placed on the ducks head abolished this response (3). Menaker
et al. demonstrated that injection of India ink under the scalp of
a sparrow also abolishes testicular recrudescence, whereas pinealectomy does not interfere with this response (4). The Japanese
quail (Coturnix japonica) is an excellent model to study these
processes because of its rapid and robust response to changes in
the photoperiod (5, 6). Removing the eyes and/or pineal gland
from quail does not inuence the photoperiodic response (7). In
contrast, local illumination of the mediobasal hypothalamus
(MBH) or septal region of the telencephalon leads to gonadal
growth, suggesting the existence of deep brain photoreceptors in
these regions (1, 8). In 1985, Foster et al. and Foster and Follett
reported an action spectrum for the photoperiodic responses of
quail and suggested the possible involvement of rhodopsin-like
photoreceptors (9, 10). Indeed, gene expression and immunoreactivity for rhodopsin have been reported in the septal region of
the pigeon (11, 12). Surprisingly, in several studies, rhodopsin-like

1526415268 | PNAS | August 24, 2010 | vol. 107 | no. 34

immunoreactivity has not been successfully detected in the quail

(13, 14). Recently, mRNA expression of melanopsin (Opn4) in
the chick septal region and VA opsin-like immunoreactivity in the
chicken and quail anterior hypothalamus have been reported, and
these opsins are suggested to be candidate deep brain photoreceptors (15, 16). However, the functional signicance of these
candidate photopigments remains unknown, along with the identity of the deep brain photoreceptors.
In this study, we identied expression of a novel mammalian
neural tissue opsin (Opsin 5) within the quail MBH. Functional
characterization demonstrated that Opsin 5 is a violet-sensitive
photopigment. Furthermore, short-wavelength light induced testicular growth in eye-patched, pinealectomized quail.
Results
Localization of Opsin 5 in the PVO and Its Projections to the External
Zone of the Median Eminence. The recently available chicken ge-

nome sequence has facilitated comprehensive expression analyses in quail, because these species are both galliformes with high
interspecies DNA sequence conservation (17). To identify opsin
genes that are expressed in the MBH and/or the septal region,
we rst performed a database search to identify DNA sequences
of opsin superfamily members; these sequences were used to
create 33P-labeled oligonucleotide probes for in situ hybridizations
(Table S1). Among the 12 opsin genes examined, the expression of
only OPN5 (Opsin 5: also known as Gpr136, Neuropsin, PGR12
and TMEM13) was detected in the PVO (Fig. 1A and Fig. S1). We
did not detect the expression of any of the examined genes in the
septal region (Fig. S1). The PVO is composed of three layers of
neurons arranged in parallel to the surface of the third ventricle
(18). The neuronal perikarya of the rst layer are located either in
the ependyma or just underneath it (i.e., the subependyma). The
perikarya of the second and third neuronal layer are located farther away from the ependyma. OPN5 expression in the PVO was
further conrmed using a digoxigenin (DIG)labeled riboprobe,
which produced a strong signal in the subependymal layer of the
PVO (Fig. 1B and Fig. S2).
Of note, bipolar subependymal neurons in the PVO that contact the cerebrospinal uid (CSF) have been considered as candidate deep brain photoreceptors for several decades, because

Author contributions: T.Y. designed research; Y.N., K.I., H.O., N.Y., S.Y., K.H., Y.K., and T.Y.
performed research; S.E. contributed new reagents/analytic tools; Y.N., K.I., H.O., N.Y.,
S.Y., K.H., Y.K., and T.Y. analyzed data; and Y.N. and T.Y. wrote the paper.
The authors declare no conict of interest.
This article is a PNAS Direct Submission.
Data deposition: The sequence reported in this paper has been deposited in the GenBank
database (accession no. AB547151).
1

To whom correspondence should be addressed. E-mail: takashiy@agr.nagoya-u.ac.jp.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.

1073/pnas.1006393107/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1006393107

ingly, projections from the PVO to the median eminence and the
pituitary gland have been reported in several other species (18).
Functional Characterization of Opsin 5 as a Photopigment. Vertebrate rhodopsin and cone opsins signal through transducin (Gt),
whereas many invertebrate opsins signal through Gq protein.
Sequence analysis of the opsin superfamily has shown that Opsin 5
is related to invertebrate opsins (22, 23). In addition, we detected
the expression of the Gq protein (GNAQ) gene in the PVO (Fig.
S5). We therefore predicted that Opsin 5 signals through Gq
protein. Xenopus oocytes are known to possess an endogenous
Ca2+-Cl channel that is activated through Gq protein. To test the
ability of Opsin 5 to form a functional sensory photopigment, we
expressed quail Opsin 5 in Xenopus oocytes and analyzed the
resulting current under voltage-clamp conditions (24). Illumination with bright white light (> 1,000 lx) induced light-dependent
activation of membrane currents in oocytes injected with OPN5
5-capped cRNA (Fig. 2 A and C). This photocurrent was not
observed in noninjected oocytes (Fig. 2 B and D). These results
clearly demonstrated that Opsin 5 is a functional photopigment.

these neurons structurally resemble modied pineal photoreceptors and photoreceptor cells in the developing retina (19). To
further detail the localization of Opsin 5, we generated various
anti-quail Opsin 5 antibodies. Among them, one that recognized
a C-terminal region of Opsin 5 was highly specic for quail Opsin 5.
Western blot analysis of quail MBH extracts revealed an immunoreactive band of the expected size (42 kDa) (Fig. S3A). The
1.9-kDa synthetic Opsin 5 peptide used to produce the antibody
was also detected (Fig. S3A). Most of the cells labeled with this
antibody were CSF-contacting neurons in the rst layer (i.e., neurons with a cell body localized in the subependymal layer and
dendritic process with a knob-like terminal that penetrates the
ependyma) and a few immunopositive cells were also observed in
the second layer (Fig. 1 C and D and Fig. S3C). These immunopositive signals were eliminated after the antibody was preincubated
with the synthetic Opsin 5 peptide (Figs. S3 B and D). We also observed Opsin 5-positive bers in the external layer of the median
eminence adjacent to the pars tuberalis of the pituitary gland (Fig.
1E). Recently, long-day induction of TSH expression in the pars
tuberalis was shown to trigger the photoperiodic response, and the
pars tuberalis was reported to be the key organ that relays photoperiodic information to a neuroendocrine output (17, 20, 21). To
examine neural projections in this area, a neural tracer (DiI) was
applied to the PVO. DiI-labeled bers were observed in the external zone of the median eminence (Fig. 1F and Fig. S4). InterestNakane et al.

PHYSIOLOGY

Fig. 1. Localization of Opsin 5 in the PVO and its projections to the external
zone of the median eminence. (A) Schematic drawing of the quail mediobasal hypothalamus. (B) In situ hybridization of OPN5 mRNA in the PVO.
(CE) Representative image of Opsin 5-like immunoreactivity in PVO neurons
that contact the CSF (C and D) and bers in the external zone of median
eminence (ME) (arrowhead) adjacent to the pars tuberalis (PT) of the pituitary gland (E). Note that the angles of the sections are slightly different
between B and C (Fig. S7). (F) The neural tracer DiI was applied to the PVO
(arrow) and labeled bers in the external zone of the ME (arrowheads).
(Scale bars: 10 m, D; 100 m, B, C, and E; and 200 m, F.) 3V, third ventricle.

Fig. 2. Functional characterization of Opsin 5 as a photopigment. Current

recordings in Opsin 5-expressing oocytes (A) and noninjected oocytes (B)
before and after light exposure. (C and D) The time course of the response is
shown by plotting the current amplitudes measured at the end of the
depolarizing pulses over time. Arrows indicate the timing of light exposure.
(E) Irradiance-response curves for monochromatic light pulses. Bars indicate
mean SEM (n = 37). (F) Action spectrum for the Opsin 5-mediated photocurrent. Half-activation values derived from sigmoidal ts of irradianceresponse curves were plotted versus the wavelength and t with a curve for
a retinal1-based photopigment.

PNAS | August 24, 2010 | vol. 107 | no. 34 | 15265

We next examined the spectral sensitivity of the Opsin 5-mediated

photocurrent in oocytes. The effects of monochromatic light
pulses were examined and the irradianceresponse relationships
for different wavelengths were measured (Fig. 2E). Relative
quantum sensitivity to each wavelength was determined from the
half-maximum response and an action spectrum was generated
(25, 26). The action spectrum closely matched that predicted for
a retinal1-based pigment with a max of 420 nm (419 nm and 418
nm in two independent experiments) (Fig. 2F).
Effects of Short-Wavelength Light on Testicular Growth in EyePatched, Pinealectomized Quail. We examined testicular growth in

eye-patched, pinealectomized quail exposed to 2 wk of photostimulation with UV-B, UV-A, or blue light (Fig. S6). The
light intensity for each wavelength (UV-B, 0.2 mW/cm2; UV-A,
2 mW/cm2; blue, 1 mW/cm2) was less than that observed under
direct sunlight in Japan, the natural habitat of the wild quail (UVB, 0.2 mW/cm2; UV-A, 5 mW/cm2; blue, 13 mW/cm2). As
shown in Fig. 3, long-day stimulus with each of the short wavelengths induced testicular growth, the magnitude of which was
similar to that observed in birds exposed to white light (600 lx).
Discussion
In this paper, we have described the identication of a previously
uncharacterized photoreceptive molecule, Opsin 5, in CSF-contacting neurons of the PVO. We also found projection of Opsin 5
neuron to the external zone of the median eminence adjacent to
the pars tuberalis of the pituitary gland. Thus, we propose that
the Opsin 5-positive PVO neurons comprise one of the longsought deep brain photoreceptors that mediate seasonal reproduction in birds (Fig. 4).
Our present ndings may have implications for evolutionary
and developmental biology, because the retinal and pineal photosensory cells structurally resemble these neuronse.g., they
send dendritic processes into the pineal lumen and the photoreceptor space of the retina, and both are derived from diverticles of
the third ventricle of the diencephalon (18). Although immunoreactivities for most of the opsins are generally observed in the
photoreceptor outer segments or cilia-like structures of the
modied photoreceptor cells, immunoreactivity of Opsin 5 was
also observed in the axons of the external zone of the median
eminence as well as in the cell bodies of the PVO. As Opsin 5
appears to activate the Ca2+-Cl channel via the Gq protein, it is
possible that not only Opsin 5 localized in the cell bodies but also
Opsin 5 localized in the axons activate membrane currents. It is
well established that the CSF-contacting neurons in the PVO

Fig. 3. Effects of short-wavelength light on testicular growth in eyepatched, pinealectomized quail. Quail were subjected to 2 wk of photostimulation with UV-B, UV-A, blue, or white light, and testicular growth was
examined. Values are mean + SEM (P < 0.01, ANOVA, F4,37 = 72.6, n = 710;
*P < 0.01, Scheff post hoc test). (Scale bar, 1 cm.) LD, long-day; SD, short-day.

15266 | www.pnas.org/cgi/doi/10.1073/pnas.1006393107

Fig. 4. Model of photoperiodic signal transduction pathway in birds. Light

detected by Opsin 5-positive PVO neurons that contact the CSF is transmitted to the pars tuberalis (PT) of the pituitary gland and induces thyroidstimulating hormone (TSH) expression in the PT. PT TSH induces expression
of type 2 deiodinase (DIO2) in tanycytes lining the ventrolateral walls of the
third ventricle (3V) (17). DIO2 converts prohormone T4 to bioactive T3 (6).
Long-dayinduced T3 in the MBH causes morphologic changes in GnRH nerve
terminals and glial processes and induces GnRH secretion.

contain serotonin or dopamine (18). Neurotransmitters used by

Opsin 5 neurons remain to be identied in the future.
Photons with longer wavelengths are generally thought to penetrate into the brain more effectively than photons with shorter
wavelengths (10, 27, 28), which seems to contradict our results.
Foster et al., Foster and Follett, and Halford et al. reported an
action spectrum for the photoperiodic response in quail with a max
of 483492 nm (9, 10, 16). Careful analysis of their results, however, revealed that light with a wavelength of 410 nm also caused
a photoperiodic response. Furthermore, Oishi used a large spectrograph (National Institute for Basic Biology) to show that light
with a wavelength of 350 nm also induced testicular growth (29).
This result seems reasonable because UV light reaches the quail
hypothalamus (10, 28), and feathers, skin, bone, and brain tissue
are known to autouoresce in the range from UV to blue light in
response to UV irradiation (3032). Although another unidentied
UV photoreceptor may be present in the quail brain, Opsin 5 in the
PVO appears to cover a relatively wide range of short-wavelength
light from UV to blue light. The effects of short-wavelength light on
avian photoperiodism have not been extensively examined because
of the low transmission efciency. However, it is reasonable to
hypothesize that birds also use short-wavelength light in their natural habitats, because UV and blue light are known to regulate
photoperiodism in mammals and insects (33, 34).
Although we failed to detect opsin gene expression in the septal
region, it is important to bear in mind that local illumination of
the septal region of the telencephalon also induces testicular
growth (1, 8). Because nucleotide sequences are higly conserved
between chicken and quail, chicken and quail probes are widely
used for both species in methods such as Northern hybridization,
Southern hybridization, in situ hybridization, chromosomal mapping using FISH, and microarrays (17, 3540). Therefore, we used
chicken DNA sequences for in situ hybridization with 33P-labeled
Nakane et al.

Materials and Methods

Animals. Japanese quail (Coturnix japonica) and Xenopus laevis were used in
this study, which was approved by the Animal Experiment Committee of
Nagoya University and the National Institute for Physiological Sciences.
In Situ Hybridization. Nonperfused frozen sections were examined with 33Plabeled oligonucleotide probes as previously described (40). The probe
sequences are shown in Table S1. Parafn sections were examined with DIGlabeled riboprobes as previously described (47). The entire ORF of OPN5 was
used to generate the DIG-labeled riboprobe. BM purple AP substrate (Roche)
was used to visualize hybridization, which was followed by counterstaining
with nuclear fast red (Merck).
Antibodies. We generated rabbit polyclonal antibodies against a C-terminal
peptide from quail Opsin 5 (CISSHRDSAALSETQLEV). Puried peptides (Opsin
5-KLH) were dissolved in 8 M urea containing 1,3-diaza-2,4-cyclopentadiene
(imidazole) and used as an immunogen. After injecting the immunogen into
each rabbit six times, blood was collected and IgG molecules were puried
using peptide-Sepharose afnity chromatography. Puried antibodies were
dialyzed in PBS and concentrated. IgG in PBS containing 50% glycerol was
stored at 30 C. Antibodies were diluted at 1:5005,000 for Western blotting
and 1:1004,000 for immunohistochemistry.
Western Blotting. Western blotting was performed as previously reported
(48). Brain slices (3 mm) were generated by a mouse brain matrix (ASI
Instruments), and the MBH, including the PVO (2.5-mm diameter), was
punched out (6). Membranes were incubated with anti-quail Opsin 5 antibodies (1:5,000; nal concentration of 0.278 g/mL). To ascertain the specicity of the immunostaining, immunoadsorption analysis was performed

Nakane et al.

with a solution in which the molar ratio of anti-quail Opsin 5 antibodies and
the indicated antigen peptides was 1:20.
Immunohistochemistry. Immunohistochemistry was performed using antiquail Opsin 5 antibodies (1:1,000) and a Vectastain Elite ABC rabbit IgG kit
(Vector Laboratories) with a standard protocol (48).
Carbocyanine Dye (DiI) Experiments. Following deep anesthesia with pentobarbital, quail were perfused with 0.75% saline followed by 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4). The brain was postxed
in fresh xative for 2 d. After postxation, the brain was cut in half along
the midsagittal plane with a razor blade. A small carbocyanine dye (1,1dioctadecyl-3,3,3,3-tetramethylindocarbocyanine perchlorate: DiI) crystal
(Molecular Probes) was placed onto the paraventricular organ. The insertion
site was covered with 5% gelatin and stored in the same paraformaldehyde
xative at 37 C in darkness for 14 d. Brains were then sectioned coronally
using a microslicer (D.S.K., DTK-3000) at 50 m. Sections were observed under
a BZ-9000 microscope (Keyence).
Preparation of Xenopus Oocytes. Xenopus oocytes were collected from frogs
anesthetized in water containing 0.15% tricaine (24). 5-capped cRNA was
prepared from quail OPN5 inserted in the pGEMHE vector using an in vitro
transcription kit (mMESSAGE mMACHINE kit, Ambion), and the yield was
estimated by loading cRNA onto an agarose gel. The isolated oocytes were
treated with 2 mg/mL collagenase type 1 (Sigma-Aldrich) for 6 h to remove
follicular cells. Oocytes were injected with 55 nL cRNA solution and incubated in standard frog saline [88 mM NaCl, 1 mM KCl, 0.3 mM Ca(NO3)2,
0.41 mM CaCl2, 0.8 mM MgSO4, 2.4 mM NaHCO3, and 15 mM Hepes at
pH 7.6] at 17 C overnight in a dark box.
Electrophysiology. Oocytes were positioned and impaled for recordings under
dim light,and recordings were carried out under dark conditions. Oocytes were
incubated in 40 M 11-cis retinal (a gift from R.K. Crouch, Medical University
of South Carolina, Charleston, SC) in standard frog saline supplemented with
0.1% penicillinstreptomycin (Sigma-Aldrich) for 1 h in a dark box at 17 C
before recording. Recordings were made in Ca2+-free saline (96 mM NaCl, 2
mM KCl, 3 mM MgCl2, and 5 mM Hepes at pH 7.4) to reduce the basal Ca2+-Cl
current (24). The Ca2+-Cl current was monitored by applying a 175-ms
depolarizing pulse to 60 mV from a holding potential of 80 mV every 2 s and
light irradiation began 8 s after recording was initiated.
Spectral Analysis. Monochromatic light was produced using an interference
lter (half-bandwidth, 7.014.2 nm; Vacuum Optics Co.) and the light intensity was adjusted using neutral density lters (25). A halogen lamp (NPI
PCS-UNX; 400, 450, 475, and 500 nm) and UV LED (375 nm; Nitride Semiconductors Co.) were used as light sources. Light intensities (W/cm2) were
measured using a radiometer (S371, UDT Instruments; UVR-300, Topcon).
Measurements at 375 nm were tted to the Hill function using the leastsquares method, wherein the mean amplitude (22 A) of the saturated response evoked by full-strength white light was used as the peak value for
the tting. Based on the parallel nature of these curves, the Hill coefcient
for the data collected at 375 nm was used for curve tting data obtained for
other wavelengths (25). Relative quantum sensitivity to each wavelength
was determined from the half-maximum response and t with a retinal1based photopigment template function (26).
Effect of Short-Wavelength Light on Testicular Growth in Eye-Patched, Pinealectomized Quail. Four-week-old male quail were kept under short-day
conditions (6 h/18 h light/dark cycle) for 4 wk in light-tight boxes. At 7 wk of
age, the pineal gland was removed. At 8 wk of age, both eyes from each
pinealectomized quail were covered with eye patches to block light. Eye
patches were made by cutting the adhesive part of a Band-Aid (10 mm in
diameter), and pasting black tape on the nonadhesive side. After feathers from
the area surrounding the eye were removed, rubber cement was applied both
around the eye and around the edge of the patch, and the patch was placed
over the eye. These quail had no difculty feeding. To assess the effects of 2 wk
of photostimulation on testicular growth, long-day quail were transferred
from short-day to long-day conditions (20 h/4 h light/dark cycle) for 2 wk,
whereas short-day quail were kept under short-day conditions. Photic stimuli
were produced using low-pressure mercury vapor uorescent lamps (Philips
UV-B, TL/12; UV-A, TL/08; blue, TL/52) (Fig. S6). The light intensity for each
wavelength (UV-B, 0.2 mW/cm2; UV-A, 2 mW/cm2; blue, 1 mW/cm2) was less
than that observed under direct sunlight in Japan, the natural habitat of the
wild quail (UV-B, 0.2 mW/cm2; UV-A, 5 mW/cm2; blue, 13 mW/cm2).

PNAS | August 24, 2010 | vol. 107 | no. 34 | 15267

PHYSIOLOGY

riboprobes. However, we cannot exclude the possibility that some

of the chicken probes did not work in quail. In addition, it is
reported that expression of some opsin genes is rhythmically
regulated (15, 41). Because we collected brains in the middle of
the day, the expression of some genes may have been too low for
detection at that time point. It is also possible that the density of
opsin-expressing cells and/or the levels of opsin mRNA are below
the limits of detection with our current methods. Multiple photoreceptors are likely involved in the photoperiodic response, as
has been reported for circadian photoreception, which involves
both visual photoreceptors (rod and cones) and nonvisual photoreceptors (melanopsin) (42, 43). Although rhodopsin-like immunoreactivity has been observed in the pigeon septal region (11,
12), multiple independent studies failed to detect similar signals
in the quail brain (13, 14). Expression of melanopsin has been
demonstrated in the lateral septal region as well as the retina and
pineal gland in chicks (15). Recently, vertebrate ancient (VA)
opsin-like immunoreactivity was detected in the avian anterior
hypothalamus (16). Although spectral sensitivities of avian melanopsin and VA opsin remain to be determined, both mammalian
melanopsin and teleost VA opsin have a max of 480 nm (44, 45).
This max is consistent with the action spectrum for photoinduced
LH release, suggesting possible involvement of melanopsin and
VA opsin in the photoperiodic response. However, neural projections from melanopsin- and VA opsin- expressing neurons to
the pars tuberalis, and their physiological signicance remains to
be claried. It is particularly noteworthy that local illumination of
MBH, including PVO, induces testicular growth, and PVO
lesions block the photoperiodic response of the gonads; this
highlights the physiological role of the PVO in the regulation of
seasonal reproduction (8, 46). The gene-targeting technique is
still unavailable for research in avian species. Analyses of the
effect of Opsin 5 and/or melanopsin and/or VA opsin knock-outs
on the photoperiodic response are required for conclusive identication of avian deep brain photoreceptors in future.
The eye is believed to be the only photoreceptive organ in
mammals. The mammalian ortholog of OPN5 is reportedly
expressed in the neural tissues (retina, brain, and spinal cord) and
the testis (22). The photoresponsiveness and physiological roles of
mammalian Opsin 5 should be addressed in future studies.

ACKNOWLEDGMENTS. We thank Nobuhiro Nakao, Naoki Takai, Tsuyoshi

Shimmura, Yuta Hoshino, Itaru Murayama, Eriko Takarada, Tomomi Yamamoto, Mamiko Sukeno, Hideki Abe, Hideki Ukai, and Hiroki R. Ueda for
technical support and helpful discussions. We are grateful to R. K. Crouch for
the gift of 11-cis retinal. We thank Nagoya University Radioisotope Center
for use of facilities. This work was supported in part by Grant-in-Aid for

Young Scientists (S) 19678002, Grant-in-Aid for Scientic Research on Innovative Areas, Regulatory Mechanism of Gamete Stem Cells 21116504,
Japan Society for the Promotion of Science Research Fellow, the Global COE
program Advanced Systems-Biology from the Ministry of Education, Culture, Sports, Science and Technology of Japan, Mitsubishi Foundation, and
Toyoaki Scholarship Foundation.

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