1. MELANOPSIN EXPRESSION IN DOPAMINE-MELATONIN NEURONS OF
THE PREMAMMILLARY NUCLEUS OF THE HYPOTHALAMUS AND
SEASONAL REPRODUCTION IN BIRDS
S. W. KANG,a
B. LECLERC,a
S. KOSONSIRILUK,a
L. J. MAURO,a
A. IWASAWAb
AND
M. E. EL HALAWANIa
*
a
Department of Animal Science, University of Minnesota, St Paul, MN
55108, USA
b
Laboratory of Comparative Biochemistry, Graduate School of Applied
Biological Science, Gifu University, Gifu 501-1193, Japan
Abstract—Melanopsin (OPN4) is a photoreceptive molecule
regulating circadian systems in mammals. Previous studies
from our laboratory have shown that co-localized dopamine-
melatonin (DA-MEL) neurons in the hypothalamic premam-
millary nucleus (PMM) are putatively photosensitive and ex-
hibit circadian rhythms in DAergic and MELergic activities.
This study investigates turkey OPN4x (tOPN4x) mRNA distri-
bution in the hypothalamus and brainstem, and characterizes
its expression in PMM DA-MEL neurons, using in situ hybrid-
ization (ISH), immunocytochemistry (ICC), double-label ISH/
ICC, and real time-PCR. The mRNA encoding tOPN4x was
found in anatomically discrete areas in or near the hypothal-
amus and the brainstem, including nucleus preopticus medi-
alis (POM), nucleus septalis lateralis (SL), PMM and the pineal
gland. Double ICC, using tyrosine hydroxylase (TH, the rate
limiting enzyme in DA synthesis)—and OPN4x antibodies,
confirmed the existence of OPN4x protein in DA-MEL neu-
rons. Also, tOPN4x mRNA expression was verified with dou-
ble ISH/ICC using tOPN4x mRNA and TH immunoreactivity.
PMM and pineal gland tOPN4x mRNA expression levels were
diurnally high during the night and low during the day. A light
pulse provided to short day photosensitive hens during the
photosensitive phase at night significantly down-regulated
tOPN4x expression. The expression level of tOPN4x mRNA in
PMM DA-MEL neurons of photorefractory hens was signifi-
cantly lower as compared with that of short or long day
photosensitive hens. The results implicate tOPN4x in hypo-
thalamic PMM DA-MEL neurons as an important component
of the photoreceptive system regulating reproductive activity
in temperate zone birds. Published by Elsevier Ltd on behalf
of IBRO.
Key words: melanopsin, dopamine, turkey, avian reproduc-
tion, in situ hybridization, real time-PCR.
Birds and many other non-mammalian vertebrates have
shown the capacity for extra-retinal photoreception. In
these species, the skin, several deep brain nuclei, and
the pineal gland contain functional photopigments that
are photosensitive and can mediate the effects of light in
the absence of eyes (Groos, 1982). Melanopsin (OPN4) is
one of these photopigments first identified in photosensi-
tive dermal melanophores, deep brain nuclei, and the iris
of Xenopus (Provencio et al., 1998). Subsequently, OPN4
was found in the retinal pigment epithelium (RPE) cells of
the inner retina of frogs and fish where it appears to
function as a phototransducing opsin (Provencio et al.,
1998). In mammals, an ocular output arises from retinal
ganglion cells and projects to the suprachiasmatic nucleus
(SCN) (Moore et al., 1995; Pickard et al., 2002) to entrain
circadian rhythm activity (Yamazaki et al., 2002). Most of
the ganglion cells projecting to the SCN contain OPN4
(Hannibal and Fahrenkrug, 2002; Hattar et al., 2002) and
are intrinsically photoreceptive, responding to light inde-
pendently of the rod and cone photoreceptors used for
vision (Berson et al., 2002). Knockout of OPN4 in mice
results in a loss of intrinsic photosensitivity of these cells
(Lucas et al., 2003). Likewise, in amphibian melanophores,
over-expression of OPN4 augments the photic response
(Rollag et al., 2003). In addition, OPN4 mRNA in the
light-sensitive chick pineal gland displays a daily rhythm
with a peak in the transitional phase from light to dark,
(Holthues et al., 2005). These studies support the concept
that OPN4 mediates photoresponsiveness and modulates
circadian rhythms in diverse cell types in several verte-
brate species.
Although xenopus OPN4 (OPN4x) has a role in avian
pineal photoreception (Chaurasia et al., 2005; Tomonari et
al., 2007), its photoreceptive role in the regulation of re-
productive seasonality in birds has not been investigated.
The existence of internal circadian clocks located in the
mediobasal hypothalamus (MBH) that respond to chang-
ing day length via encephalic (aka extra-retinal or “deep
brain”) photoreceptors has been described in birds (Benoit,
1935; Menaker, 1968). Neurons immunoreactive for vari-
ous opsins occur in the walls of brain ventricles in the avian
hypothalamus and septum (Vigh et al., 2002). Based on
such anatomical evidence, these cells have been assumed
to act as extra-retinal photoreceptors. However, we do not
know whether the opsins of these putative photoreceptor
cells act as functional photopigments and we do not know
what type of phototransduction cascade might be involved.
Recent findings suggest that seasonal reproductive
neuroendocrine activity in the turkey is regulated by puta-
*Corresponding author. Tel: ϩ1-612-624-9744; fax: ϩ1-612-625-
2743.
E-mail address: elhal001@umn.edu (M. E. El Halawani).
Abbreviations: cOPN4x, chicken OPN4x; DA-MEL, dopamine-melato-
nin; Dio2, type 2 deiodinase; GnRH-I, gonadotropin-releasing hor-
mone; ICC, immunocytochemistry; ISH, in situ hybridization; LD, long
day; LH, luteinizing hormone; OPN4x, xenopus OPN4; PBS, phos-
phate-buffered saline; PMM, premammillary nucleus; PRL, prolactin;
PT, pars tuberalis; RF, photorefractory; SCN, suprachiasmatic nu-
cleus; SD, short day.
Neuroscience 170 (2010) 200–213
0306-4522/10 $ - see front matter. Published by Elsevier Ltd on behalf of IBRO.
doi:10.1016/j.neuroscience.2010.06.082
200