BRAIN

RESEARCH
ELSEVIER Brain Research 708 (1996) 177-181

Short communication

Circadian regulation of melatonin production in cultured zebrafish pineal and

retina
Gregory M. Cahill *
Department of Biology, University of Houston, 4800 Calhoun, Houston, TX 77204-5513, USA
Accepted 24 October 1995

Abstract

Melatonin release was measured from zebrafish pineal organs and retinas maintained in flow-through culture. Pineal organs released
melatonin in a strong circadian rhythm through 5 days in constant darkness, and the phase of this rhythm was reset by in vitro exposure to
phase-shifted light cycles. In contrast, the retinal melatonin rhythm rapidly damped out in constant darkness, even in the presence of
(phase-shifted) light cycles. The zebrafish pineal should be useful for in vitro studies of vertebrate circadian clock mechanisms.

Keywords: Circadian regulation; Oscillator; Melatonin; Pineal; Retina; Photoreception; Danio (Brachydanio) rerio; Organ culture

The goal of this initial examination of the zebrafish
(Danio rerio) circadian system was to determine whether
this species might be a useful model system for genetic
analysis of vertebrate circadian clock mechanisms. This
study was inspired by the recent rapid progress in develop-
ment of genetic tools and information for zebrafish
[8,22,25,34]. Genetic studies of circadian rhythmicity in
other species have led to significant recent progress in
understanding circadian clocks. Mutations that affect circa-
dian rhythms have been recovered in organisms ranging
from cyanobacteria to mammals [9,14,17,21,27,33].
Cloning and manipulation of genes identified by mutation
in Drosophila and Neurospora have resulted in identifica-
tion of molecular clock components and development of
new models of clock mechanisms [1,7,14,18]. These devel-
opments underscore the value of genetic approaches in
circadian clock investigations. If zebrafish express strong
circadian rhythms, they could be a useful vertebrate model
system for a genetic approach. Rhythms that are measur-
able from cultured clock tissues would be particularly
useful in determining the cellular effects of circadian
mutations.
In some vertebrates, the pineal organ and retina contain
circadian oscillators that regulate rhythmic melatonin pro-
* Corresponding author. Fax: (1) (713) 743-2636;
gcahill@uh.edu
duction in vitro [6,12,30]. However, the capacity of these
tissues to generate sustained rhythmicity and their roles in
organismal circadian systems vary, even among closely
related species [19,32]. Therefore, I investigated whether
cultured zebrafish pineal organ and retina contain circadian
oscillators capable of driving rhythms of melatonin release.
Light resets circadian oscillator phase and suppresses
melatonin synthesis in cultured pineal and retina of other
non-mammalian species [4,11,12,30,36]. Therefore, the ef-
fects of light cycles in vitro on zebrafish pineal and retinal
melatonin rhythms were also characterized.
Adult zebrafish of both sexes were obtained from Car-
olina Biological Supply (Burlington, NC), kept for at least
2 weeks in aquaria with biological filters at 21C under
cycles of 12 h light and 12 h dark, and fed once per day
with commercial flake food. Fish were killed by decapita-
tion during the light period, and the pineals and retinas
(including the whole neural retina and the attached pig-
ment epithelium) were isolated and placed in superfusion
culture near the normal light offset time. The culture
apparatus is similar to that used previously in studies of
Xenopus photoreceptor rhythms [5]. Pineals and retinas
were cultured individually in microtiter plate wells. Each
well was sealed with a stopper penetrated by inflow and
outflow tubing. The stoppered plates were submerged in
custom-designed water jackets, and the temperature was
E-mail: maintained at 21.1 + 0.1C by a refrigerated circulator. In
vitro light exposure was from a 20 W fluorescent lamp,

0006-8993/96/$15.00 1996 Elsevier Science B.V. All rights reserved

SSDI 0 0 0 6 - 8 9 9 3 ( 9 5 ) 0 1 3 6 5 - 2
178 G.M. Cahill / Brain Research 708 (1996) 177-181

and was directed at the bottom of the culture wells via a 5

plexiglass mirror, through a clear plastic window (1.3 cm
thick) and 1.3 cm of water. The cultures were otherwise
maintained in total darkness. Culture medium was deliv-
4
ered continuously (1 m l / h ) from a 20-channel syringe
pump via thick-walled teflon tubing (Spectrum, Houston,
TX). Superfusate from each well was collected during each
2 h interval of the experiment, starting at the normal
light-offset time. t-
The defined culture medium used in these experiments
was identical to that used previously for superfusion cul- v

ture of Xenopus eyecups [4]. It consists of salts balanced A '

for amphibian tissue, a mixture of 14 amino acids, 35 mM
.ll) ....
0 1 5
NaHCO3, 0.1 mM ascorbic acid, penicillin, streptomycin
-
and 0.1 mM 5-hydroxy-L-tryptophan, which enhances
melatonin production by increasing precursor availability e- Retinas
o 0.6
[3,4]. The medium was saturated with 95% 0 2 / 5 % CO 2. r--q Lighi
In preliminary short-term culture experiments (not shown), ~_ ~ ~, I Dark
zebrafish retinas cultured in this medium produced more 0.4 ~.., ~,~ ,
melatonin and maintained more normal cellular morphol-
ogy than retinas cultured in L15 medium (Gibco, Grand
Island, NY), which has been used previously for culture of
zebrafish cells [34].
0.2 ~
Melatonin content of unextracted superfusate samples
was measured by a modification of the radioimmunoassay
B
~ , , I . I , ' - I , , , I i , , I , , ,

(RIA) of Rollag and Niswender [26,28]. This RIA has been O. 0 1 2 3 4 5

validated for measurement of melatonin released into cul-
ture medium by chick and lizard pineals and Xenopus
eyecups [3,20,28]. Data from light cycle experiments were
normalized to facilitate comparison of rhythms from or-
gans that produced different overall melatonin levels: The
amount of melatonin in each sample from an organ was
divided by the average of all samples from that organ.
These normalized values were then averaged for each
treatment group.
Zebrafish pineals, cultured in constant darkness, re-
leased melatonin in a robust circadian rhythm through the
entire culture period of 4 or 5 days (Fig. 1A). This
demonstrates that the pineal contains a self-sustaining cir-
cadian oscillator. Pineal melatonin release peaked during
the subjective night and returned to low levels during the
subjective day. Melatonin release during the first peak was
consistently 1.5- to 2-fold higher than during the second
peak.
Melatonin release by isolated retinas cultured in con-
stant darkness consistently increased to a peak during the
first night, declined to a trough during the first subjective
day in darkness and rose to a second night time peak, but
then the rhythms damped out (Fig. 1B). The retinas contin-
ued to release melatonin at intermediate to low levels
through 4 days in culture. Similar results (not shown) were
obtained when retinas were cultured in L15 medium equili-
brated with air, in L15 with 35 mM sodium bicarbonate
added and equilibrated with 95%O2/5%CO2, or in a
balanced salt solution developed for goldfish retina [15]. In
histological sections of retinas after 4 days in culture,
Days in Culture
Fig. 1. Circadian rhythms of melatonin release from individual zebrafish
pineals and retinas cultured in constant darkness. Each trace shows
release from a single pineal (A) or retina (B). Each graph shows
representative examplesfrom 2 experiments.Tissues were isolated during
the light period (indicated on the bar above each graph) and placed
individually in flow-through culture chambers at the beginning of the
normal dark period (Time 0). Melatonin release during each 2 h period
was measuredby RIA.
photoreceptor cells appeared essentially normal, but pig-
ment epithelium cells were rounded up and separated from
one another, the inner retina was swollen, and many inner
retinal cell nuclei were pyknotic (data not shown).
Pineal melatonin production was suppressed acutely
during light exposure, and rhythms were reset to opposite
phases after exposure to opposing light cycles in vitro (Fig.
2A). Melatonin release declined rapidly and remained at
low levels throughout each 12 h light exposure, regardless
of the phase of the rhythm when the light treatment was
initiated. Opposing light cycles drove the initially syn-
chronous pineal melatonin rhythms to opposite phases, and
the rhythms persisted in antiphase for 3 cycles in constant
darkness, indicating that the underlying circadian oscilla-
tors were reset. These oscillator phase shifts reflect the
entrainment mechanisms through which endogenously gen-
erated rhythms are synchronized with daily environmental
cycles, and they are common to all intact circadian systems
[24].
 G.M. Cahill / Brain Research 708 (1996) 177-181
41 t~ , , Light
e-
~1) 0 1 2 3 4 5
E 5
._>
Retinas
4 oscillator that can drive one full cycle of melatonin synthe-
n- ~--~lLight
3 Dark
2 quate culture conditions. Retinas continued to produce
0 ~
(11 0 1 2 3 4 5
Days in Culture
Fig. 2. Effects of phase-shifted light cycles on melatonin release rhythms
of cultured zebrafish pineals (A) and retinas (B). Tissues were isolated
during the light period (indicated on the bar above each graph) and placed
individually in flow-through culture chambers at the beginning of the
normal dark period (Time 0). The lighting regimen for each group is
indicated on the bar with the corresponding plot symbol. One group was
treated during 12 h periods beginning 6 and 30 h (filled squares) after
normal light-offset, and the other group was treated during 12 h periods
beginning 18 and 42 h (filled squares) after normal light-offset. After two
12 h light exposures, the tissues were maintained in constant darkness.
Means + S.E.M. of normalized melatonin release are plotted; n = 2 in the
pineal group exposed to advanced cycles, n = 4 - 6 for other groups.
Melatonin production by cultured retinas was sup-
pressed acutely during light exposure during the first 2
days in culture (Fig. 2B). Opposite light cycles thus drove
antiphase rhythms of melatonin. However, peak levels of
melatonin declined during this time, and rhythmicity did
not persist during subsequent exposure to constant dark-
ness. Therefore, it is not clear whether a light-sensitive
oscillator existed in these retinas.
The data presented above demonstrate that the zebrafish
pineal contains a self-sustaining circadian oscillator that
regulates melatonin synthesis, as well as phototransduction
mechanisms sufficient for entrainment of the oscillator.
These are all of the basic components of a circadian clock
[10]. This type of local circadian regulation of melatonin
exists in many non-mammalian vertebrate pineals, includ-
ing pineals of birds [19,28,30,37], Anolis lizard [20] and
179
teleosts, including pike [11], goldfish [16], and white sucker
[36]. However, pineal melatonin rhythms from most of
these species damp out within 5 days of culture in constant
darkness. Melatonin production by trout pineal does not
appear to be controlled by a local circadian oscillator at all,
but it is suppressed by light [12,13]. In contrast, zebrafish
pineal melatonin rhythms are robust through at least 5
cycles in constant darkness, comparable to rhythms mea-
sured from Anolis pineal [20]. Furthermore, although there
is considerable interindividual variability in the total
amount of melatonin produced by zebrafish pineals, there
is little variability in the period of the rhythms (Fig. 1).
These features make the zebrafish pineal an attractive
model system for in vitro studies of circadian oscillator
mechanisms.
The zebrafish retina appears to contain a circadian
sis in vitro, but rhythmicity then damps out. It is unclear to
what extents this damping is due to inherent instability of
the retinal oscillator or to pathology resulting from inade-
melatonin for up to 4 days in culture and were responsive
to light for at least 2 days, indicating that some functions
persist in vitro. Furthermore, the photoreceptor cells, which
are candidate clock cells in the retina of other species [5,6],
appeared morphologically normal after 4 days in culture.
However, degeneration was observed in the pigment ep-
ithelium (which is necessary for maximal retinal melatonin
production [5]), and in many inner retinal cells that might
also have roles in retinal rhythmicity. In chick pineal cells,
exposure to light cycles prevents damping of melatonin
rhythms and resynchronizes them after damping of the
rhythm in constant darkness [30,37], but this was not true
of the zebrafish retina. This suggests that the damping of
the retinal rhythm results largely from degeneration of the
tissue in culture. Several aspects of retinal function are
rhythmic in teleosts, including melatonin synthesis,
dopamine synthesis, retinomotor movements, ultrastruc-
tural features of outer plexiform layer synapses, and visual
response threshold, all measured in vivo [6]. Indeed, an in
vivo circadian retinomotor rhythm has been observed in
zebrafish [2]. However, persistent circadian rhythmicity
has not been measured from any teleost retina in culture,
although it has been observed in cultured preparations of
amphibian and avian retina [4,5,23].
Most approaches to investigation of circadian clock
mechanisms require estimation of oscillator phase and/or
period from measurements of an output rhythm that is
controlled by the oscillator. The persistent high amplitude
of the zebrafish pineal melatonin rhythm facilitates precise
measurement of the phase and period of the underlying
oscillator. The pineal should therefore be useful for 'per-
turbation analysis', in which the role of a candidate mecha-
nism is deduced by altering it and measuring the effect on
oscillator timing [29,30,37]. The pineal rhythm in vitro
should also be useful for analysis of the cellular conse-
180 G.M. Cahill / Bram Research 708 (1996) 177-181
quences of genetic clock mutations, when they become
available. It does not, however, constitute a particularly
efficient measure for screening large populations for mu-
tant circadian clock phenotypes. To be useful in clock
mutant screening, a rhythm should be precise and repeat-
able, easily measurable from a large number of animals
and non-invasive [31]. The pineal and melatonin have been
implicated in regulation of development, metabolism, be-
havior and pigmentation in other fish species [35]. One or
more of these may be regulated as a circadian rhythm that
can be measured easily in a clock mutant screen.
Acknowledgements
This research was carried out at the University of
Kansas Medical Center, Kansas City. I thank Joseph C.
Besharse for access to his laboratory and equipment and
for valuable discussions, Sandra Parsons for technical as-
sistance and Carla B. Green and Arnold Eskin for valuable
discussions and critical comments on the manuscript. This
work was supported by NIH Grant MH49757 and AFOSR
Grant F49620-94-1-0314.
References
[1] Aronson, B.D., Johnson, K.A., Loros, J.J. and Dunlap, J.C., Nega-
tive feedback defining a circadian clock: autoregulation of the clock
gene frequency, Science, 263 (1994) 1578-1584.
[2] Burnside, B., McCormack, C.A., Hoover, K. and McDonnell, M.T.,
Zebrafish cones exhibit circadian- and dopamine-regulated retinomo-
tor movements, Invest. OphthalmoL Vis. Sci., (Suppl.) 34 (1993)
1063.
[3] Cahill, G.M. and Besharse, J.C., Circadian regulation of melatonin
in the retina of Xenopus laevis: limitation by serotonin availability,
J. Neurochem., 54 (1990) 716-719.
[4] Cahill, G.M. and Besharse, J.C., Resetting the circadian clock in
cultured Xenopus eyecups: regulation of retinal melatonin rhythms
by light and D 2 dopamine receptors, J. Neurosci., 11 (1991) 2959-
2971.
[5] Cahill, G.M. and Besharse, J.C., Circadian clock functions localized
in Xenopus retinal photoreceptors, Neuron, 10 (1993) 573-577.
[6] Cahill, G.M. and Besharse, J.C., Circadian rhythmicity in vertebrate
retinas: regulation by a photoreceptor oscillator, Prog. Retinal Eye
Res., 14 (1995) 267-291.
[7] Curtin, K.D., Huang, Z.J. and Rosbash, M., Temporally regulated
nuclear entry of the Drosophila period protein contributes to the
circadian clock, Neuron, 14 (1995) 365-372.
[8] Driever, W., Stemple, D., Schier, A. and Solnica-Krezel, L., Ze-
brafish: genetic tools for studying vertebrate development, Trends
Genet., 10 (1994) 152-159.
[9] Dunlap, J.C., Genetic analysis of circadian clocks, Annu. Rev.
Physiol., 55 (1993) 683-728.
[10] Eskin, A., Identification and physiology of circadian pacemakers,
Fed. Proc., 38(12) (1979) 2570-2572.
[11] Falc6n, J., Marmillon, J.B., Claustrat, B. and Collin, J.-P., Regula-
tion of melatonin secretion in a photoreceptive pineal organ: an in
vitro study in the pike, J. Neurosci., 9(6) (1989) 1943-1950.
[12] Falc6n, J., Thibault, C., Begay, V., Zachmann, A. and Collin, J.-P.,
Regulation of the rhythmic melatonin secretion by fish pineal pho-
toreceptor cells. In M.A. Ali (Ed.), Rhythms in Fishes, Plenum
Press, New York, 1992, pp. 167-198.
[13] Gem, W.A. and Greenhouse, S.S., Examination of in vitro melatonin
secretion from superfused trout (Salmo gairdneri) pineal organs
maintained under diel illumination of continuous darkness, Gen.
Comp. Endocrinol., 71 (1988) 163-174.
[14] Hall, J.C., Tripping along the trail to the molecular mechanisms of
biological clocks, Trends Neurosci., 18 (1995) 230-240.
[15] Harsanyi, K. and Mangel, S.C., Activation of a D 2 receptor in-
creases electrical coupling between retinal horizontal cells by in-
hibiting dopamine release, Proc. Natl. Acad. Sci. USA, 89 (1992)
9220-9224.
[16] Iigo, M., Kezuka, H., Aida, K. and Hanyu, I., Circadian rhythms of
melatonin secretion from superfused goldfish (Carassius aurams)
pineal glands in vitro, Gen. Comp. Endocrinol., 83 (1991) 152-158.
[17] Kondo, T., Tsinoremas, N.F., Golden, S.S., Johnson, C.H., Kutsuna,
S. and lshiura, M., Circadian clock mutants of cyanobacteria, Sci-
ence, 266 (1994) 1233-1236.
[18] Loros, J., The molecular basis of the Neurospora clock, Semin.
Neurosci., 7 (1995) 3-13.
[19] Menaker, M., The search for principles of physiological organization
in vertebrate circadian systems. In J. Aschoff, S. Daan and G. Groos
(Eds.), Vertebrate Circadian Systems, Springer-Verlag,
Berlin/Heidelberg, 1982, pp. 1-11.
[20] Menaker, M. and Wisher, S., Temperature-compensated circadian
clock in the pineal of Anolis, Proc. Natl. Acad. Sci. USA, 80 (1983)
6119-6121.
[21] Millar, A.J., Carr6, I.A., Strayer, C.A., Chua, N.-H. and Kay, S.A.,
Circadian clock mutants in Arabidopsis identified by luciferase
imaging, Science, 267 (1995) 1161-1163.
[22] Mullins, M.C., Hammerschmidt, M., Haffter, P. and Niisslein-
Volhard, C., Large-scale mutagenesis in the zebrafish: in search of
genes controlling development in a vertebrate, Curr. Biol., 4 (1994)
189-202.
[23] Pierce, M.E., Sheshberadaran, H., Zhang, Z., Fox, L.E., Applebury,
M.L. and Takahashi, J.S., Circadian regulation of iodopsin gene
expression in embryonic photoreceptors in retinal cell culture, Neu-
ron, 10 (1993) 579-584.
[24] Pittendrigh, C.S., Circadian systems: Entrainment. In J. Aschoff
(Ed.), Handbook of Behavioral Neurobiology, Vol. 4, Biological
Rhythms, Plenum, New York, 1981, pp. 95-124.
[25] Postlethwait, J.H., Johnson, S.L., Midson, C.N., Talbot, W.S, Gates,
M., Ballinger, E.W., Africa, D., Andrews, R., Carl, T., Eisen, J.S.,
Horne, S., Kimmel, C.B., Hutchinson, M., Johnson, M. and Ro-
driguez, A., A genetic linkage map for the zebrafish, Science, 264
(1994) 699-703.
[26] Rollag, M.D. and Niswender, G.D., Radioimmunoassay of serum
concentrations of melatonin in sheep exposed to different lighting
regimens, Endocrinology, 98 (1976) 482-489.
[27] Sehgal, A., Price, J.L., Man, B. and Young, M.W., Loss of circadian
behavioral rhythms and per RNA oscillations in the Drosophila
mutant timeless, Science, 263 (1994) 1603-1606.
[28] Takahashi, J.S., Hamm, H. and Menaker, M., Circadian rhythms of
melatonin release from individual superfused chicken pineal glands
in vitro, Proc. Natl. Acad. Sci. USA, 77 (1980) 2319-2322.
[29] Takahashi, J.S., Kornhauser, J.M., Koumenis, C. and Eskin, A.,
Molecular approaches to understanding circadian oscillations, Annu.
Rev. Physiol., 55 (1993) 729-753.
[30] Takahashi, J.S., Murakami, N., Nikaido, S.S., Pratt, B.L. and
Robertson, L.M., The avian pineal, a vertebrate model system of the
circadian oscillator: cellular regulation of circadian rhythms by light,
second messengers, and macromolecular synthesis, Recent Prog.
Horm. Res., 45 (1989) 279-352.
[31] Takahashi, J.S., Pinto, L.H. and Hotz Vitaterna, M., Forward and
reverse genetic approaches to behavior in the mouse, Science, 264
(1994) 1724-1733.
[32] Underwood, H. and Goldman, B.D., Vertebrate circadian and pho-
 G.M. Cahill / Brain Research 708 (1996) 177-181
toperiodic systems: role of the pineal gland and melatonin, J. Biol.
Rhythms, 2(4) (1987) 279-315.
[33] Vitaterna, M.H., King, D.P., Chang, A.-M., Kornhauser, J.M.,
Lowrey, P.L., McDonald, J.D., Dove, W.F., Pinto, L.H., Turek,
F.W. and Takahashi, J.S., Mutagenesis and mapping of a mouse
gene, Clock, essential for circadian behavior, Science, 264 (1994)
719-725.
[34] Westerfield, M., The Zebrafish Book, University of Oregon Press,
Eugene, OR, 1993,
[35] Zachmann, A., Ali, M.A. and Falc6n, J., Melatonin and its effects in
181
fishes: an overview. In M.A. Ali (Ed.), Rhythms in Fishes, Plenum,
New York, 1992, pp, 149-165.
[36] Zachmann, A., Falc6n, J., Knijff, S.C.M., Bolliet, V. and Ali, M.A.,
Effects of photoperiod and temperature on rhythmic melatonin secre-
tion from the pineal organ of the white sucker (Catostomus commer-
soni) in vitro, Gen. Comp. Endocrinol., 86 (1992) 26-33.
[37] Zatz, M., Light and norepinephrine similarly prevent damping of the
melatonin rhythm in cultured chick pineal cells: Regulation of
coupling between the pacemaker and overt rhythms? J. Biol.
Rhythms, 6 (1991) 137-147.