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The Journal of Neuroscience, February 1, 1998, 18(3):1105-1114
Multioscillatory Circadian Organization in a Vertebrate,
Iguana iguana
Gianluca
Tosini and
Michael
Menaker
Department of Biology and National Science Foundation Center for
Biological Timing, Gilmer Hall, University of Virginia,
Charlottesville, Virginia 22903
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ABSTRACT |
The lizard Iguana iguana when kept in constant
ambient temperature displays endogenously generated circadian rhythms
of body temperature and locomotor activity. Although surgical removal of the parietal eye has only slight effects on overt circadian rhythmicity, subsequent pinealectomy completely abolishes the rhythm of
body temperature. However, the rhythm of locomotor activity is only
slightly affected by parietalectomy plus pinealectomy. Our results
demonstrate that the pineal complex is centrally involved in the
generation and control of the circadian rhythm of body temperature but
is only marginally involved in locomotor rhythmicity. Plasma melatonin
levels are not significantly reduced by parietalectomy, whereas
pinealectomy dramatically lowers the level and completely eliminates
the circadian rhythm of melatonin in the circulation. Isolated parietal
eye, pineal, and retina all synthesize melatonin with robust circadian
rhythmicity when maintained for  4 d in culture, although in the
intact animal all or almost all of the circulating melatonin comes from
the pineal. The circadian system of I. iguana is
composed of multiple circadian oscillators that reside in different
tissues and have specific and different roles.
Key words:
circadian rhythms; body temperature; locomotor activity; lizards; melatonin; pineal; parietal eye; retina
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INTRODUCTION |
Although a great deal of
experimental evidence indicates that circadian systems of multicellular
organisms are composed of several discrete circadian oscillators,
circadian organization, by which is meant the interactions
of these oscillators with the environment, with each other, and with
the rest of the organism, is poorly understood. This is attributable in
part to lack of experimental attention and in part to emphasis on the
use of mammalian models in which such organization is particularly
difficult to dissect. Nonmammalian vertebrates can provide useful
alternatives with which to develop the principles that underlie
circadian organization, and the green iguana, the circadian system of
which is described here, appears to offer particular advantages.
The pineal complex forms part of the diencephalic roof of the brain of
most vertebrates; it may be directly photosensitive, as in fish,
amphibians, reptiles, and birds, or it may receive photic information
via sympathetic neural pathways, as in mammals. Many reptile species
possess several circadian photoreceptors: a photoreceptive pineal
gland, an extracranial parapineal organ (parietal eye), and deep brain
photoreceptors (Engbretson, 1992 ; Solessio and Engbretson, 1993; Grace
et al., 1996).
The pineal gland is a central component in the circadian organization
of many nonmammalian vertebrates. In some Agnatha (Lampetra japonica) pinealectomy abolishes the circadian rhythm of locomotor activity (Morita et al., 1992) and has the same effect in some species
of lizards (Underwood, 1983; Molina-Borja, 1996); in others it produces
marked changes in the free-running period and circadian activity time
(Underwood 1977, 1981, 1992; Foà, 1991; Innocenti et al., 1996);
however, in Dipsosauros dorsalis it has no obvious effects
(Janik and Menaker, 1990). Pinealectomy abolishes circadian body
temperature and locomotor activity rhythms in the house sparrow Passer domesticus and disrupts or abolishes rhythmicity in
other passerine birds (Gaston and Menaker, 1968; Binkley et al., 1971; McMillan, 1972; Gwinner, 1978; Ebihara and Kawamura, 1981; Fuchs, 1983). In pigeons (Columbia livia) the circadian rhythms of
locomotor activity and body temperature are abolished by removal of the pineal and the retinas, both sources of circulating melatonin (Ebihara
et al., 1984), and can be restored by rhythmic infusions of exogenous
melatonin (Chabot and Menaker, 1992).
The isolated pineals of some birds, lizards, and fish, when cultured
in vitro, can maintain circadian rhythms of melatonin synthesis for many days in constant darkness (Takahashi et al., 1980;
Menaker and Wisner, 1983; Falcon et al., 1987; Barrett and Takahashi,
1995; Bolliet et al., 1996). The lateral eyes of the frog Xenopus
laevis, chicken, quail, fish (Esox lucius), and mammals also contain self-sustained circadian oscillators that regulate melatonin synthesis (for review, see Cahill and Besharse, 1995; also
see Menaker and Tosini, 1996; Tosini and Menaker, 1996a). Although
there is evidence suggesting that the parietal eye of some lizards may
synthesize melatonin (Quay, 1965; Firth and Kennaway, 1987), this
structure has never been studied in vitro to test for the
capacity to synthesize melatonin and for the presence of circadian
oscillators.
We have recently shown that the lizard Iguana iguana when
kept in constant ambient temperature displays endogenously generated circadian rhythms of body temperature and locomotor activity, and that
these two rhythms may free run with different periods. These data
suggest that spontaneous internal desynchronization between these two
rhythms may occur in this species under some laboratory conditions.
This result, which is unique among circadian responses of animals
(although a similar response has been observed in humans; Aschoff and
Wever, 1976), suggests that the two rhythms may be generated by two
separate circadian oscillators (Tosini and Menaker, 1995). We report
here the results of experiments designed to clarify the roles played by
the retina, pineal, and parietal eye in the circadian organization of
this species.
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MATERIALS AND METHODS |
Animals
Seven adults (weight range, 973-1985 gm) and 25 juveniles
(weight range, 105-150 gm) of I. iguana were obtained from
an authorized dealer (Safari Pet and Supply, San Diego, CA). They were
housed in groups of two or three in large enclosures (130 × 70 × 40 cm). Heat for thermoregulation was furnished by a thermal
pad placed beneath the floor of the enclosures; the air temperature was
26°C, and the light cycle was 12 hr light/12 hr dark (light from 7 A.M.-7 P.M. Eastern Standard Time). Lizards were fed with a diet of
lettuce and fruit combined with Zu/Preem marmoset food and
multivitamin-protein complex (Vitalife). Water was available ad
libitum.
Temperature and activity recording
For experiments lizards were housed individually in plastic
cages (measuring 48 × 26 × 20 cm) inside an environmental
chamber at 27 ± 0.2°C with no daily component (see Tosini and
Menaker, 1995). Body temperature and level of activity were measured by telemetry using implanted transmitters (models VM-FH in adults and
XM-FM in juveniles; Mini-Mitter Co., Sunriver, OR). A radio receiver
(model RA-1010, Mini-Mitter Co.) coupled to a data acquisition system
(Dataquest III; Data Sciences Inc., St. Paul, MN) was placed under the
plastic cage inside the environmental chamber. Body temperature and
activity (as number of movements) were recorded in 6 min bins and saved
on disk for later analysis. Cages were cleaned once per week, and
animals were fed three times per week. Because the experiments were
conducted in constant darkness, lizards inside the environmental
chamber were fed with the aid of an infrared viewer (FJW Optical
Systems, Elgin, IL) to avoid exposing them to visible light.
Surgery
Surgery was performed during the animal's day (6-9 hr after
the onset of light). To implant a transmitter in the abdominal cavity,
animals were first cooled in a refrigerator (1-4°C) until immobilized and then embedded in crushed ice. A small cut (1-2 cm) was
made on the lateral-ventral surface, and a transmitter was implanted
in the abdominal cavity. The operation required 10-15 min. Animals
were allowed to recover for 24 hr before recording began. For
parietalectomy, animals were first cooled until immobilized and then
embedded in crushed ice, and the parietal eye was removed using a
microcurette (diameter, 0.5 mm). Sham surgery (sham PAR-X) was
performed using the same procedure, except that instead of removing the
parietal eye, a scale behind it was removed. The whole procedure lasted
<1 hr. For pinealectomy the lizards were cooled and immobilized, and a
square flap of skin above the pineal was reflected to expose the skull.
Using a dental drill, a small circle of bone was cut out of the skull
above the pineal exposing the pigmented dura. The pigmented layer was
removed, the meninges covering the pineal were cut, and the pineal was
grasped with fine spring forceps. After the pineal was removed, the
small circle of bone was replaced, the wound was packed with Gelfoam,
and the flap of skin was fixed in place with cyanoacrylate glue. Sham operations (sham PIN-X) were performed in the same manner, except that
the pineal was not grasped and removed. During the surgery it was
evident that the pineal was completely removed, because it came out
easily as an intact structure. In some animals we also performed
postmortem histological analysis to confirm the absence of pineal
tissue. In all the animals examined no pineal tissue was found. After
the operation lizards were allowed to recover for 6-8 hr before
returning them to the environmental chamber.
Unfortunately it has not been possible for us or, for that matter, for
other researchers (Foà, 1991; Innocenti et al., 1993) to remove
the pineal gland without seriously damaging the parietal eye. Because
we thought that data from completely parietalectomized animals would be
easier to interpret than data from animals with uncontrolled damage to
the parietal eye, we first parietalectomized and then pinealectomized
all of the animals in which the role of the pineal was to be tested
(also see Discussion).
The eyes of iguanas are large and heavily vascularized. Although it
might be possible to remove them, we chose to avoid this trauma in the
present study, because we thought that our data would be clearly
interpretable as they stand.
Plasma extraction and radioimmunoassay
Fifty microliters of plasma from each blood sample (see below)
were extracted in 2 ml of chloroform and washed first with 0.1 M sodium carbonate buffer, pH 10.25, and then with water. A
1 ml aliquot of extract was dried under nitrogen, resuspended in 0.5 ml
of phosphate buffer with 0.9% NaCl and 1% gelatin, and washed with 2 ml of ethyl alcohol (100%). The extract was then assayed for melatonin
according to a modification of the method of Rollag and Niswender
(1976). This assay has been already validated for I. iguana
(Tosini and Menaker, 1996b). The intra-assay coefficients of variation
for pooled culture medium quality control samples containing low (15.6 pg), medium (31.2 pg), and high (125 pg) levels of melatonin were 12.2, 10.4, and 8.5%, respectively.
Flow-through culture technique
Parietal eyes, retinas, and pineals were maintained individually
in flow-through perifusion chambers constructed from ELISA plates (see
Tosini and Menaker, 1996a) and were placed inside a light-tight
incubator at the constant temperature of 27°C. Culture medium
[medium 199 (Life Technologies, Gaithersburg, MD) with HBSS and
L-glutamine] was delivered (1 ml/hr) to the superfusion chambers from a multichannel syringe pump via Teflon tubing. Each organ
was placed in a well of the ELISA plate and sealed by a plastic plug.
Two stainless steel tubes penetrated the plastic plug into the well;
one delivered culture medium to the well, and the other carried medium
from the chamber to a fraction collector via flexible tubing. Samples
were collected at 2 hr intervals and stored at 4°C until assayed at
the end of the experiment.
Melatonin levels in the medium were measured by radioimmunoassay in a
modification of the method of Rollag and Niswender (1976). Because we
were primarily interested in relative differences in melatonin (or
methoxyindole) concentrations rather than in absolute levels, we
validated the assay without extraction of the samples. Melatonin (0, 10, 25, 50, 100, 250, and 500 pg/tube; four replicates at each level)
added to pooled perifusate collected from each of the organs at night
was quantitatively recovered. Dilution curves of pooled day and pooled
night medium 199 perifusate samples from each of the organs were
parallel to those of standard melatonin, indicating equivalent binding
kinetics in unknowns and standards. The lower and upper limits of the
assay were 2.5 and 500 pg/tube, respectively.
Data analysis
To determine the free-running period ( ) and robustness for
both the circadian rhythm of activity (CRA) and the circadian rhythm of
body temperature (CRT), we used the  2 period periodogram
procedure (Sokolove and Bushell, 1978). The recorded time series data
for both body temperature and locomotor activity, which showed a
probability level for circadian rhythmicity of p > 95%, were considered as statistically significant rhythms. The
robustness of these rhythms was numerically defined as the value of the
Sokolove-Bushell QP statistic (see Refinetti et
al., 1994). To determine the effects of PAR-X and PIN-X on the CRT and
CRA, statistical analysis (see Results) was performed on data from the
10 d before parietalectomy and the 10 d after parietalectomy and pinealectomy.
Analyses of the melatonin rhythms from cultured parietal eyes, pineals,
and retinas were performed using an iterative, coupled Fourier
transform-nonlinear least squares estimation procedure developed by
Dr. Marty Straume (National Science Foundation Center for Biological
Timing, University of Virginia) (see Tosini and Menaker, 1996a; Plautz
et al., 1997). This method is capable of accommodating temporal
nonstationarities in time series data (e.g., drifting observable and/or
nonconstant oscillatory amplitudes) and maintaining an ability to
detect underlying rhythmic behavior. This analysis provides a means for
estimating amplitude, period, phase, and significance level of rhythms
in time series data. This software is available from the National
Science Foundation Center for Biological Timing.
Experimental design
CRA and CRT in constant darkness and effects of
parietalectomy and pinealectomy. Lizards were moved from group
enclosures to individual cages placed inside the environmental chamber
at the constant temperature of 27°C and were allowed to free run in
constant darkness (DD) for at least 12 d. After this initial period, 11 animals (four adults and seven juveniles) were
parietalectomized, whereas five animals (three adults and two
juveniles) were subjected to sham surgery. After the surgery the
animals were returned to the environmental chamber and allowed to free
run for at least 12 d. After this, lizards that had been
parietalectomized previously were pinealectomized, whereas animals that
had been sham parietalectomized were sham pinealectomized and were
returned to the environmental chamber. Animals were then allowed to
free run for an additional 12 d.
Circulating melatonin levels. Twelve juveniles were used in
this experiment. Each lizard was kept in an individual plastic cage
(48 × 26 × 20 cm), which was located inside a light-tight wooden box. The temperature inside the wooden box was maintained at a
constant level of 27 ± 0.3°C. Food was provided three times per
week, and water was available ad libitum. Lighting was
provided by an overhead fluorescent bulb (Vita-lite 40 W), giving an
average light intensity of 200 lux at the level of the lizard's head. All lizards were held for 3 months under a 12 hr light/12 hr dark cycle
(lights on at 7 A.M., lights off at 7 P.M.). Blood samples were taken
from the lizards over a 24 hr period. The schedule of sampling is shown
in Figure 1. Each test in DD started at 9 A.M. of the second day the lizard had spent in DD (39 hr after the
light went off). The LD cycle was reinitiated with lights on at 7 A.M.
of the fourth day. The short interval between the start of the DD
treatment and the test was chosen to reduce variability (circadian
oscillators are expected to free run with different periods in
different individuals, thus increasing asynchrony among lizards as more
time is spent in DD). Phase shift of the melatonin rhythm or
suppression of melatonin synthesis by light was avoided by collecting
the blood with the aid of an infrared viewer (FJW Optical Systems) when
the collection interval fell during darkness. Sampling was accomplished
by drawing ~120 µl of blood from the blood vessel located on the
midventral surface at the base of the tail with a 1 cc heparinized
syringe. The blood was then transferred to heparinized tubes and
centrifuged for 45 min at 4000 rpm, and the plasma was transferred to
another tube and stored at  80°C until assayed.
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Figure 1.
Blood-sampling schedule. Twelve lizards
(A-N) were subdivided into three groups
(circles, triangles, diamonds) of four lizards each.
Blood samples were drawn at 12 times (every 2 hr) during a 24 hr period
(4 times from each group during the 24 hr) following the time schedule
and alternation among groups shown. This schedule of sampling and the
same grouping of lizards was used in both LD and DD (for results, see
Fig. 5).
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Melatonin release from cultured parietal eyes, pineals, and
retinas. Parietal eyes, pineals, and retinas were removed from the
lizards under anesthesia (ketamine, 100 mg/kg body weight) 30 min
before the offset of the light (7 P.M.) and within 10-20 min were
cultured individually at a constant temperature of 27°C either in a
12 hr light/12 hr dark cycle or in DD.
A fiber-optic light powered by a 150 W tungsten-quartz halogen lamp
was used in the experiments in which the cultured organs were exposed
to LD cycles. The source of the light was located outside the
incubator, whereas the end of the fiber-optic probe was located inside
the incubator about 15 cm from the plate containing the cultures. Light
intensities at the level of the cultured organs were estimated by
placing the irradiance detector of a United Detector Technology
(Hawthorne, CA) UDT 350 radiometer-photometer at the location of the
culture chambers during the experiments. The irradiance level was
estimated to be 800 µW/cm2.
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RESULTS |
CRT and CRA in DD and the effects of parietalectomy
and pinealectomy
All 16 intact lizards (seven adults and nine juveniles) tested in
DD displayed significant circadian rhythms of body temperature and
activity (Tables 1,
2). The mean amplitudes of CRTs were 0.8 ± 0.2 and 0.5 ± 0.1°C in adults and juveniles,
respectively (Figs. 2, 3). The difference
in amplitude with age (i.e., size) was significant, but there were no
differences among animals in the same age class (one-way ANOVA,
p > 0.1). Comparisons of CRT and CRA showed that the
CRA is more robust (i.e., the QP values are
greater) than the CRT (Tables 1, 2, Fig. 4).
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Table 1.
Period estimates from periodogram analysis of I. iguana body temperature time series data recorded during 10 d in DD in intact, parietalectomized, and pinealectomized animals
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Table 2.
Period estimates from periodogram analysis of I. iguana locomotor activity time series data recorded during
10 d in DD in intact, parietalectomized, and pinealectomized
animals
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Figure 2.
Locomotor activity (top) and body
temperature (bottom) records of an individual of
I. iguana (IO 16) showing effects
on these rhythms of sham PAR-X and sham PIN-X in constant temperature
and darkness. The arrows indicate the days on which the
animal was released in DD, sham PAR-X, and sham PIN-X. Estimates of the
free-running period for these data sets are reported in Tables 1 and
2.
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Eleven of the sixteen lizards free running in DD were PAR-X and five
were sham PAR-X. All of these animals continued to display CRT and CRA
(Tables 1, 2). Although parietalectomy did not affect the amplitude of
the CRT (Fig. 3), 10 of 11 lizards showed
changes in the free-running period (the free-running period lengthened between 0.3 and 0.6 hr), and the robustness of this rhythm was somewhat
reduced (Table 1, Fig. 4). After
parietalectomy, three lizards showed changes of CRA free-running
period; however, the robustness of the CRA was not affected (Table 2,
Fig. 4). Sham PAR-X lizards did not show any differences with respect
to intact animals (Tables 1, 2, Fig. 3). After parietalectomy, but not before, QP values of the CRA were higher than
QP values of the CRT (Fig. 4).
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Figure 3.
Locomotor activity (top) and body
temperature (bottom) records of an individual of
I. iguana (IO 4) showing effects
on these rhythms of PAR-X and PIN-X in constant temperature and
darkness. The arrows indicate the days on which the
animal was released into DD, PAR-X, and PIN-X. Estimates of the
free-running period for these data sets are reported in Tables 1 and
2.
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Figure 4.
Effects of PAR-X and PIN-X on the robustness of
the body temperature and locomotor activity rhythms of I.
iguana. Each bar corresponds to the median of 11 animals. The line indicates the 0.05 level of
significance, values below this line indicate
arrhythmicity.
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All of the lizards survived parietalectomy and sham parietalectomy and
were then PIN-X or sham PIN-X. Pinealectomy produced a dramatic effect
on the CRT (Table 1, Fig. 3). In 10 (91%) of 11 lizards the CRT was
completely abolished. CRA was not abolished by pinealectomy. However,
in eight lizards significant changes in (three shortened and five
lengthened ; Table 2) were observed. There was no effect on the
robustness of the CRA with respect either to the intact animals or to
the PAR-X ones (Kruskal-Wallis test, p > 0.1) (Fig.
4). Sham pinealectomy did not produce any significant effects either on
the CRT or on the CRA (Kruskal-Wallis tests, p > 0.1)
(Tables 1, 2, Fig. 2).
Circulating melatonin levels
There was a robust daily rhythm of melatonin in the plasma
of intact lizards in the LD cycle (Fig.
5A); melatonin concentration increased rapidly after the offset of the lights (7 P.M.), reached a
peak at 1 A.M., and began to decrease before the onset of the lights.
After parietalectomy lizards did not show any significant differences
in the levels of circulating melatonin with respect to intact lizards
(t tests, p > 0.1). Pinealectomy abolished
the normal circadian rhythm in melatonin level; levels throughout the
24 hr period were relatively constant and were not significantly different from levels measured during the day for intact animals (t test, p > 0.1).
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Figure 5.
Mean serum melatonin profiles for 12 individuals
of I. iguana under a 12 hr light/12 hr dark cycle
(A) and mean serum melatonin profiles in constant
darkness (B). Each point
represents the mean value of serum melatonin of four individuals. Each
individual was sampled every 6 hr (Fig. 1). Error bars represent SEM.
If error bars are lacking, the SEM is encompassed by the symbol
representing the mean values of intact ( ), PAR-X ( ), PIN-X ( )
lizards. Each of the 12 lizards was subjected to sampling under six
conditions: as intact in LD and DD, as parietalectomized in LD and DD,
and finally as pinealectomized (and parietalectomized) in LD and DD. See Figure 1 for sampling schedule.
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In constant darkness, both intact and PAR-X lizards showed clear
rhythms in plasma melatonin level (Fig. 5B), with a somewhat broader peak than in the 12 hr LD cycle (Fig. 5, compare A,
B). Melatonin levels of PIN-X lizards in DD were low throughout
the 24 hr and were not different from those recorded in PIN-X lizards in the LD cycle (t test, p > 0.1).
Melatonin synthesis in cultured parietal eyes, pineals,
and retinas
Individually cultured parietal eyes, pineals, and
retinas "released" melatonin into the perifusate rhythmically and
were entrained to the LD cycles with the normal phase relationship;
i.e., melatonin was high during the dark phase of the cycle and low
during the light phase (Fig. 6). (Release
is used here in the general rather than the technical sense, because
all available evidence indicates that melatonin diffuses out of cells
as it is synthesized and is not subject to controlled release).
Although there are quantitative differences in the amplitude of the
rhythms within and between the different structures, the general
pattern is remarkably stable and uniform. In all cases the decrease in
melatonin release preceded the lights-on transition, suggesting that
the rhythm is not directly driven by the light itself but rather shows
the anticipatory behavior that may occur when a self-sustained
oscillator is entrained. To test for the influence of self-sustained
circadian oscillators on melatonin rhythmicity in the parietal eye, the
pineal, and the retina, the release of melatonin by these structures
was examined in DD for 4 d. Figure 7
shows 4 d of melatonin release in DD from simultaneously but
separately cultured parietal eye, pineal gland, and retina from the
same animal. In all three structures the circadian rhythms of melatonin
release free ran with a period close to but different from 24 hr (Table
3).
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Figure 6.
In vitro melatonin rhythms recorded
under a 12 hr light/12 hr dark cycle from parietal eye
(A), pineal (B), and retina
(C) collected from the same individual and placed
simultaneously in the culture apparatus. The black bars
at the top represent periods of darkness.
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Figure 7.
In vitro circadian rhythms of
melatonin release during 4 d in DD from parietal eye
(A), pineal (B), and retina
(C) collected from the same individual and placed
simultaneously in the culture apparatus.
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Table 3.
Average free-running periods (see Materials and Methods) of
circadian rhythms of melatonin release by cultured parietal eye, pineal, and retina of I. iguana held in constant darkness
for 4 d and mean melatonin release (i.e., the total amount of
melatonin released during the entire experiment divided by the number
of samples) and averaged peak level
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DISCUSSION |
We have demonstrated directly that an individual I. iguana possesses four self-sustained circadian oscillators (one in
the pineal organ, one in the parietal eye, and one in each lateral eye). Furthermore, it is likely that each of these organs actually contains a large number of individual cellular circadian oscillators, as has been demonstrated for the chicken pineal (Takahashi and Menaker,
1984), and that the locomotor rhythm is driven by a fifth set of
self-sustained circadian oscillators in the hypothalamus [suprachiasmatic nucleus (SCN)] as in D. dorsalis and
Podarcis sicula (Janik et al., 1990; Minutini et al., 1995;
see also Underwood and Edmonds, 1995). Each of the structures that we
have demonstrated to contain circadian oscillators is also
photoreceptive and therefore has direct access to environmental light
cycles. In addition, deep brain photoreceptors with input to the
circadian system exist in most nonmammalian vertebrates (for review,
see Foster et al., 1994) and have been tentatively identified in
I. iguana (Grace et al., 1996).
I. iguana is perhaps the best example of what has by now
become a generalization to which there are no clearly documented exceptions; circadian rhythms in behavior and physiology of all vertebrates (including mammals) are generated and controlled, not by a
single biological clock, but by a circadian system composed of several
photoreceptors, several self-sustained oscillators, and their
interconnections. These complexities raise questions on at least three
levels: (1) how circadian systems are organized (i.e., how the
environment affects the components of the system, how the components
affect each other, and how the system regulates its outputs); (2) how
the circadian organization of a particular species relates to the
environmental niche that it occupies; and (3) how vertebrate circadian
systems have evolved, in particular, what selection pressures have
produced the variability that we observe (Menaker and Tosini, 1996).
This formidable set of questions cannot be approached other than by
detailed analysis of the circadian systems of several different
vertebrate species. For reasons that are clear from the data reported
above, I. iguana is an excellent candidate for one such
model species.
The endogenously generated circadian rhythm of body temperature change
(in itself remarkable in such a small ectotherm; Bartholomew, 1982)
depends on the presence of the pineal gland, although our experimental
design does not rigorously exclude the possibility that the effects
that we are attributing to pinealectomy are in fact the results of
pinealectomy plus parietalectomy. By implication (although not by
direct demonstration) the body temperature rhythm is a consequence of
the circadian rhythm of circulating melatonin for which the pineal is
solely responsible. Melatonin levels are normally high in the
circulation at night when the body temperature is low; therefore, it is
likely that melatonin suppresses heat production (i.e., basal metabolic
rate), increases heat loss, or both. Its effect on body temperature is
not an indirect result of an effect on activity, because pinealectomy
does not abolish the circadian activity rhythm or markedly reduce the
level of activity (Fig. 3). However, pinealectomy did have small but
significant effects on the free-running period of the activity rhythms
of 8 of 11 lizards, suggesting that the pineal oscillator is weakly coupled to whatever circadian oscillator (SCN?) is driving the activity
rhythm. The observation that an identified circadian oscillator (in
this case the pineal) drives one circadian output rhythm but not
another is unique in the circadian literature and suggests the need to
reevaluate the interpretation of experiments in which lesion of a
suspected circadian oscillator has no effect on a single endpoint
(e.g., our own studies of pinealectomy in D. dorsalis; Janik
and Menaker, 1990).
Very little is known about the function of the parietal eye of lizards.
Although it has previously been suspected of producing melatonin (Quay,
1965; Firth and Kennaway, 1987), ours is the first unequivocal
demonstration of this capacity. The observation that melatonin is
synthesized under the control of circadian oscillators within the
parietal eye is also unique. Rhythmic synthesis of melatonin by
cultured retinas has now been shown in amphibians (for review, see
Cahill and Besharse, 1995), reptiles (this paper), and mammals (Tosini
and Menaker, 1996a), and the photoreceptive pineals of all classes of
nonmammalian vertebrates also contain circadian oscillators that
regulate melatonin synthesis (Underwood, 1990; Takahashi et al., 1989).
Therefore, it appears that all vertebrate photoreceptors synthesize
melatonin rhythmically (although the deep brain photoreceptors
possessed by most nonmammalian vertebrates have not yet been tested
either for rhythmicity or for the capacity to synthesize melatonin).
This generalization supports the suggestion (Gern, 1982) that melatonin
must fulfill unknown but critically important functions in
photoreception itself, perhaps in light and/or dark adaptation.
Elsewhere (Menaker and Tosini, 1996) we have speculated that its role
in photoreception may have been its original function and that it was
converted into a circulating hormone by a simple amplification of the
synthetic capacity of the pineal and/or suppression of the enzymatic
pathway for its rapid degradation. These speculations suggest an
explanation for the role of melatonin rhythmicity in the parietal and
lateral eyes of I. iguana; it may fulfill a local function
involved in some way with photoreceptor adaptation to the environmental
light/dark cycle and might play either no role or only a small one in
circadian regulation at the level of the whole organism. However, even
if correct, this explanation is far from complete, because it does not
deal with the several ways in which the retina and parietal eye might
be coupled to the rest of the circadian system, either as sources of
photic information or as recipients of circadian signals. Nor is such
an explanation applicable to all nonmammalian vertebrates, because
there are at least two well documented cases among birds in which the
retinas exert circadian influence at the organismal level (Konishi et
al., 1985; Oshima et al., 1989; Underwood et al., 1990). Viewed in this
way, the circadian system of I. iguana may conform rather
closely to the general neuroendocrine loop model proposed by Cassone
and Menaker (1984) if one assumes that the locomotor activity rhythm is
driven by the SCN. Revisions of this model required by our new
information are limited to (1) removal of the uncertainty about the
existence of a retinal circadian oscillator, (2) addition of a parietal
eye circadian oscillator, and (3) recognition that each oscillator in
the system may regulate different rhythmic outputs. Although the model
of Cassone and Menaker (1984) remains a useful framework on which to
hang new facts about vertebrate circadian organization, it is well to
remember that it has been assembled using data from several different
organisms and contains important untested assumptions.
| |
FOOTNOTES |
Received July 14, 1997; revised Nov. 3, 1997; accepted Nov. 7, 1997.
This work was supported by International Human Frontier Science Program
Fellowship LT-145/93 to G.T. and by Air Force Office of Scientific
Research Grant F49620-97-1-0012 to M.M.
Correspondence should be addressed to Michael Menaker, Department of
Biology, Gilmer Hall, University of Virginia, Charlottesville, VA
22903.
| |
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Abstract
Introduction
