The circadian system of reptiles: A multioscillatory and multiphotoreceptive system

Abstract

Many parameters exhibited by organisms show daily fluctuations that may persist when the organisms are held in constant environmental conditions. Rhythms that persist in constant conditions with a period close to 24 h are called circadian. Although nowadays most research in this field is focused on the molecular and genetic aspects--and therefore mostly on two animal models (Drosophila and mouse)--the study of alternative animal models still represent a useful approach to understanding how the vertebrate circadian system is organized, and how this fascinating time-keeping system has changed throughout the evolution of vertebrates. The present paper summarizes the current knowledge of the circadian organization of Reptiles. The circadian organization of reptiles is multioscillatory in nature. The retinas, the pineal, and the parietal eye (and, possibly, the suprachiasmatic nuclei of the hypothalamus, SCN) contain circadian clocks. Of particular interest is the observation that the role these structures play in the circadian organization varies considerably among species and within the same species in different seasons. Another remarkable feature of this class is the redundancy of circadian photoreceptors: retinas of the lateral eyes, pineal, parietal eye, and the brain all contain photoreceptors.

Full text

The circadian system of reptiles: a multioscillatory and
multiphotoreceptive system
Gianluca Tosini
a,
*, Cristiano Bertolucci
b
, Augusto Foa
Á
b
a
Neuroscience Institute, Morehouse School of Medicine, 720 Westview Drive, SW, Atlanta, GA 30310-1495, USA
b
Department of Biology, University of Ferrara, Via Borsari 46, Ferrara, Italy
Received 10 May 2000; received in revised form 14 September 2000; accepted 17 October 2000
Abstract
Many parameters exhibited by organisms show daily fluctuations that may persist when the organisms are held in constant environmental
conditions. Rhythms that persist in constant conditions with a period close to 24 h are called circadian. Although nowadays most research in
this field is focused on the molecular and genetic aspects Ð and therefore mostly on two animal models (Drosophila and mouse) Ð the study
of alternative animal models still represent a useful approach to understanding how the vertebrate circadian system is organized, and how this
fascinating time-keeping system has changed throughout the evolution of vertebrates. The present paper summarizes the current knowledge
of the circadian organization of Reptiles. The circadian organization of reptiles is multioscillatory in nature. The retinas, the pineal, and the
parietal eye (and, possibly, the suprachiasmatic nuclei of the hypothalamus, SCN) contain circadian clocks. Of particular interest is the
observation that the role these structures play in the circadian organization varies considerably among species and within the same species in
different seasons. Another remarkable feature of this class is the redundancy of circadian photoreceptors: retinas of the lateral eyes, pineal,
parietal eye, and the brain all contain photoreceptors. D2001 Elsevier Science Inc. All rights reserved.
Keywords: Pineal; Parietal eye; Melatonin; Retina; Circadian rhythms; Reptiles; Vertebrate; SCN
1. Introduction
Many biochemical, physiological, and behavioral para-
meters exhibited by organisms show daily fluctuations, and
most of these fluctuations persist when the organisms are
maintained in constant environmental conditions, thus
demonstrating that they are driven by an endogenous
oscillator. Rhythms that persist in constant conditions with
a period close to 24-h are called circadian. Circadian
rhythms have been now described from bacteria to humans
and a large amount of information about the physiological,
cellular, and molecular mechanisms responsible for the
generation of circadian rhythmicity is now available. Circa-
dian rhythms are controlled by endogenous clocks that,
now, have been localized to discrete neural anatomical
structures within the nervous system. In vertebrates there
are some structures the removal of which has significant
effects on the behavioral circadian rhythmicity, and there-
fore they can be considered as part of the circadian system.
These structures are the suprachiasmatic nuclei of the
hypothalamus (SCN), the lateral eyes, and the pineal com-
plex. This set of organs constitutes what is now called the
``Vertebrates Circadian Axis'' [37]. Although these struc-
tures are present in all the vertebrates, their contribution to
the circadian system may vary considerably among classes
and even within the same class. The SCN, for example, are
the central circadian pacemaker in mammals, and their
lesion abolish almost all circadian rhythms (the only known
exceptions are some circadian rhythms within the retinas),
while in nonmammalian vertebrates the evidence for SCN
involvement in circadian rhythmicity is far less extensive.
The lateral eyes (retinas) contain self-sustained circadian
oscillators in all vertebrate classes and their removal may
affect physiological and/or behavioral rhythms in amphi-
bians, reptiles, birds, and also in mammals. In addition, in
mammals the eyes are the only structures capable of
perceiving light and thus necessary for circadian entrain-
ment [69].
The pineal gland is a central component in the regulation
of circadian rhythmicity of reptiles and other nonmamma-
* Corresponding author. Tel.: +1-404-756-5214; fax: +1-404-752-
1041.
E-mail address: tosinig@msm.edu (G. Tosini).
Physiology & Behavior 72 (2001) 461 ± 471
0031-9384/01/$ ± see front matter D2001 Elsevier Science Inc. All rights reserved.
PII: S 0031-9384(00)00423-6\

lian vertebrates [62], whereas the role the gland plays in the
circadian organization of mammals is marginal, since its
removal has little or no effects on overt rhythms [6].
Because of their phylogenetic position and ecology
reptiles have provided Ð and still provide Ð the
circadian field with some of the most interesting models
for understanding circadian organization, its evolution,
and its variability.
2. The pineal complex
The pineal complex (pineal gland and parietal eye) is a
morphologically and functionally related set of organs that
arises as an evagination of the roof of the diencephalon. The
pineal organ is present in almost all vertebrates (alligator
and owl have only a very rudimentary pineal organ) whereas
the parietal eye is present only in some lizards species and in
the tuatara (Sphenodon punctatus).
In Reptiles the pineal gland contains photosensory cells
with secretory activity. The major product of these secretory
cells is the hormone melatonin, and this hormone is believed
to play an important role in the circadian system of reptiles
(see below). Melatonin is synthesized from the amino acid
tryptophan via a well-known biosynthetic pathway. Because
of its capability to respond to changes in illumination and
temperature, the pineal gland is considered to be the photo-
thermoendocrine transducer (via the action of the hormone
melatonin) of changes in photoperiod and environmental
temperature [62].
The parietal eye has a lens, cornea, and retina; the
parietal eye retina is very simple (i.e. is made of photo-
receptors and ganglion cells only) and the photoreceptors
synapse directly onto the ganglion cells, the axons of which
form the parietal nerve. The parietal eye nerve innervates
several areas of the brain (but does not project to the visual
part). The parietal eye seems to be involved in many
physiological functions of lizards (thermoregulation, repro-
duction, and orientation), but, in general, its role seems
marginal or redundant. Almost unknown is the relationships
between the parietal eye and the pineal gland. The parietal
eye synthesizes melatonin [10,52], but in much lower
quantity with respect to the pineal gland. It is likely that
melatonin may simply fulfill a local function within the
parietal eye.
3. The pineal gland as circadian clock: in vitro studies
In most vertebrates melatonin levels (pineal and blood)
show a clear daily rhythmicity, and reptiles are not an
exception to this general pattern. For example, in Testudo
hermanni Ð during the activity season Ð melatonin is high
at night and low during the day [68]. Clear daily rhythms in
melatonin levels are present in the snake Nerodia rhombi-
fera [48], and in S. punctatus [11], in the lizard Anolis
carolinensis [59], Dipsosaurus dorsalis [27], Trachydo-
saurus rugosus [8± 10], Tiliqua rugosa [12], and Iguana
iguana [51,52]. In T. rugosus [8], A. carolinensis [59], D.
dorsalis [27], Podarcis sicula [14], and I. iguana [52] the
melatonin rhythm persisted also when the animals were held
in constant darkness and temperature, demonstrating there-
fore its true circadian nature.
However, the presence of a circadian melatonin rhythm
per se cannot be used as a reliable indicator of the presence
of a self-sustained oscillator, since the rhythmic melatonin
synthesis/release could be driven by circadian oscillators
located outside the pineal, as it occurs in mammals.
An easy way to demonstrate the presence or absence of a
circadian clock in the pineal is that of preparing an in vitro
culture of the gland for a few days (at least three), while
simultaneously measuring melatonin release at fixed inter-
vals of time. This approach was firstly pioneered in chicken
pineal [31] and since then has been applied to the pineal (but
also to the retina) of many other animals. In the lizards A.
carolinensis,Sceloporus occidentalis,D. dorsalis,I. iguana
(Iguanidae), and Christinus marmoratus (Gekkonidae) mel-
atonin synthesis and release persisted in isolated cultured
pineals for several days, and the synthesis/release was
rhythmic under light/dark cycles [27,35,36,42,44,52].
However, only the pineal of A. carolinensis,S. occiden-
talis, and I. iguana showed a persistent rhythm in melatonin
release when cultured in constant darkness and temperature,
thus demonstrating the presence of a circadian oscillator
Fig. 1. In vitro pattern of melatonin release (as percent of the mean) from
the pineal glands of three different species of iguanid lizards. A circadian
rhythm of melatonin release is present in A. carolinensis and I. iguana, but
not in D. dorsalis. The figures are redrawn from the following sources: A.
carolinensis [36], D. dorsalis [27], and I. iguana [52].
G. Tosini et al. / Physiology & Behavior 72 (2001) 461±471462

within the pineal itself (Fig. 1). On the other hand, cultured
D. dorsalis pineals secreted melatonin in large Ð but not
rhythmic Ð quantities (Fig. 1). Exposing the cultured pineal
to bright illumination greatly reduced melatonin synthesis
and/or release and abolished rhythmicity in I. iguana [49]
and in C. marmoratus [42].
In A. carolinensis and I. iguana the circadian rhythm of
melatonin synthesis/release from cultured pineal has been
shown to be temperature compensated [36,53] (Fig. 2).
Recent studies have also shown that the parietal eye may
contain a circadian clock controlling the synthesis/release of
melatonin [49,52].
Fig. 2. In vitro temperature compensation of melatonin circadian rhythm from cultured pineal gland of (A) A. carolinensis and (B) I. iguana. Although the
period of the rhythm is affected by the temperature, the rhythm is temperature compensated, since the Q
10
(1.1 ± 1.2, respectively) is in the range 0.8 ± 1.3
(redrawn from Menaker and Wisner [36] and Tosini and Menaker, unpublished data).
Table 1
Effects that manipulation of the circadian axis causes on reptiles' circadian rhythms
Species Manipulation Effects Reference(s)
A. carolinensis PINX abolishes, CRL, affects ELR [47,58]
S. olivaceus PARX no effect on CRL [55]
S. olivaceus PINX changes in CRL aand t[55]
S. olivaceus ENU changes in CRL t[65]
S. occidentalis PINX changes in CRL t[57]
S. occidentalis ENU changes in CRL t[57]
D. dorsalis PINX no effects on CRL [27]
D. dorsalis ENU changes in CRL t[27]
D. dorsalis SCNX abolishes CRL [28]
I. iguana PARX changes in tof CRL [52]
I. iguana PINX no effect on CRL, abolishes CRT [52]
I. iguana PINX affects BTS and ELR [40,51]
I. iguana ENU no effect [2]
G. galloti PINX abolishes CRL [41]
P. sicula PARX abolishes BTS for 1 week [24]
P. sicula PINX changes in t[13]
P. sicula PINX the effect changes with the season [25,26]
P. sicula PINX abolishes BTS for 2 ± 3 weeks [24]
P. sicula RETX marked changes in CRL t[13]
P. sicula ONX marked changes in CRL t[38]
P. sicula SCNX abolishes CRL [39]
PINX: pinealectomy; PARX: parietalectomy; RETX: retinalectomy; ENU: bilateral enucleation; ONX: optic nerve lesion; SCNX= electrolytic lesion to the
SCN. CRL = circadian rhythm of locomotor activity; CRT = circadian rhythms of body temperature; BTS = circadian rhythm of behavioral temperature
selection; ELR = ciracadian rhythm in the electroretinogram.
G. Tosini et al. / Physiology & Behavior 72 (2001) 461±471 463

4. The pineal and melatonin in the regulation of
circadian rhythms
The pineal gland is considered the neuroendocrine trans-
ducer of variation in environmental (light and temperature)
conditions, and such action is likely to be mediated via the
hormone melatonin. An easy way to address the role that the
pineal plays in the circadian organization is to remove this
gland and observe the effect that this removal has on the
circadian rhythms (see Table 1).
Circadian rhythms in locomotor activity have been
reported for several species of reptiles (see Ref. [62]).
Removal of the pineal gland abolishes circadian rhythms
of locomotor activity in A. carolinensis and in some
individuals of S. olivaceous [55,58], Gallotia galloti [41],
affects the period of the rhythm in S. olivaceous [55], S.
occidentalis [57], P. sicula [13,26], and has no effect in D.
dorsalis [27] or in I. iguana [52]. Pineal transplantation in
previously pinealectomized P. sicula induced significant
changes in the free-running period of locomotor activity
rhythms [18]. In I. iguana a circadian rhythm in body
temperature has also been demonstrated [50], and the
pineal organ is centrally involved in the generation and
control of this rhythm, since rhythmicity disappears after
its removal [52].
Circadian rhythms in behavioral thermoregulation have
been reported for several reptiles [7,30,46]. Pinealectomy
temporarily abolished the circadian rhythms in behavioral
thermoregulation in P. sicula [24], and reduced the ampli-
tude of these rhythms in I. iguana [51].
Finally, a circadian rhythm in electroretinogram has also
been reported in several lizards (review in Ref. [47]. Pine-
alectomy affected the amplitude of the circadian rhythm of
electroretinographic response in A. carolinensis and I.
iguana, suggesting an involvement of the pineal in the
modulation of this rhythm [40,47]
The behavioral effects of pinealectomy (see Table 1) are
likely to be mediated by melatonin, because of the following
observations: (i) pinealectomy greatly reduces the amount of
circulating melatonin and abolishes its circadian rhythmicity
(review in Ref. [49]); (ii) daily injections of exogenous
melatonin can entrain locomotor rhythms [4,66]; (iii) daily
12-h melatonin infusions that in S. occidentalis closely
mimic the normal, rhythmic pattern of pineal melatonin
secretion in this species entrain locomotor rhythms of both
pineal-intact and pinealectomized lizards [21]; (iv) chronic
administration of melatonin (in silastic capsules) lengthens
the period of circadian rhythms in S. olivaceous and S.
occidentalis [56,57], in D. dorsalis [27], and in P. sicula
[15]; (v) a phase response curve to melatonin has been
described in lizards (S. occidentalis) [61], and (vi) finally,
melatonin administration altered the circadian rhythm of
body temperature selection in I. iguana [51].
Parietalectomy (see Table 1) did not affect locomotor
rhythms in A. carolinensis and S. olivaceous [55,58] and in
P. sicula [24], while in I. iguana it produces slight changes
in the circadian rhythms of locomotor activity and body
temperature [52]. In P. sicula parietalectomy temporarily
abolishes (1 week) the circadian rhythm in behavioral
thermoregulation [24].
5. The pineal gland as seasonal clock
Investigations in the lizards A. carolinensis and T.
rugosa demonstrated that 24 h cycles of both light and
temperature can entrain the pineal melatonin rhythm and
that differences in length of daily photoperiod or thermo-
period affect the phase, amplitude, and duration of this
rhythm [12,59,67]. Hence, the current ambient lighting and
Fig. 3. Seasonal differences in the behavioral effects of pinealectomy in the
lacertid lizard P. sicula. (A,B) Means (  S.E.M.) of the absolute changes in
the free-running period of locomotor rhythms |Dt|) and circadian activity
time (|Da|) induced by pinealectomy (PIN-X) in different seasons and by
sham pinealectomy (SHAM). Pinealectomy was effective in altering the
free-running period (t) in all seasons. Changes in twere significantly
greater in summer than in winter, spring, and autumn. Circadian activity
time (a) was found to change significantly in response to pinealectomy
only in spring and summer. Since no seasonal differences in |Dt| and |Da|
were found among the four seasonal groups of SHAM, the data were
pooled. (C) Locomotor activity record of one lizard subjected to
pinealectomy (PIN-X) in summer. Each horizontal line is a record of 1
day's activity, and consecutive days are mounted one below the other.
Pinealectomy markedly lengthens t, shortens a, and abolishes the bimodal
locomotor pattern (redrawn from Refs. [25,26]).
G. Tosini et al. / Physiology & Behavior 72 (2001) 461±471464

temperature conditions (and their seasonal change) are
readily translated into an internal cue in the form of the
pineal melatonin rhythm. This cue can be used to regulate
both the daily and annual physiology of lizards [63]. In the
Fig. 4. Circadian locomotor activity of lacertid lizards P. sicula free-running in constant temperature (29°C) and darkness (DD). Lizards were collected and
subjected to daily melatonin injections in autumn (A), winter (B), spring (C), and summer (D), respectively. (A ± C) Starting and ending dates of melatonin
treatment are shown on the left of each record. The vertical line drawn through each record shows the time of day of melatonin injections during the whole
injection period. (D) On July 31st the time schedule of melatonin injections was advanced (Shift) from 7:00 p.m. to 3:00 p.m. On September 4th melatonin was
replaced with vehicle solution. Although the locomotor rhythms of the lizards tested in summer entrain successfully to the 24-h period of melatonin injections,
the locomotor rhythms of lizards tested in all other seasons do not entrain to the 24-h period of the injections (redrawn from Ref. [4]).
G. Tosini et al. / Physiology & Behavior 72 (2001) 461±471 465

Lacertid lizard P. s i c u l a the pineal was shown to be
involved in the seasonal reorganization of the circadian
system that is typical of this lizard [17,25]. In constant
temperature and constant darkness pinealectomy in P.
sicula actually induces an immediate transition from the
typical circadian locomotor behavior of summer, character-
ized by a marked bimodal pattern, short free-running
period (t) and long circadian activity time (a), to the
typical circadian locomotor behavior of autumn, character-
ized by an unimodal pattern, a long tand short a(Fig.
3C). Again, the behavioral effects of chronic implants of
exogenous melatonin (in silastic capsules) were found to
be the same as those of pinealectomy in summer: the
abolition of the bimodal pattern after application of the
implants was always associated with a lengthening in t
and shortening in a[15]. Robust circadian rhythms of
blood-borne melatonin expressed by intact P. sicula in late
summer become heavily disrupted or abolished in response
to either pinealectomy or melatonin implants [14]. Taken
together, these results strongly support the view that the
transition from a summer locomotor pattern to an autumn ±
winter one in response to both pinealectomy and melatonin
implants is due to the concomitant suppression of circadian
melatonin rhythms in the blood. Accordingly, in contrast to
the situation in summer, in autumn and winter circadian
rhythms of blood-borne melatonin do not seem to be
required for the expression of the locomotor pattern typical
of these seasons, and therefore in autumn± winter the
behavioral effects of pinealectomy are expected to be
substantially reduced with respect to those observed in
summer. For instance, in the tortoise T. hermanni annual
changes in melatonin rhythms have actually been shown to
occur under natural conditions, with maximal amplitude of
these rhythms in summer and their complete disappearance
in winter [68]. The results of an investigation that com-
pared systematically the effects of pinealectomy on circa-
dian locomotor behavior of P. sicula at different times of
the year actually confirmed the existence of marked annual
changes in the role of the pineal in the circadian organiza-
tion of this lizard [26]. Seasonal differences in the beha-
vioral effects of pinealectomy have been reported in one
other nonmammalian species, the burbot Lota lota [32]. In
the burbot, pinealectomy was found to induce a drastic
lengthening in tin winter, and a comparably drastic
shortening in tin summer. Hence, unlike the case of the
lizard P. sicula, the pineal in burbots is centrally involved
in determining circadian organization both in winter and in
summer. Further investigations demonstrated that daily
injections of exogenous melatonin are capable of entrain-
ing circadian locomotor rhythms of P. sicula exclusively
during the summer (Fig. 4) [4]. Altogether, these findings
demonstrate that the pineal Ð via its hormonal product
melatonin Ð is centrally involved in determining the
circadian organization of the Lacertid lizard P. sicula in
summer, while it is only marginally (or not at all) involved
in the other seasons. As mentioned before, the effects of
pinealectomy on circadian locomotor behavior of lizards
may vary consistently depending on the species (arrhyth-
micity, period changes, no effects). On the other hand,
because of the seasonal differences in the behavioral
effects of pinealectomy we have found in P. sicula,it
seems reasonable to doubt that the differences among
lizards are completely interspecific in nature. Instead they
may, at least in part, depend on the particular season in
which the behavioral effects of pinealectomy have been
examined in each different species. The Lacertidae, as well
as many Iguanidae, inhabit temperate zones, i.e. zones in
which seasonal changes in circadian organization are likely
to have evolved in response to the regular seasonal
fluctuations in photoperiod and/or thermoperiod experi-
enced by the lizards throughout the year [17,63]. Hence,
before deciding about interspecific differences, one should
verify whether, for instance, A. carolinensis and G. galloti
were tested in a season when the behavioral effects of
pinealectomy are maximal and whether D. dorsalis was
tested in a season when these effects are minimal.
6. The role of the retinas in the circadian system
The retinas of reptiles can participate in circadian func-
tion not only as photosensory inputs to the clock, but also as
loci of circadian oscillators: in I. iguana the retina isolated
in culture drives circadian rhythms of melatonin synthesis
[52]. Bilateral ocular enucleation under constant bright light
(LL) was found to induce a marked shortening in tin S.
olivaceus,S. occidentalis, and in some case arrhythmicity in
S. olivaceus [57,65]. Enucleation has a modest effect on the
circadian rhythms of body temperature and locomotor
activity in I. iguana, however enucleation plus pinealectomy
abolished both rhythms in 30% of the animals tested [2].
Bilateral retinalectomy induces a marked shortening in tin
P. sicula kept in constant darkness (DD). These data suggest
that the retinae may either (S. olivaceus) be a component of
the primary pacemaker that drives locomotor rhythms, or (S.
occidentalis,P. sicula) at least play an important modulating
role, that is independent of light perception (P. sicula), on
this primary pacemaker [13,34,57]. In P. sicula electrolytic
lesions of both optic nerves at the level of the optic chiasm
in DD produce the same behavioral effects as bilateral
retinalectomy [13,38]. This demonstrates that the influence
of the retinae on the circadian system of P. sicula is neurally
mediated. Accordingly, in the iguanid lizard S. occidentalis
the influence of the retinae on the circadian system appears
to be neurally mediated, since bilateral optic nerve section
induces marked changes in shape of the phase ± response
curve to light [60].
The retinas play also a role in entrainment of circadian
rhythms to LD cycles, since the light threshold for entrain-
ment is lower in sighted than in blinded S. olivaceus [54].
Several investigations made it clear that extraretinal
photoreceptors participate in mediating entrainment of cir-
G. Tosini et al. / Physiology & Behavior 72 (2001) 461±471466

cadian rhythms of reptiles to LD cycles. In nine species of
lizards, representing five different taxonomic families (Igua-
nidae, Gekkonidae, Eublepharidae, Xantusidae, and Lacer-
tidae) the locomotor rhythms can be entrained to LD cycles
after enucleation of the lateral eyes [23,54,60,64,65]. Hence,
intact retinas are not necessary for entraining behavioral
rhythms of lizards to LD cycles.
Studies carried out in the Iguanid lizards S. olivaceus and
the Lacertid lizard P. sicula showed that ablation of all
known photoreceptive structures (lateral eyes, pineal, and
parietal eye) in the same individual animal does not prevent
entrainment of their circadian rhythms of locomotor activity
to light [16,65]. Furthermore, shielding the brains of
blinded± pinealectomized S. olivaceus entrained to LD
cycles causes them to free-run. Taken together, these data
demonstrate the existence of brain photoreceptors mediating
entrainment of locomotor rhythms to LD cycles. Reptile
extraretinal photoreceptors must be quite sensitive, because
blind lizards can be entrained to an LD cycle as dim as 1 lx
[54]. Brain photoreceptors mediating entrainment have been
documented in Alligator missisipiensis [33].
Recent attempts to localize photoreceptors in the deep
brain by using antibodies were successful. In the Iguanid
lizards A. carolinensis and I. iguana anti-opsins antibodies
labeled neurons in the basal region of the lateral ventricles
[19,20]. A brain rhodopsin was recently cloned in P. sicula,
but its location in the brain is unknown [43].
Two different mechanisms, but not mutually exclusive,
have been proposed for entrainment [1,45]: in one, only
the transitions from light to dark and from dark to light
are considered effective for entrainment to 24-h LD
cycles (nonparametric entrainment); in the other, light
and darkness are assumed to exert a more or less
continuous action on the velocity of circadian oscillators
(parametric entrainment). The observation that the velo-
city of oscillators changes by changing the intensity of
LL (Aschoff's rule) supports the model of parametric
entrainment. In diurnal animals, for example, the light
portion of the 24 h LD cycle may increase the velocity of
the oscillators and the dark portion decrease their velocity,
with the net effect of entraining period of the zeitgeber.
Hence, it may be interesting to examine what array of
photoreceptors mediate the response of the circadian
system to changes in LL intensities. Underwood and
Menaker [65] investigated this aspect of circadian orga-
nization in S. olivaceus (Iguanidae) and P. sicula (Lacer-
tidae), by testing the locomotor behavior of these diurnal
lizards exposed to different levels of LL intensities after
bilateral enucleation. When intact, both S. olivaceus and
P. sicula obey Aschoff's rule for diurnal animals [22,65].
After blinding, S. olivaceus continuestoobeyto
Aschoff's rule, while P. sicula cannot discriminate among
different levels of LL and between LL and DD [65]. This
led to the conclusion that in S. olivaceus extraretinal
photoreceptors can mediate the response of the circadian
system to changes in level of LL, while in P. sicula this
function is accomplished only by the retinas of the lateral
eyes. In contrast with this, new investigations in P. sicula
showed clearly the existence in this lizard of extraretinal
photoreceptors that are capable of mediating the effects of
changing in level of LL on circadian locomotor behavior
(Fig. 5) [16]. Such a disagreement may depend on genetic
differences between animals, since the P. sicula used in
the experiments by Underwood and Menaker were col-
Fig. 5. Locomotor activity record of a Lacertid lizard P. sicula subjected to
pinealectomy (PIN-X) and then to retinalectomy (RET-X) under LL 30 lx.
This lizard can discriminate between different levels of LL (Aschoff's rule)
after PIN-X-RET-X: tshortens under LL 600 lx and lengthens under LL 30
lx. The final part of the record shows entrainment of the activity rhythm of
the PIN-X-RET-X lizard to a 24-h light cycle. These data show the
existence of extrapineal ± extraretinal photoreceptors in P. sicula (redrawn
from Ref. [16]).
G. Tosini et al. / Physiology & Behavior 72 (2001) 461±471 467

lected in north-east Italy and Croatia, whereas the P.
sicula used by Foa
Áet al. were collected in central Italy
(about 500± 700 km apart). Even if this interpretation is
correct, it is still unclear why some lizards can use
extraretinal photoreception to discriminate among different
intensities of LL, while others have only the retinas
available to accomplish this function.
7. The SCN and their role in the circadian organization
In mammals, namely rodents, the SCN have been
shown to contain a circadian multioscillator system that
acts as the primary (master) pacemaker for a host of
physiological and behavioral rhythms. During the last
decade the SCN have also been recognized to play a role
in the circadian system of lizards [28,39]. First of all, the
lizard SCN are topographically similar to the SCN of
rodents, and receive a direct retinal projection. As in
rodents, the lizard SCN lies just dorsal to the optic chiasm
and adjacent to the third ventricle, in the region of
transition from the preoptic area to the hypothalamus
(Fig. 6) [5]. Furthermore, as in rodents, the SCN of the
Iguanid lizard D. dorsalis were shown to bind antibodies
raised against neuropeptide Y [29]. Collectively, these
data strongly support the contention that the SCN of
lizards are homologous to the SCN of mammals. Electro-
lytic lesions to 90% or more of the SCN (SCN-X) were
found to abolish circadian rhythms of locomotor activity
both in D. dorsalis and P. sicula (Figs. 6C and 7A)
[28,39]. Except SCN lesions, no experimental treatment or
lesion has so far succeeded in abolishing circadian loco-
motor rhythmicity in both P. sicula and D. dorsalis. This
evidence supports the contention that in both species the
SCN contain the primary circadian pacemaker driving
locomotor rhythms.
Fig. 6. (A) Schematic reconstruction of a transverse brain section at the level of the SCN of the lizard P. sicula. The SCN lies just dorsal to the optic chiasm and
adjacent to the third ventricle, in the region of transition from the preoptic area to the hypothalamus. Dotted lines encompass the area of the section containing
the SCN. Magnifications of this area are presented in the photomicrographs of transverse brain sections reported in the right panel. Sections (B) and (C) are
stained with cresyl violet. Scale bar 40 mm. (B) Brain section of a lizard in which the SCN (indicated by arrows) remained intact. (C) Brain section of a lizard in
which the SCN (indicated by arrows) were completely lesioned. Abbreviations: cpp, posterior pallial commissure; Cxl, cortex lateralis; Cxm, cortex medialis;
DVR, dorsal ventricular ridge; lfb, lateral forebrain bundle; mfb, medial forebrain bundle; OC, optic chiasm; PH, nucleus periventricularis hypothalami; V, III
ventriculus (redrawn from Ref. [39]).
G. Tosini et al. / Physiology & Behavior 72 (2001) 461±471468

Other experiments confirmed the role of the SCN as
primary pacemaker in the P. sicula circadian system. Daily
injections of exogenous melatonin entrain locomotor
rhythms of intact, pinealectomized, and unilaterally SCN-
lesioned P. sicula, but are incapable of restoring rhythmicity
in subjects previously rendered arrhythmic by SCN-X (Fig.
7B) [4] Furthermore, the fact that the circadian rhythm of
behavioral temperature selection of P. sicula is not definitely
abolished after both parietalectomy and pinealectomy sug-
gests that the SCN or neighboring hypothalamic areas may
also be involved in driving this rhythm [24]. Interestingly, in
D. dorsalis the daily bout of voluntary hypothermia dis-
appears after lesion to the periventricular preoptic area of
the hypothalamus [3].
The SCN may be important to the circadian organization
of lizard species besides P. sicula and D. dorsalis. In both S.
occidentalis and I. iguana the removal of all known circa-
dian components (retinas, pineal, and parietal eye) does not
abolish circadian rhythms of locomotor activity [52,57],
demonstrating the existence of oscillators elsewhere in the
system. In A. carolinensis pinealectomy abolishes locomo-
tor rhythms in constant conditions, but the entrained phase
of these rhythms in pinealectomized individuals shows the
existence of oscillators elsewhere [58]. The data in P. sicula
and D. dorsalis suggest that such oscillators in S. occiden-
talis,I. iguana, and A. carolinensis may be contained within
the SCN.
8. Conclusions and perspectives
Reptiles because of their phylogenetic position and
ecology still provide the circadian field with some of the
most interesting model to understand circadian organiza-
tion, its evolution, and its variability. In lizards the pineal
complex, the retinas of the lateral eyes, and more recently
the SCN were all shown to participate in the control of
circadian rhythms. Noteworthy, the role these structures
play in circadian organization may vary interspecifically, as
it is the case of the pineal. However, the profound seasonal
differences in the behavioral effects of pinealectomy dis-
covered in P. sicula suggest that some of the interspecific
differences reported so far among lizards may, at least in
part depend on the particular season in which the beha-
vioral effects of pinealectomy have been examined in each
different species. Systematic studies of pinealectomized
lizards in different seasons across several species will
certainly solve the problem. Due to the central role of
the SCN in the circadian organization of two lizards (D.
dorsalis and P. sicula), similar studies should be extended
to further species of reptiles and to other aspects of
circadian behavior, such as, for instance, photic entrain-
ment. This will improve and extend phylogenetic compar-
isons among vertebrates concerning the circadian role of
the SCN. Again, several functional aspects of the redun-
dancy of circadian photoreceptors in reptiles (lateral eyes,
pineal, parietal eye, and deep brain photoreceptors) await
clarification. As regards deep brain photoreceptors, future
investigations in reptiles should be aimed at establishing
the precise location(s) of those brain photoreceptors that
effectively mediate photic entrainment, their characteriza-
Fig. 7. Effects of complete electrolytic lesions to the SCN (SCN-X) on
circadian locomotor rhythms in the Lacertid lizard P. sicula. Locomotor
activity records were double-plotted to aid in interpretation. (A) Record of a
lizard free-running in constant darkness (DD), which became arrhythmic in
response to SCN-X. (B) While intact, a lizard tested during the summer
entrained to the 24-h period of melatonin injections. After SCN-X this
lizard became behaviorally arrhythmic. Melatonin injections continued after
surgery, and their schedule was shifted on September 7th from 11:00 a.m. to
03:00 p.m. The SCN-X lizard remained arrhythmic during the whole
injection period (redrawn from Refs. [4,39]).
G. Tosini et al. / Physiology & Behavior 72 (2001) 461±471 469

tion at the molecular level, and the pathway(s) from them
to the rest of the circadian system.
Acknowledgments
Work in the A. Foa
Áand C. Bertolucci laboratory was
supported by grants from the Ministero dell'Universita' e
della Ricerca Scientifica. Work in the G. Tosini laboratory
was supported by grants from the National Institute of
Neurological Disorders and Stroke.
References
[1] Aschoff J. Exogenous and endogenous components in circadian
rhythms. Cold Spring Harbor Symp Quant Biol 1960;25:11 ± 28.
[2] Bartell P, Miranda-Anaya M, Menaker M. Effects of light, pinealect-
omy and enucleation on the circadian organization of the green igua-
na. Int Congr Chronobiol Abstr, 1999;49.
[3] Berk ML, Heath JE. Effects of preoptic, hypothalamic and telence-
phalic lesion on thermoregulation in lizards, Dipsosaurus dorsalis.J
Therm Biol 1975;1:65 ± 78.
[4] Bertolucci C, Foa
ÁA. Seasonality and role of the SCN in entrainment
of lizard circadian locomotor rhythms to daily melatonin injections.
Am J Physiol 1998;274:R1004 ± 14.
[5] Casini G, Petrini P, Foa
ÁA, Bagnoli P. Pattern of organization of
primary visual pathways in the European lizard Podarcis sicula Ra-
finesque. J Hirnforsch 1993;34:361 ± 74.
[6] Cassone VM. Effects of melatonin on vertebrate circadian systems.
Trends Neurosci 1990;13:457 ± 64.
[7] Cowgell G, Underwood H. Behavioral thermoregulation in lizards:
circadian rhythm. J Exp Zool 1979;210:189 ± 94.
[8] Firth BT, Kennaway DJ, Rozenbilds MAM. Plasma melatonin in the
scincid lizard, Thachydosaurus rugosus: diel rhythms, seasonality,
and the effects of constant light and constant darkness. Gen Comp
Endocrinol 1979;37:493 ± 500.
[9] Firth BT, Kennaway DJ. Plasma melatonin levels in the scincid lizard
Thachydosaurus rugosus: The effects of paristal eye and lateral eye
impairment. J. Exp. Biol. 1980;85:311 ± 21.
[10] Firth BT, Kennaway DJ. Melatonin content of pineal, parietal eye and
blood plasma of the lizard, Thachydosaurus rugosus: effect of con-
stant and fluctuating temperature. Brain Res 1987;404:313 ± 8.
[11] Firth BT, Thompson MB, Kennaway DJ. Thermal sensitivity of repti-
lian melatonin rhythms ``cold'' tuatara vs. ``warm'' skink. Am J Phy-
siol 1989;256:R1160± 3.
[12] Firth BT, Belan I, Kennaway DJ, Moyer RW. Thermocyclic entrain-
ment of lizard blood plasma melatonin rhythms in constant and cyclic
photic environments. Am J Physiol 1999;277:R1620 ± 6.
[13] Foa
ÁA. The role of the pineal and the retinae in the expression of
circadian locomotor rhythmicity in the ruin lizard, Podarcis sicula.J
Comp Physiol, A 1991;169:201 ± 7.
[14] Foa
ÁA, Janik D, Minutini L. Circadian rhythms of plasma melatonin in
the ruin lizard Podarcis sicula: effects of pinealectomy. J Pineal Res
1992;12:109 ± 13.
[15] Foa
ÁA, Minutini L, Innocenti A. Melatonin: a coupling device be-
tween oscillators in the circadian system of the ruin lizard Podarcis
sicula. Comp Biochem Physiol 1992;103A:719 ± 23.
[16] Foa
ÁA, Flamini M, Innocenti A, Minutini L, Monteforti G. The role of
extraretinal photoreception in the circadian system of the ruin lizard
Podarcis sicula. Comp Biochem Physiol 1993;105A:223 ± 30.
[17] Foa
ÁA, Monteforti G, Minutini L, Innocenti A, Quaglieri C, Flamini
M. Seasonal changes of locomotor activity patterns in ruin lizards
Podarcis sicula: I. Endogenous control by the circadian system. Be-
hav Ecol Sociobiol 1994;34:227 ± 74.
[18] Foa
ÁA, Bertolucci C, Marsanich A, Innocenti A. Pineal transplantation
to the brain of pinealectomized lizards: effects on circadian rhythms of
locomotor activity. Behav Neurosci 1997;111:1123± 32.
[19] Foster RG, Garcia-Fernandez JM, Provencio I, DeGrip WJ. Opsin
localization and chromophore retinoids identified within the basal
brain of the lizard Anolis carolinensis. J Comp Physiol, A
1993;172:33 ± 45.
[20] Grace MS, Alones V, Menaker M, Foster RG. Light perception in the
vertebrate brain: an ultrastructural analysis of opsin- and vasoactive
intestinal polypeptide-immunoreactive neurons in iguanid lizards. J
Comp Neurol 1996;367:575 ± 94.
[21] Hyde LL, Underwood H. Daily melatonin infusions entrain the loco-
motor activity of pinealectomized lizards. Physiol Behav
1995;58:951 ± 3.
[22] Hoffmann K. Versuche zur Analyse der Tagesperiodik: I. Der Einfluss
der Lichtintensitaet. Z Vgl Physiol 1960;43:544 ± 66.
[23] Hoffmann K. Zur Syncronisation biologischer Rhythmen. Verh Dtsch
Zool Ges 1970;25:166 ± 273.
[24] Innocenti A, Minutini L, Foa
ÁA. The pineal and circadian rhythms of
temperature selection and locomotion in lizards. Physiol Behav
1993;53:911± 5.
[25] Innocenti A, Minutini L, Foa
ÁA. Seasonal changes of locomotor ac-
tivity patterns in ruin lizards Podarcis sicula: II. Involvement of the
pineal. Behav Ecol Sociobiol 1994;35:27 ± 32.
[26] Innocenti A, Bertolucci C, Minutini L, Foa
ÁA. Seasonal variations of
pineal involvement in the circadian organization of ruin lizards Po-
darcis sicula. J Exp Biol 1996;199:1189± 94.
[27] Janik DS, Menaker M. Circadian locomotor rhythms in the desert
iguana: I. The role of the eyes and the pineal. J Comp Physiol, A
1990;166:803 ± 10.
[28] Janik DS, Pickard GE, Menaker M. Circadian locomotor rhythms in
the desert iguana: II. Effects of electrolytic lesions to the hypothala-
mus. J Comp Physiol, A 1990;166:811 ± 6.
[29] Janik DS, Cassone VM, Pickard GE, Menaker M. Retinohypothala-
mic projections and immunocytochemical analysis of the suprachias-
matic region of the desert iguana Dipsosaurus dorsalis. Cell Tissue
Res 1994;275:399 ± 406.
[30] Jarling C, Scarperi M, Bleichert A. Circadian rhythm in the tempera-
ture preference of the turtle, Chrysemys (=Pseudemys)scripta ele-
gans, in a thermal gradient. J Therm Biol 1989;14:173 ± 8.
[31] Kasal C, Menaker M, Perez-Polo R. Circadian clock in culture: N-
acetyltransferase activity of chick pineal glands oscillates in vitro.
Science 1979;203:656 ± 8.
[32] Kavaliers M. Circadian locomotor activity rhythms of the burbot Lota
lota: seasonal differences in period length and the effect of pinealect-
omy. J Comp Physiol 1980;136:215 ± 8.
[33] Kavaliers M, Ralph CL. Encephalic photoreceptor involvement in the
entrainment and control of circadian activity of young American alli-
gators. Physiol Behav 1981;26:413 ± 8.
[34] Menaker M. The search for principles of physiological organization in
vertebrate circadian system. In: Aschoff J, Daan S, Groos GA, editors.
Vertebrate circadian system. Berlin: Springer-Verlag, 1982. pp. 1 ± 12.
[35] Menaker M. Eyes Ð the second (and third) pineal gland? In:
Evered D, Clark S, editors. Photoperiodism, melatonin and
pineal. London: Pitman, 1985. pp. 39 ± 52.
[36] Menaker M, Wisner S. Temperature-compensated circadian clock in
the pineal of Anolis. Proc Natl Acad Sci USA 1983;80:6119 ± 21.
[37] Menaker M, Tosini G. The evolution of vertebrate circadian system.
In: Honma K, Honma S, editors. Circadian organization and oscilla-
tory coupling. Sapporo: Hokkaido Univ. Press, 1996. pp. 39 ± 52.
[38] Minutini L, Innocenti A, Bertolucci C, Foa
ÁA. Electrolytic lesions to
the optic chiasm affect circadian locomotor rhythms in lizard. Neu-
roReport 1994;5:525 ± 7.
[39] Minutini L, Innocenti A, Bertolucci C, Foa
ÁA. Circadian organization
in the ruin lizard Podarcis sicula: the role of the suprachiasmatic
nuclei of hypothalamus. J Comp Physiol, A 1995;76:281 ± 8.
[40] Miranda-Anaya M, Bartell P, Yamasaki S, Menaker M. Circadian
G. Tosini et al. / Physiology & Behavior 72 (2001) 461±471470

rhythm of ERG in Iguana iguana: role of the pineal. J Biol Rhythms
2000;15:163 ± 71.
[41] Molina-Borja M. Pineal-gland and circadian locomotor-activity
rhythm in the lacertid Gallotia galloti eisentrauti, pinealectomy in-
duces arrhythmicity. Biol Rhythm Res 1996;27:1 ± 11.
[42] Moyer RB, Firth BT, Kennaway DJ. Effect of constant temperatures,
darkness and light on the secretion of melatonin by pineal explants
and retinas in the gecko Christinus marmoratus.BrainRes
1995;675:345 ± 8.
[43] Pasqualetti M, Innocenti A, Foa
ÁA, Nardi I. Cloning of a brain opsin
from lizard Podarcis sicula. Biol Rhythm Res 1997;28:127.
[44] Pickard GE, Tang WX. Individual pineal cells exhibit a circadian
rhythm in melatonin secretion. Brain Res 1993;627:141 ± 6.
[45] Pittendrigh CS. Circadian system: entrainment. In: Aschoff J, editor.
Biological rhythms. Handbook of behavioral neurobiology, vol. 4.
New York: Plenum, 1981. pp. 95± 124.
[46] Refinetti R, Susalka SJ. Circadian rhythm of temperature selection in a
nocturnal lizard. Physiol Behav 1997;62:331 ± 6.
[47] Shaw AP, Collazo CR, Easterling K, Young CD, Karwoski CJ. Cir-
cadian rhythm in the visual system of the lizard Anolis carolinensis.J
Biol Rhythms 1993;8:107 ± 24.
[48] Tilden AR, Hutchison VH. Influence of photoperiod and temperature
on serum melatonin in the diamondback water snake, Nerodia rhom-
bifera. Gen Comp Endocrinol 1993;92:347 ± 54.
[49] Tosini G. The pineal complex of reptiles: physiological and behavioral
roles. Ethol Ecol Evol 1997;9:313 ± 33.
[50] Tosini G, Menaker M. Circadian rhythm of body temperature in an
ectotherm (Iguana iguana). J Biol Rhythms 1995;10:248 ± 55.
[51] Tosini G, Menaker M. Pineal complex and melatonin affect the daily
rhythm of temperature selection in the green iguana. J Comp Physiol,
A 1996;179:135 ± 42.
[52] Tosini G, Menaker M. Multioscillatory circadian organization in a
vertebrate, Iguana iguana. J Neurosci 1998;18:1105± 14.
[53] Tosini G, Moreira LF, Bartell P, Menaker M. Temperature compensa-
tion of circadian rhythms in a multioscillatory system. SRBR Meeting
Abstr 1998;6:175.
[54] Underwood H. Retinal and extraretinal photoreceptors mediate en-
trainment of the circadian locomotor rhythm in lizard. J Comp Phy-
siol 1973;83:187 ± 222.
[55] Underwood H. Circadian organization in lizards: the role of the pineal
organ. Science 1977;195:587 ± 9.
[56] Underwood H. Melatonin affects circadian rhythmicity in lizard. J
Comp Physiol 1979;130:317 ± 23.
[57] Underwood H. Circadian organization in the lizard, Sceloporus occi-
dentalis: the effects of blinding, pinealectomy and melatonin. J Comp
Physiol 1981;141:537 ± 47.
[58] Underwood H. Circadian organization in the lizard Anolis carolinen-
sis: a multioscillatory system. J Comp Physiol 1983;152:265 ± 74.
[59] Underwood H. Pineal melatonin rhythms in the lizard Anolis caroli-
nensis: effects of light and temperature cycles. J Comp Physiol, A
1985a;157:57 ± 66.
[60] Underwood H. Extraretinal photoreception in the lizard Scelopo-
rus occidentalis: phase response curve. Am J Physiol 1985b;248:
R407 ± 14.
[61] Underwood H. Circadian rhythms in lizards: phase response curve for
melatonin. J Pineal Res 1986;3:187 ± 96.
[62] Underwood H. The pineal and melatonin: regulators of circadian
function in lower vertebrates. Experientia 1990;46:120 ± 8.
[63] Underwood H. Endogenous rhythms. In: Gans C, Crews D, editors.
Biology of reptilia: hormones, brain and behavior, vol. 18. Chicago:
University of Chicago Press, 1992. pp. 229 ± 97.
[64] Underwood H, Menaker M. Extraretinal light perception: entrainment
of the biological clock controlling lizard locomotor activity. Science
1970;170:190 ± 3.
[65] Underwood H, Menaker M. Extraretinal photoreception in lizard.
Photochem Photobiol 1976;24:241 ± 77.
[66] Underwood H, Harless M. Entrainment of the circadian activity
rhythm of a lizard to melatonin injection. Physiol Behav
1985;35:267 ± 70.
[67] Underwood H, Calaban M. Pineal melatonin rhythms in lizards Anolis
carolinensis: I. Response to light and temperature cycles. J Biol
Rhythms 1987;2:179 ± 93.
[68] Vivien-Roels B, Arendt J, Bradtke J. Circadian and circannual fluc-
tuations of pineal indoleamines (serotonin and melatonin) in Testudo
hermanni Gmelin (Reptilia, Chelonia): I. Under natural conditions of
photoperiod and temperature. Gen Comp Endocrinol 1979;37:197 ±
210.
[69] Yamazaki S, Goto M, Menaker M. No evidence for extraocular photo-
receptors in the circadian system of the Syrian hamster. J Biol
Rhythms 1999;14:197 ± 201.
G. Tosini et al. / Physiology & Behavior 72 (2001) 461±471 471