Brain Research Reviews, 18 (1993) 315-333
0 1993 Elsevier Science Publishers I3.V, All rights reserved ~16S”~l~3~93/S~.~
Circadian rhythms *
Benjamin D. Aronson, Deborah Bell-Pedersen, Gene D. Block, Nice P.A. Bos, Jay C. Dunlap,
Arnold &kin, Norman Y. Garceau, Michael E, Geusz, Keith A. Johnson, Sat Bir S. I&&a,
Gerdien C. Koster-Van Hoffen, Costas Koumenis, Theresa M. Lee, Joseph LeSauter,
Kristin M. Lindgren, Qiuyun Liu, Jennifer J. Lures, Stephan H, Michel, Majid Mirmiran,
Robert Y, Moore, Norman F. Ruby, Rae Silver, Fred W. Turek, Martin Zatz and Irving Zucker
(Accepted 2 April 1993)
Key wrzrds: Suprachiasmatic nucleus; Retina; Transplant; Pineal gland; Melatonin; Ap[ysia; Neurospora; Pacemaker
Circadian rhythms are a ubiquitous adaptation of eukaryotic organisms to the most reliable and predictable of environmental changes, the daily
cycles of light and temperature. Prominent daily rhythms in behavior, physiology, hormone levels and biochemistry &eluding gene expression)
are not merely responses to these environmental cycles, however, but embody the organism’s ability to keep and tell time. At the core of
circadian systems is a mysterious mechanism, located in the brain (actually the suprachiasmatic nucleus of the hypothalamus) of mammals, buf
present even in unicellular organisms, that functions as a clock. This clock drives circadian rhythms. It is independent of, but remains responsive
to, environmental cycles (especially light). The interest in temporal regulation - its organization, mechanism and consequences
investigators in diverse disciplines studying otherwise disparate systems. This diversity is reflected in the brief reviews that summarize the
~~sen~a~jons at a meeting on circadian rhythms held in New York City on October 31,1992. The meeting was sponsored by the Fandation pour
I’Etude du Systime Nerveux (FESN) and followed a larger meeting heId 18 manths eartier in Geneva, whose proceedings have been published
(M. Katz (Ed,), Report of the Ninth FESN Study Group on ‘Circadian Rhythms’, Disc~&~~ in Newosc&ceJ
Amsterdam, 1992% Some speakers described progress made in the interim, while others addressed aspects of the field not previously covered.
Vd KY& Mm. 2 + 3, Elsevier,
Summary,.., .,.> I . . . . . . . . . . . . . . . . . . . . . . ..t.........~ . ...* 4 + .,.. . . ...*. a .........n...l..+ .f I........._
Temperature, seasonal@ and circadian rhythms
Aging, feedback and the circadian clock
Functional organization of the circadian timing system
Majid Mirmiran, Nice P.A. E?as and Gerdien C. Koster-Van Hoffen .................................................
4.1. Introduction .....................................................................................
and neuraph~~a~io~ of rhe ~~prac~~as~tic nucleus in culture
Room 2A-17, Bethesda, MD 20692, USA. Fax: fl) (30’1) 4964103.
* These manuscripts were presented at a l%SN Study Group Follow-up Meeting on ‘Circadian rhythms’, held in New York City on October
** Far a list of participants of this FESN Study Group and the authors’ addresses, please see page 330 (at the end of this artide).
M. Zatz, Section on Biochemical Fha~a~lo~, Laboratory of Cell Siology, Nationai Institute of Mental Health, Buiiding 36,
4.2. Experimental evidence in favor of the pacemaker hypothesis
4.3. Experimental evidence in favor of the network hypothesis
5. What do suprachiasmatic nucleus transplants do’?
Rae Silver and Joseph LeSauter ..........................................................................
5.1. Introduction .....................................................................................
5.2. Where are the pacemakers? ..........................................................................
5.3. What responses are restored by suprachiasmatic nucleus grafts? ................................................
5.4. Responses of grafted animals to pharmacological agents. .....................................................
5.5. Transplanted suprachiasmatic nuclei vs those in situ .................................
5.6. Output signals of the suprachiasmatic nucleus .............................................................
5.7. What do transplants really do? ........................................................................
5.8. Summary ................................
. . ... . .................. . ................ . .............
Cellular studies of a retinal biological clock
Gene D. Block, Michael E. Geusz, Sat Bir S. Kbalsa and Stephan H. Michel ...................
6.2. Entrainment .....................................................................................
6.3. Expression ......................................................................................
6.4. Rhythmgeneration ................................................................................
6.5. Pacemakermodel .................................................................................
Convergence and divergence in chick pineal regulation
Putative oscillator proteins in the Aplysiu eye circadian system
Genetic and molecular analysis of the Neurospora circadian clock
Jay C. Dunlap, Jennifer J. Lores, Keith A. Johnson, Kristin M. Lindgren, Benjamin D. Aronson, Deborah Bell-Pedersen, Qiuyun Liu
1. Temperature, seasonality and circadian rhythms
Irving Zucker, Theresa M. Lee and Norman F. Ruby
(CRs) have been documented
mals. The period (7) of the free-running
and the times of activity onset relative to dawn or dusk
change markedly over the course of the year36. One
might suppose that seasonal variations in day length,
temperature, humidity and food availability induce
these responses. Chronobiology
with counterintuitive causalities: seasonal changes in
some CRs are endogenous;
years even when environmental
During the spring and summer,
ground squirrels are homeothermic
temperatures CT,> of 37°C; during the autumn and
winter heterothermic phase, Tb declines to l-2°C above
ambient temperature CT,). Squirrels kept at a T, of
23°C maintain a Tb of 37°C for half the year and
decrease their Tbs intermittently
seasonal changes in circadian rhythms
in fish, birds and mam-
is, however, replete
they persist for several
conditions are held
and defend body
to 24°C during the
remaining 6 months 37 A constant
compatible with marked fluctuations in Tb. We sought
to explain seasonal variations in CRs within this frame-
work. Specifically, we asked whether seasonal changes
in r of the free-running CR are caused by endogenous
seasonal reductions in Tb. This conjecture was upheld:
when T, was increased and decreased by 10°C during
the squirrels’ heterothermic
CR became shorter and longer, respectively (Fig. D3’.
Identical changes in T, were without effect on T during
the squirrels’ homeothermic phase. T, appears to influ-
ence squirrel CRs when it effects changes in Tb. A
similar relation obtained in Siberian hamsters: r of the
CR changed only when animals displayed
(Thomas, Jewett and Zucker,
When tissue temperature
rates of many biological processes double or triple, but
circadian time measurement
T, is, therefore,
phase, the T of the activity
increases by 10°C reaction
changes relatively little
Fig. 1. Decreases and increases in circadian r in response to 10°C
changes in T, of a squirrel kept in constant dim light. The heavy
lines on the activity record were added to facilitate visual estimation
of 7 (ref. 37).
rized above do not challenge this principle; they do
indicate, however, that when fluctuations in T, induce
marked changes in Tb, the impact on CRs is substan-
tial. It has not yet been determined whether the circa-
dian pacemaker in the suprachiasmatic nucleus (SCN)
is directly responsive to temperature.
of temperature as a significant influence on CRs is
unjustified and based on misinterpretation
The importance of the SCN for circadian organiza-
tion of mammals is widely acknowledged;
generation of the Tb CR has been both championed
and disputed on the basis of ablation studies in rats.
Species that display daily torpor provide a favorable
model system to address this question; the amplitude
compensation)72. The findings summa-
its role in
Fig. 2. Elimination of daily torpor in a hamster after ablation of the
SCN (* ); torpor was reinstated during food restriction (FR) and
absent during resumption of ad libitum (AL) feeding63.
of their Tb CRs is an order of magnitude greater than
that of homeothermic mammals.
Siberian hamsters typically endure for 4-8 h, during
which Tb declines from 37” to 20°C. Torpor bouts are
entrained by the light-dark (LD) cycle and free-run in
constant illumination64. Torpor, which typically is man-
ifested during the winter, can be induced by maintain-
ing animals for several months in short day lengths or
at any time of year by curtailing food availability.
Ablation of the SCN (SCN,) eliminated torpor in short
day hamsters (Fig. 2)63. Torpor was, however, rein-
stated when animals were food restricted
The temporal structure of torpor bouts was severely
disrupted in SCN, hamsters; entrainment
Torpor bouts of
to the LD
4 8 12 16
. . . *. ' . . . I
. . ' . " .
0 4 12
Ti m e
of Day (h)
Fig. 3. Representative preoperative (top) and postoperative Tas from
a food-restricted SCN, hamster that expressed multiple torpor bouts
per 24 h after surgery. Lights were on from 08.00 to 16.00 h.
cycle and the coherence
were eliminated. The occurrence of several torpor bouts
per day in SCN, but not intact animals was notabIe
of the free-running rhythm
(Fig. 3jh’. The SCN is not essential for the expression
of torpor but plays a crucial role in its temporal organi-
2. Aging, feedback and the circadian clock
Fred W. Turek
Age related changes have been documented
docrine, metabolic and behavioral circadian rhythms in
a variety of animal species, including humans’, These
changes in&de alterations of the period length, ampli-
tude and/or phase of many circadian rhythms. Support
for the hypothesis that age-related changes in the ex-
pression of circadian rhythms reflect, at least in part,
the aging of the circadian clock itself come from stud-
ies demonstrating that metabolic and neuropeptidergic
changes occur in advanced age in the suprachiasmatic
nucleus (SCN) of the hypothalamus, the location of a
master circadian pacemaker in mammals71~78.
The underlying causes for age-related
the circadian system remain clown.
demonstrating that changes in the activity-rest
can have major feedback effects on the circadian clock
that regulates this rhythm74, coupled with the observa-
tions that the activity-rest
age’, have led us to investigate whether the feedback
effects of the activity-rest cycle on the circadian clock
may be altered in advanced age.
Acute exposure of old hamsters (16-28 months of
age) to two different stimuli (i.e., benzodiazepines
dark pulse on a background of constant light), which
are known to induce phase shifts in the circadian clock
of young hamsters (2-6 months of age) by inducing
activity at times when the animals are normally inactive
or showing little activity, has very little, if any, phase-
shifting effect on the circadian clock (Fig. 4). In con-
trast, old hamsters remain sensitive to the phase shift-
ing effects of stimuli that are not associated with any
change in the activity-rest
thesis inhibitors or pulses of light. In addition, we have
recently found that while the circadian rhythm of loco-
motor activity of young hamsters can be entrained by
daily injections of a short-acting benzodiazepine,
response is lost or attenuated in old animals.
Taken together, these results indicate that the circa-
dian system of old animals becomes selectively unre-
sponsive to synchronizing signals mediated by the activ-
ity-rest state. Previous age-related
dian rh~hmici~ that have been observed in mammals,
cycle is disrupted in old
state, such as protein syn-
changes in circa-
including humans, might be related to weakened cou-
pling or feedback between the activity-rest
the circadian clock.
- -- _-~
Fig. 4. Mean ( f S.E.M.) phase shifts in the activity rhythm of young
and old hamsters maintained in LL that were subjected, beginning at
either CT 6 (left panel) or CT 18 (right panel) to a 6-h dark pulse. A
value above the solid line indicates an advance in the onset of
locomotor activity, a value below indicates a delay. The values in
parentheses indicate the number of trials for each group of animals.
The mean phase shift in the activity rhythm in response to the dark
pulse was significantly greater (P < 0.001) in young than in old
hamsters at both circadian times tested. Bottom: representative
sections from the wheel-running activity records of two young (left
panets) and two old (right panels) hamsters housed in LL before and
after they were subjected to a 6-h dark pulse beginning at CT 6 (top
panels) or CT 18 (bottom panels). The day of the dark pulse is
designated by ‘DP’ at the left of each record with the exact beginning
and end of the dark pulse designated by two stars. Reproduced with
permission from 0. Van Reeth et a1.76.
3. Functional organization of the circadian timing system
Robert Y. Moore
The mammalian circadian timing system (CIS) is a
set of neural structures that functions to provide a
temporal organization for physiological processes and
behavior to promote adaptation and survival. The com-
ponents of the CTS are as follows: (1) photoreceptors
and projections of retinal ganglion cells that form
entrainment pathways; (2) circadian pacemakers; and
(3) efferent pathways that couple the pacemakers
effector systems displaying circadian function.
3.2. Entrainment pathways
Light is the principal
Light affects a specific subset of retinal photorecep-
tors18. These photoreceptors
of ganglion cells that projects as a retinohypothalamic
tract (RI-IT) to the suprachiasmatic nucleus (SCN), the
adjacent anterior hypothalamic
matic area and to the lateral hypothalamic areaz. The
RHT is sufficient to maintain entrainment; transection
of the RHT results in a loss of entrainment”.
RHT forms as collaterals of optic nerve axons that
traverse the optic chiasm and optic tract to innervate a
subdivision of the thalamic lateral geniculate’complex,
the intergeniculate leaflet (IGL). The IGL contains a
population of neuropeptide
projects in a separate pathway, the geniculohypothala-
mic tract (GHT), to the SCN5*. IGL neurons are also
GABA-containing (Fig. 5j51.
What is the function of these pathways? Stimulation
of the RHT produces changes in the phase of free-run-
ning rhythms with a phase response curve (PRC) very
similar to that of light .
45 In contrast, stimulation of the
GHT or direct application of NPY to the SCN, results
Zeitgeber for entrainment.
are connected to a subset
area and retrochias-
Y (NPY) neurons that
Fig. 5. Diagram showing the organization of the circadian timing
system. Retinal ganglion cells, thought to contain glutamate, project
to both the SCN and IGL. One population of SCN neurons, contain-
ing VIP and GABA, receives input from both the retina and the
IGL. A second population contains VP and GABA and is recipro-
cally connected with the VIP-GABA neurons. Both project mainly to
the hypothalamus with some projections
to basal forebrain and
in phase changes with a PRC that is quite different
from light or RHT stimulation5*. This PRC also can be
obtained from a series of disparate stimuli that share
the common feature of inducing locomotor activity5*.
Ablation of the IGL eliminates activity-induced phase
changes5* indicating that the IGL-GHT
volved in mediating feedback regulation of the circa-
dian pacemaker. This suggests that, contrary to gener-
ally held views, pacemaker
plished by an interaction of visual and feedback input
to the pacemaker. Much of the feedback regulation
system is in-
homeostasis is accom-
Fig. 6. Photomicrographs
the intergeniculate leaflet (Cl of the lateral geniculate but not to other primary visual nuclei. The Bartha strain of the pseudorabies virus is
injected into the eye and is taken up by the selected population of ganglion cells that transmits the virus to the SCN and IGL. Marker bars, 20
pm (A), 150 pm (B), 200 pm (0.
showing viral labeling of a population of retina1 ganglion cells (A) that projects to the suprachiasmatic nucleus (B) and
appears to be accomplished
pathway but other, direct inputs to the SCN undoubt-
edly contribute as well.
Although it has appeared
ganglion cell projection to the SCN and IGL derives
from a separate subset of ganglion cells that do not
project to other major visual centers, this heretofore
has not been demonstrated
neurotrophic herpesvirus, we have found labeling of a
subset of retinal ganglion cells with a mean perikaryal
diameter of about 15 pm that projects selectively to
the SCN and IGL (Fig. 619. On this basis we would
conclude that these ganglion cells are components of
through the IGL-GHT
likely that the retinal
directly. Using a specific
The SCN are pacemakers in the mammalian brain45.
The evidence for this conclusion is as follows: the RHT
terminates in the SCNz. Ablation of the SCN abol-
ishes most, if not all, circadian function45. In isolation,
both in vivo and in vitro, the SCN maintain a circadian
rhythm in neuronal activity45. Transplantation
SCN into the third ventricle of arrhythmic, SCN-le-
sioned hosts restores circadian rhythmicity with a pe-
riod characteristic of the donor59. The SCN contains
two subdivisions: one is characterized
of vasoactive intestinal polypeptide-producing
and receives most of the RHT and GHT input; the
second is characterized by a population of vasopressin-
by a population
input4”. Both sets of SCN peptide-producing
also appear to be GABA-producing
SCN subdivisions appear to be comprised of individual
circadian oscillators that become coupled by intercon-
nections to form a network with pacemaker function.
neurons and receives very limited visual
(Fig. 5)“‘. The
3.4. Efferent pathways
The efferent projections of the SCN are largely to
adjacent anterior hypothalamic
restricted projections to basal forebrain and midline
thalamus (Fig. 5)77.
areas with relatively
3.5. Circadian timing system organization
The CIS has three identified sets of component
nuclei, the retina, SCN and IGL. The retinal ganglion
cells projecting to the SCN and IGL are a distinct
subpopulation of ganglion cells that does not project to
other visual centers’. Like other ganglion cells, how-
ever, they appear to produce glutamate or a closely
related excitatory transmitter (Fig. 5)45. IGL neurons
produce either GABA and NPY and project to the
SCN or GABA and enkephalin
contralateral IGL5*. The IGLGHT
integrate photic with non-photic
participate in pacemaker homeostasis5*. The neurons
of the SCN are also GABA-producing51
the output of the CTS provides a cyclic inhibitory
influence on systems that express circadian function.
and project to the
system appears to
entraining input to
4. Neurophysiology and neuropharmacology
nucleus in culture
of the suprachiasmatic
Majid Mirmiran, Nice P.A. Bos and Gerdien C. Koster-Van Hoffen
An overwhelming body of evidence indicates that
the suprachiasmatic nucleus (SCN; the so-called bio-
logical clock) of the anterior hypothalamus is involved
in the generation and synchronization
rhythms32,45,81. The following results suggest that the
SCN is indeed capable of endogenous rhythm genera-
tion with a periodicity of around 24 h in the absence of
neuronal input/output connectivities: (1) in viva, circa-
dian rhythms of electrical activity remain in the neu-
ronally isolated SCN island; (2) after SCN transplanta-
tion it only takes a few days before the circadian
rhythms reappear in an arhythmic SCN-lesioned ani-
mal (this is not long enough for the reestablishment
the neuronal input/output
the rhythm period is defined by the donor SCN and
not by the original inherited host rhythm; (3) in vitro,
neuronal discharges, 2-deoxyglucose
synthesis and peptide production/release
dian rhythms in acute or chronic SCN explants.
Earlier studies using acute SCN slices taken from
animals sacrificed during the subjective day and night
showed that neuronal discharges are high by day (peak
at CT 6-8) and low at night6’. In a semi-chronic
were able to show high- and
low-rhythmic levels of vasopressin release from SCN
explants in the medium over several days. A recent
study suggests that such rhythms are even Present in
dissociated SCN cells taken from newborn rad3.
One of the d~~iculties of i~te~reting
in vitro from acute adult SCN slices is the presence of
degeneration and acute hypoxia. In order to overcome
this problem and develop a suitable model for studying
neuronal mechanisms underlying the generation of cir-
cadian ~~~~rns within the SCN, we cultured organ-
otypic SCN explants (taken from lZday=old rat pups)
in a chemically defined medium over a period of sev-
eral weeks’. The morphology of the SCN remains more
or less intact in this preparation except for the elimina-
tion of retinoh~thalami~
input/output from the rest of the brain. Starting l-4
weeks after culturing, we were able for the first time to
record continuously from single SCN neurons over a
period of 36-66 h. Twenty-two
(each from a different explant) and 17 neurons showed
high- and low-abide leveis with a periodic&y ranging
from 16-32 h. Eleven of these cells had a circadian
period of 20-Z h5. There was no indication, however,
of a connection between high/low firing levels and the
time of day (Fig. 7).
In a recent microiontophoretic
SCN we were also able to characterize
SCN neurons: spontaneously
sive), irregular and regular. We could not find any
specific distribution pattern of these different types6.
Glutamate excited the majority of the regular and 60%
of the irregular cells. GA3A inhibited both the sponta-
neous and the glutamate-evoked
90% of all three types of SCN neurons. MK-801 par-
tiaily blocked glutamate responses. Of the nine SCN
neurons tested for the effects of MK-801 on sponta-
neous activity only one showed a reduction in firing
rate, This suggests that even if the neuronal discharges
within the SCN are synaptically mediated, their sponta-
neous activity does not depend on activation of NMDA
receptors. Another interesting finding of this microion-
tophoreti~ study was that the magnitude of glut~ate
response varies inversely as a function of the sponta-
neous firing frequency of regular SCN neurons. It was
also found that N-acetylaspartylglutamate
rectly increases firing rate or potentiates
responses in several of the SCN neurons studied (Figs.
8 and ref. 6).
Assuming that the SCN is capable of generating
circadian rhythms independently
brain and/or body, it is of interest to know how this is
done. There are several hypotheses: (1) the ~~ce~ker
~~thesis: ~dependen~y and/or
of pacemaker cells generate the clock output rhythm;
and (2) the network hypothesis: the interaction
fibers and the SCN
cells were recorded
study of our cultured
three types of
dent (glutamate respon-
activity of more than
of the rest of the
in concert a group
factor in the generation of a precise cicadian rhea
within the SCN. It is important to keep the contribu-
of SCN cells are pacemakers)
cells (a small
is the key
CLOCK TIME (HOURS)
discharges (HZ) of cultured neurons continu-
au& recorded as a function of time of day are shown (A-H). Each
record shows a single SCN neuron continuously recorded from a
different explant, except for B, which is the multiple unit recording
of the same explant as in C. The recording shown in H is also a
multiple recording of another SCN explant. Note that apart from the
main circadian rh~hrnic~~ also activity peaks with a periodic&y
lower or higher than the mean are found’. This is very pronounced
when the multiple neuronal recording in E is compared with the
single neuronal data discriminated from the same record in C. These
results are in accordance with the multipk sub-cwcillators model of
the SCN in which due to a lack of synchronization (by light or other
internal Zeitgebers) the pacemaker and non-pacemaker cells gradu-
ally drift apart as a function of in vitro culturing. This hypothesis is
supported by the fact that 8-h samplings on several successive days
(using 5-min ~egistrat~o~s~ gave no indication of high- or low-activity
Ievels as a function of the time of day (I). fn I, individual regular
SCN neuronal firing rates are indicated as dots, while irregular cells
are shown as open squares.
Fig. 7. S~ntaneous
400 600 600 700
Fig. 8. Inhibition of a spontaneously active SCN neuron by GABA
and excitation of another single (spontaneously silent) SCN cell upon
microiontophoretically applied glutamate and/or
tion of the glia cells in mind when determining whether
(1) and/or (2) is/are correct.
4.2. Experimental evidence in favor of the pacemaker
1. In vivo infusion of ‘ITX into the SCN disrupts
circadian rhythm expression. However, upon drug with-
drawal the rhythm phase corresponds to the original
phase before drug delivery as if the information about
circadian rhythms remains intact in the pacemaker(s)
although it is not expressed”‘.
2. The presence of circadian rhythms in neuronal
discharges of the SCN taken from fetal rat on the last
day of gestation despite a very low synapse leve16’.
3. In vitro the circadian rhythm of intracellular pro-
tein synthesis within the SCN remains intact in the
presence of TTX68.
4.3. Experimental evidence in favor of the network hy-
1. Lack of circadian rhythms in 2-DG uptake in the
SCN in high Mg2+ and low Ca2+ in vitro69.
2. Dissociation of SCN neurons (50%) from two
different hamster strains with two different genetically
determined circadian rhythms (20 and 24 h) and trans-
plantation to an arhythmic SCN-lesioned hamster re-
sult in recovery of the rhythm (around 22 h) in the host
which is the mean of the two (and not independent
expression of two different rhythm@‘.
3. No circadian rhythm is found in rat 2-DG until
2-3 weeks after birth (when the synaptic density of the
SCN has matured)67,69.
With regard to the pacemaker
potheses, it is important to record SCN neuronal dis-
charges in the absence of synaptic transmission and
also to use patch clamp techniques. Furthermore,
only dissociated SCN neurons or glia makes it easier to
determine the rhythm generator. As far as the manner
in which circadian rhythms are generated within the
SCN is concerned, it is also important to know what
endogenous SCN neurotransmitters
the role of, e.g., GABA, which is found abundantly in
the internal terminals of the SCN and is co-localized in
peptidergic SCN neurons? Our organotypic
ture would be a good model for studies aimed at
answering these questions.
and network hy-
really do. What is
5. What do suprachiasmatic nucleus transplants do?
Rae Silver and Joseph IkSauter
It is well established that the mammalian suprachi-
asmatic nucleus (SCN) is a biological pacemaker that
provides period and phase information modulating be-
havioral and physiological responses. The precise loca-
tion of pacemaker cells and whether or not they are
restricted to a locus (loci) within the cytoarchitectoni-
tally and peptidergically defined SCN is not known.
Evidence of extra-SCN oscillators derives from several
lines of research: (1) ultradian rhythms and spectral
energy in the circadian range survive complete lesions
of the SCN (SCN-X); (2) extra-SCN hypothalamic re-
gions receive direct retinal input; (31 some circadian
rhythms, such as anticipatory food responses, survive
total SCN-X; and (4) treatment of SCN-X rats with
rhythms. We review our attempts to understand:
which circadian rhythms’ are sustained by pacemaker
cells of the SCN itself and which are attributable
extra-SCN oscillators; (b) which functions can be re-
stored by SCN grafts; (c) responses that are similar/
different in grafted animals vs those bearing ‘SCN
islands’ vs intact animals; and (d) mechanisms whereby
SCN transplants might restore function.
restores circadian locomotor
5.2. U%ere are tlze ~cemakers?
It has been confirmed in many labs that fetal grafts
of the anterior hypothalamus which include the SCN
can restore free running locomotor rhythms following
transplantation into the third ventricle of arrhythmic
SCN-X host rats or hamsters, but effective transplants
always include extra-SCN tissue. To examine the effi-
cacy of the neural tissue grafts restricted to the SCN in
restoring circadian rhythms in SCN-X animals, we first
established the oldest effective donor tissue by using
SCN grafts taken from animals at postnatal day I, 3, 5,
7 and 106’. Restoration of locomotor rh~hmici~
lowing transplantation of postnatal day 1 grafts was as
high as that of embryonic grafts and declined there-
after. Using postnatal day 1 donor tissue, we took small
‘punches’ of the SCN from hypothalamic sections (500
km> of the anterior hypothalamus, thereby limiting the
amount of extra-SCN tissue in the graft. The results to
date indicate that such punches are sufficient to sus-
tain circadian rh~hmici~. Though a substantial effort
remains before we can determine whether any region
outside the SCN or whether pacemaker cells restricted
to any subregion within the SCN, are effective, this
work should define the locus (loci) necessary and suffi-
cient to sustain circadian rhythmicity.
Not all responses thought to be dependent
SCN are restored by neural tissue grafts. Rhythms of
locomotor activity, drinking and gnawing39 are re-
stored, but gonadal regression in constant darkness is
not. Measures of both gnawing and wheel-runn~g
tivity in SCN-X hamsters following transplantation
dicate that these behaviors reemerge simultaneously,
suggesting control by a common pacemaker.
resgonses are restored by suprachiasmatic nu-
5.4. Responses of grafted animals to pharmacological
Melatonin can set the phase of the free-running
rhythm in pups of a litter born to SCN-X mothers”,
but has little effect on adult hamsters. We used this
age difference in res~nsiveness to assess the ability of
melatonin to set the phase of locomotor
SCN-X adult hamsters
grafts containing the SCN .
lamic tissue was exposed to melatonin or vehicle at one
of two circadian phases, starting on embryonic day 8
(E8) during pregnancy and continuing until postnatal
day 7 following grafting to an adult SCN-X host. This
treatment was effective in setting the phase of the
recovered locomotor response
Une~ectedly, it also shortened the latency to recover
locomotor rh~hmici~. There were no effects of mela-
tonin administration in oil injected SCN-X grafted
animals, in intact controls, in SCN-X non-grafted ani-
mals or in SCN-X animals bearing
grafts. Maternal melatonin may normally synchronize
the circadian rhythms of the pups within a litter in a
Lithium and heavy water (*H,O) lengthen the free-
running period of circadian rhythms in a variety of
mammali~ and non-m~alian
hamsters which had recovered
following implantation of SCN grafts and in intact
controls, both of these agents lengthened the period of
the free-run without affecting amount of activity39.
Both triazolam (Tz) and exercise can phase advance
free-running locomotor activity rhythms in intact ham-
sters. Furthermore, Tz increases activity at the time of
injection and this appears to be the mechanism whereby
Tz produces phase shifts. We compared
shifting effect of Tz in grafted and intact animals8. In
both groups, increases in locomotor activity occurred at
the time of Tz injection and most intact, but no grafted
animals show a phase advance in response to Tz ad-
ministration. Given that hamsters bearing SCN grafts
have limited neural connections between the host brain
and transplanted SCN tissue, the results suggest that a
site outside the SCN, with afferents to these nuclei,
mediates the phase-shifting effect of Tz and of exer-
cise. In contrast, lithium and *H,O
directly on the pacemaker cells.
‘l Donor anterior hypotha-
in the host animals.
systems. In SCN-X
appear to act
SS. Tra~planted su~rach~smatic nuclei us. those in situ
While SCN grafts restore free-running
rhythms, there are differences
grafted animals compared to intact controls. Grafted
animals have a less precise onset of locomotor activity
and their daily amount of activity is reduced. In intact
animals the daily onset is generally more precise than
the offset, and the associated activity duration is longer
and more intense than the one at the end of the day.
In grafted animals the onset and offset of daily activity
are often difficuft to distinguish. In animals bearing a
h~thalamic island created by a knife cut around the
in the actograms of
SCN, the precision of the onset of locomotor activity is
reduced, as in lesioned animals bearing SCN grafts, but
the period of the free-run is not generally altered by
the isolation of the SCN from the rest of the hypotha-
lamus and the amount of activity is not reduced*‘. The
reduction in amount of activity in lesioned-grafted
mals is likely a function of the extent of damage to the
hypothalamus. Other differences
sioned-grafted and ‘SCN island’ animals suggest that
the SCN may regulate circadian rhythms by multiple
mechanisms and SCN grafts are sufficient to restore
some, but not all functions.
Features of the restored response can be influenced
by the condition of the graft at the time of transplanta-
tion. While animals bearing whole tissue grafts3’ usu-
ally express a free-running period that is longer than 24
h (typical of hamsters), animals bearing dissociated,
dispersed cell suspension grafts have a period shorter
than 24 h”. Possibly, coupling of pacemakers within
the graft, location of the grafted tissue or number of
transplanted pacemaker cells influences the period ex-
between intact, le-
5.6. Output signals of the suprachiasmatic nucleus
To explore the impart of extra oscillators on the
expression of circadian rhythms, we implanted SCN’s
from two fetal donor animals into intact animalss4. The
most impressive aspect of the results was the absence
of any measurable effect on the period or phase of the
free-running circadian Iocomotor rhythm or on amount
of activity, associated with the presence of 3-fold the
usual complement of SCN oscillators. Furthermore,
lesions of the host SCN subsequent to implantation of
grafts had no detectable effect on the free-running
locomotor behavior of the hamster. It is noteworthy
that less than usual complement of SCN tissue is also
compatible with free-running rhythmicity in that abla-
tion of less than 75% of both SCN does not alter
To examine the impact (if any) of SCN grafts in
intact animals, on the metabolic activity of the host and
the grafted SCN, we used radiolabelled 2-deoxyglucose
uptake to index phase 66 First, we established that we
could measure a circadian rhythm in 2-deoxyglucose
uptake in postnatal day 1 pups. Next, we housed host
and donor animals in opposite LD cycles. On the first
day after grafting, the donor clock retained its phase
indicating that surgical isolation of the SCN from the
rest of the fetal brain and implantation into the adult
host animal, did not disrupt circadian rh~hrni~i~
the donor clock. On the 14th day after grafting and
thereafter, host and donor SCN were in synchrony,
invariably with the phase of the host animal. Thus, the
host SCN sends a signal which resets the phase of the
grafted SCN and not vice versa.
5.7. What do tra~plant~ really do?
The broader question of what transplants do has
been amply addressed2*ig and the following possibilities
are offered: (a) trophic effects of the graft; (b> grafted
cells reinnervate host; (c) graft promotes regrowth in
host; (d) paracrine signal reaches host by diffusion; (e)
endocrine signal reaches host via circulatory
(Fig. 9). Each of these mechanisms have been impli-
cated in the restoration of function by either neural
and/or adrenal grafts and could mediate some of the
effects of SCN grafts. The present results, using SCN
grafts in lesioned and intact hosts suggest novel possi-
bilities: (f) axonal growth from host brain to graft; (g)
dendritic growth from graft to host; or (h) a diffusible
signal from the host brain reaches the graft.
A) Trophk effect
B) Axon sprouting from graft
C) Regrowth in hosi
E) Endocrme release F) Axonai
host to graft
graft to host
Fig. 9. Possible mechanisms of action of ventricular grafts. The dotted line demarcating the location of the SCN designates an SCN-lesioned host
animal. A solid line indicates that the host SCN is intact.
The present results indicate that the status of the
host animal and of cells within the graft influence the
recovered response. The period of the free-run differs
in animals bearing dissociated dispersed cell suspen-
sion grafts from those bearing whole tissue grafts. In
host animals lacking an SCN, grafts restore free-run-
ning activity rhythms and drugs that act directly on the
SCN influence the expression of restored rhythms. In
intact host animals bearing SCN grafts, responses at-
tributable to the transplanted
hard to detect and the phase of metabolic activity of
grafted SCN is set by the host SCN. If the condition of
the graft and of the host brain influence responses of
neural tissue grafts in other systems, as occurs with
SCN grafts, this may account for variability of out-
comes following grafting and has important
SCN tissue have been
6. Cellular studies of a retinal biological clock
Gene D. Block, Michael E. Geusz, Sat Bir S. Khalsa and Stephan H. Michel
The eyes of several mollusks contain circadian pace-
makers. Eyes in isolation express circadian rhythms in
the frequency of spontaneously
impulses. In Bulla gouldiana, the cloudy bubble snail,
the pacemaker is located among a group of approxi-
mately 100 electrically coupled neurons (basal retinal
neurons, BRNs). The hypothesis that each BRN is a
competent circadian pacemaker was based, until re-
cently, on evidence that surgically reduced retinas con-
taining only a few BRN somata continued to generate
circadian periodicities3. The single cell-pacemaker
pothesis has now received e~erimental
BRNs dispersed in cell culture reveal circadian changes
in membrane conductance that persist for at least two
cycles in culture .
47 These conductance changes are not
due to any form of communication
cells as ~divid~ cells isolated in microwell dishes
continue to exhibit rhythmic changes in conductance48.
These data provide the first direct evidence that indi-
vidual neurons are capable of generating and express-
ing a circadian rhythm.
Our Iaboratory employs three strategies to study
circadian pacemaker mechanisms: (1) we identify pro-
cesses involved in pacemaker entrainment,
trainment pathway must ultimately
pacemaker element; (2) we analyze the mechanisms
underlying pacemaker expression, as the origin of the
expression pathway is a pacemaker component; and (3)
we attempt to directly identify processes involved in
occurring optic nerve
as the en-
on a terminate
We find that light leads to membrane depolar~ation
that results in an influx of Ca2+ through voltage-de-
pendent Ca*+ channels43~26. Hyperpolarization
reduction in Ca2+ flux also generates phase shifts but
the PRC is shifted 180” on the time axis with respect to
27 Using the fluorescent calcium indica-
tor dye fura-2, we have recently confirmed that depo-
larization leads to a sustained increase in intracellular
Ca*+ concentration (Geusz et al., unpublished results).
The circadian rhythm in optic nerve impulse fre-
quency is driven by a circadian rhythm in membrane
potential@. Membrane potential is relatively hyperpo-
larized during the subjective night and depolarizes by
approximately 13 mV during the subjective day. This
rhythm is due to the m~ulation
tance58. Membrane conductance
tive dawn and increases again at subjective dusk. These
changes appear to be due to modulation of a TEA-sen-
sitive K+ channe146,48.
of membrane conduc-
decreases at subjec-
6.4. Rhythm generation
While membrane conductances are involved in both
pacemaker entrainment and expression, they do not
appear to play a critical role in rhythm generation.
Removal of Ca 2+ from the bathing medium does not
prevent the pacemaker from completing its cycle= nor
does the removal of extracellular
preparation). Removal of extracellular
the free-running period by up to 2.5 h but does not
transcription and translation appear to be critical for
rhythm generation. Inhibiting protein synthesis or tran-
scription leads to profound period lengthening and at
higher concentrations the pacemaker is held motion-
less near circadian time 0 (ref. 29).
Na+ (Khalsa, in
Cl - shortens
6.5. Pacemaker model
Our best guess for circadian pacemaker organiza-
tion is depicted in Fig. 10. Briefly, the circadian pace-
Fig. 10. Model for circadian pacemaker system within Bullu retinal
neurons. D and E represent the state variables of an intracellular
feedback loop generating the 24-h periodicity. F depicts the initial
steps of the output pathway that are as yet unidentified. C represents
the final steps of the entrainment pathway. See text for additional
maker within the basal retinal neuron consists of at
least two feedback loops. An intracellular
likely involves transcription
determining steps and is responsible for generating the
long circadian time constants. In addition to the intra-
cellular feedback loop, a transmembrane loop is formed
as rate and translation
by virtue of the fact that membrane potential is both
an input and output to the neuronal pacemaker. Envi-
ronmental synchronizing sign& act through membrane
depolarization, voltage gated Ca’+ channels, a trans-
membrane CaZf flux and a sustained increase in intra-
cellular Ca’+. On the output side, rhythm expression is
mediated by pacemaker modulation of a TEA-sensitive
K’ channel, driving a rhythm in membrane potential
that results in a circadian rhythm in impulse frequency.
We propose that depolarization-induced
during the subjective night are caused by an increase in
the level of intracellular Ca2+ and hyperpolarization-
induced phase shifts during the subjective day are due
to a reduction in a persistent diurnal Ca2+ flux2’. This
diurnal flux is caused by voltage-gated Ca”
opening during the depolarized phase of the circadian
membrane potential cycle, Some support for this pro-
posal is provided by experiments
lowered bath Ca 2+ levels lead to phase shifts similar to
those generated by membrane hyperpolarization27. Fu-
ture experiments will be directed towards further char-
acterizing the role of intracellular
entrainment and generation.
in which pulses of
Ca2+ in rhythm
7. Convergence and divergence in chick pineal regulation
The circadian rhythm of melatonin production
the pineal gland is the most robust and reliable overt
hormonal rhythm in vertebrates.
sis (and release) of melatonin (derived from tryptophan
via serotonin) is low during the day and goes up
many-fold during the night. In mammals, the rhythm of
melatonin production is unique to the pineal gland and
entirely dependent on the sympathetic
which conveys photic and circadian regulation to the
gland. The rat pineal is itself neither rhythmic nor
photosensitive, but responds to its neurotransmitter
norepinephrine (NE) by increasing the synthesis of
melatonin from serotonin3’.
pineal is itself both rhythmic and photosensitive, even
in dispersed cell culture”
transmitter NE by decreasing the synthesis of mela-
We have been using chick pineal cells to identify
and interrelate perturbations
tonin-synthesizing apparatus and to distinguish them
from those acting on the pacemaker underlying rhythm
86 Generally, agents that can acutely raise
In all classes, synthe-
In contrast, the chick
and responds to its neuro-
that act on the mela-
or lower melatonin output without ever inducing phase
shifts in the free-running rhythm are considered to act
solely on the melatonin-synthesizing
agents that can induce phase shifts are considered to
act (however indirectly) on the pacemaker mechanism.
Further experiments are then aimed at dissecting the
reiationships between the pathways converging
melatonin regulation. Results and interpretations
cerning some of these effects and relationships
summarized in Fig. 11.
Cyclic AMP is a key regulator of melatonin produc-
tion in the chick pineal, as it is in the rat pineal. Agents
that raise cyclic AMP levels, such as forskolin (FSK) or
vasoactive intestinal peptide (VIP) and cyclic AMP
analogs, such as 8-bromocyclic
acutely increase melatonin output. Agents that lower
cyclic AMP levels, such as NE or light CL), acutely
decrease melatonin output, In contrast to their effects
in certain neuronal systems, none of these agents (ex-
cept L) demonstrably affected the temporal pattern in
subsequent cycles of the melatonin rhythm; they do not
perturb the underlying circadian
SNAT -w Mel
Fig. 11. A scheme summarizing the effects of agents on melatonin
(Mel) production and their interactions. Abbreviations are explained
in the text.
guished the pathways by which L exerts its acute and
entraining effects 85 PT could block the acute in-
hibitory effect of L (or NE), but PT did not prevent the
phase shifts induced by light pulses. Other experiments
indicated that daily release of NE, which does not
entrain the melatonin rhythm, does contribute
daily L) to rhythm regulation by helping to prevent
damping and sustain a robust rhythm.
Perturbations of calcium flux through the plasma
membrane have also been shown to affect melatonin
production. Agents that increase calcium influx through
voltage sensitive calcium channels (VSCC>, such as Bay
K 8644 (Bay K) or depolarizing concentrations
cellular potassium ions (K+), increase melatonin pro-
duction, whereas agents that decrease calcium influx,
such as nitrendipine (NTR), cobalt ions or low extra-
cellular calcium concentrations
crease melatonin outputg3. Competition experimentsg8
and direct measurementsg2 indicated that these agents
act through cyclic AMP. Again, in contrast to results in
neuronal systems, none of these agents demonstrably
affected the underlying circadian pacemaker. The cir-
cadian system in chick pineal cells further differs from
neuronal systems in that perturbations
potential and cyclic GMP analogs have also failed to
induce phase shifts in the melatonin rhythm. The circa-
dian pacemaker in chick pineal cells is not, however,
impervious to perturbation.
with pertussis toxin (PT) distin-
(-Ca2’) acutely de-
In addition to pulses of
8. Putative oscillator proteins in the Aplysia eye circadian system
Arnold Eskin and Costas Koumenis
come is: “What is the molecular mechanism of circa-
in circadian biology whose time has
light and darkness, pulses of ouabain, hypertonic or
hypotonic media, protein synthesis inhibitors or tem-
perature change have been shown to induce phase
shifts in the melatonin rhythm. The mechanistic rela-
tionships among these perturbations
The importance of cyclic AMP regulation
pineal encouraged the presumption
mediates the actions of the pacemaker on melatonin
production. Recent experiments, however, strongly in-
dicate that the pacemaker
through, cyclic AMP to regulate melatonin production
in chick pineal cells 87 The key result was the demon-
stration of a persistent rhythm of melatonin output in
the constant presence of maximally effective concen-
trations of FSK (and cyclic AMP) or 8BrcAMP. The
synergy and convergence between the actions of cyclic
AMP and the circadian pacemaker are implicit in the
scheme shown in Fig. 11. At a given level of cyclic
AMP, the different states (phases) of the pacemaker
can determine different rates of melatonin synthesis
and, at a given state (phase) of the pacemaker, differ-
ent levels of cyclic AMP can determine different rates
of melatonin synthesis.
The scheme shown in Fig. 11 completely separates
and then converges the pathways for light’s acute
(‘masking’) and entraining (phase-shifting) effects. The
nature and site of convergence between these pathways
is unknown, but it is shown as occurring at the regula-
tion of serotonin ZV-acetyltransferase @NAT), the piv-
otal enzyme in melatonin
induced at night. There is precedent
and fungi l2 for dual pathways, ‘direct’ and ‘indirect’
(clock-mediated), for the regulation of specific gene
expression by light. The nature and site of divergence
between light’s pathways is also unknown; it remains to
be determined, for example, whether the acute and
phase-shifting effects of L are mediated by the same
photopigment. The acute effect of L can be partially
and plausibly explained in terms of known mechanisms
of cyclic AMP regulation and enzyme induction. The
substances and processes mediating the entraining ef-
fects of light, however, as well as those mediating
pacemaker functions, remain entirely mysterious.
that cyclic AMP
acts with, rather than
synthesis, which is itself
in both plants49
require the identification of the molecular components
Elucidation of this mechanism will
‘4 _______ _ 1
Fig. 12. Critical role of transcription and translation in the feedback
loop of the oscillator (model).
of the circadian oscillator and the dete~ination
how these molecules interact with one another to gen-
erate a circadian oscillation. Three lines of evidence
suggest that macromolecular
portant process in the circadian oscillator34~73. First,
continuous alteration of the level of translation
transcription leads to changes in the free-running
riod of circadian rhythms and brief perturbations
translation or transcription
rhythms’7*80*55. Second, inhibitors of translation block
phase-shifting effects of entraining
systemss7TD” and gene expression appears to be in-
volved in entrainment pathways in still other systems33.
Third single-gene mutations have been shown to affect
the period of circadian rhythms in several organisms.
Furthermore, a detailed study of the per mutation in
~~~0~~~~~ shows that per mRNA and protein Ievels
oscillate and that expression of the protein appears to
be essential for the oscillation of its mRNA21. These
studies on the role of macromolecular
circadian oscillation have led to a model of the oscilla-
tor (shown in Fig, 12) in which transcription and trans-
lation are critical processes in the feedback loop of the
oscillator is proposed to occur by environmental cycles
regulating the synthesis of specific mRNA or protein
com~nents of the oscillator. Critical tests of this model
require the identification of candidates for molecular
components of the pacemaker, i.e., mRNAs and pro-
synthesis may be an im-
lead to phase shifts in
agents in several
synthesis in the
entrainment of this model
teins whose synthesis is important for the normal func-
tion of the circadian oscillator.
We have used a biochemicai approach to screen for
proteins that may serve as components of the circadian
system in the eye of Aplysia. Our screening strategy is
based upon the model above, together with informa-
tion we have previously obtained concerning the en-
trainment of the circadian oscillator. The rhythm of the
eye of Aplysia is entrained over at least two pathways34.
One rather direct pathway mediates the effect of light
upon the eye while the other pathway mediates effects
of efferent nerve activity on the eye. Effects of the
efferent pathway on the rh~hm appear to be mediated
by release of the neurotransmitter,
the eye. Because light and 5-HT appear to act through
opposite actions on some element of the oscillator, the
model predicts that Iight and S-HT will have opposite
actions on the synthesis of some protein components of
the oscillator. We have searched for such proteins by
exposing eyes to radioactive amino acids in the pres-
ence of light or 5-HT and then studying individual
proteins using two-dimensional
eIectrophoresis 57,79. Also, because transcription
translation appear closely coupled in the eye circadian
system, we searched for proteins whose synthesis was
rapidly affected by a transcription inhibitor3”.
Using such criteria we have found over ten proteins
that we consider putative oscillator proteins (POPS).
The next stage in this research requires the identifica-
tion of the function of these proteins. To do this, we
have been obtaining partial amino acid sequences of
POPS. Thus far, we have been able to identify four
POPS and sequence information
other one. POP-1 belongs to the lip~ortin/annexin
family of proteinss6, POP-2 is PORIN,
POP-5 appear to be related to the heat shock family of
proteins (GRP78 and HSP70) and POP-4 may also be a
stress-related protein, for it is similar to the stringent
starvation protein of E~che~ich~ coli35. These identifi-
cations of POPS have provided reagents such as anti-
bodies and gene sequences that we are using to study
additional properties of the proteins. Also, we are
examining functions of these proteins in the circadian
system using pharmacological
functions and perturb the circadian rhythm. Finally, we
are developing techniques that will allow us to perturb
the synthesis of specific POPS or their mRNAs. A
critical future issue will be to determine whether any of
the POPS are functionally related to one another. It is
through functional reIat~onships between POPS that a
model of the molecular structure of the circadian pace-
maker will emerge.
serotonin (5HT) in
is available on an-
agents to aIter their
9. Genetic and molecular analysis of the ~~~~~~~~~~ circadian clock
Jay C. Dunlap, Jennifer J. Lores, Keith A. Johnson, Kristin M. Lindgren, Benjamin ID. Aronson,
Deborah Bell-Pedersen, Qiuyun Liu and Norman Y, Garceau
The biolagical clock is genera& believed to be corn-
prised of a feedback loop that operates, in all organ-
isms displaying a circadian rhythm, at the leve1 of the
single cell. Hence, microbial
amenable to genetic analysis, have long been appreci-
ated for the insights they can provide into the mecha-
nism of the clock and the means by which clocks act to
regulate the metabolism and behavior of ceils. The best
molecularly studied microbial clock system is the as-
comycete fungus Neurosp~ru crassa, in which a circa-
dian biological clock antrols
and development. In particular, the timing of the initi-
ation of conidiogenesis has been well studied and has
allowed genetic and molecular techniques to be used to
study the circadian clock itself from two converging
One approach has been to examine the pathways
whereby clocks act to control cellular metabolism and
behavior. Initial efforts targeted the isolation of genes
whose transcript levels are controlled by the clock. Two
such genes, designated clock controlled gene-l @g-l)
and clock-controlled gene-2 (ccg-2), were identified by
a subtractive hybridization procedure@; both are morn-
ing specific genes. Nuclear run-on assays4’ have pin-
pointed transcription as the primary point of clock
regulation. DNA sequence and gel mobility shift analy-
sis of these two clock regulated genes and their regula-
tory regions have suggested possible targets for clock
mediated regulation and preliminary experimental data
has identified a trans-acting protein factor(s), a puta-
tive clock-regulatory factors, that binds to the promoter
regiorr of ccg-2. The factor(s) appears to be present in
moving protein extracts but not in evening extracts, its
presence EoincidentaI with the time of expression of
ccg-2. Ccg-2 encodes one of the major fungal hy-
drophobin proteins required for the formation of the
hydrophobic uuter rodlet layer of conidia’. As spore
surface hydrophobici~ is a dominant determinant
pathogenicity in many fungal pathogens, a role for the
clock in determining the immediate pathogenic poten-
tial of some fungal spores is suggested. Finally, as both
ccg-1 and ccg-2 are known to be regulated by light as
well as the clock, these genes pravide excellent molecu-
lar correlates for study~g the complex interface be-
tween genes, environmental facxors and an organism’s
systems, and systems
several aspects of growth
Fig, 13. Role of frg gene in the feedback loop of the osCi&Wx.
A second approach has been to identify and charac-
terize putative clock components through the identifi-
cation, cloning and characterization
mutated, alter the expression of wild-type rh~hmiei~.
Towards this end, the period-4 and frt?qzzncy loci have
been cloned and the fatter characterized
tai142. The fieQuency locus encodes two non-overlap-
ping transcripts; the smaller transcript has little capac-
ity to code far a protein while the larger transcript has
the potential to code for a 989 amino acid protein and
appears to have an unusually long 5’ untranslated
region. Sequence analysis of the previously isolated
eight fig alleles has allowed tentative assignment of
the single amino acid substitutions that can lenghten or
shorten the period length and disrupt temperature
compensation. In general, conservative
have led to short period changes and more potentially
disruptive amino acid substitutions
lung-period mutants. Disruption of the gene has estab-
lished that it is not essential. Strong preliminary data
speak to the im~o~an~e of frq in the actual assembly
and operation of the clock. First of all, the amount of
fiq transcript is rhythmic, with a peak in the early
muming. Thus the frs gene both regulates the clock
(as exemplified by the phenotypes of the various frg
alleles) and is regulated by the clock (as exemplified by
the rhythm in frq transcript) and it appears that fq
must be placed within rather than just affecting the
feedback loop of the oscillator (Fig. 13).
of series, that when
in some de-
have resulted in
Ackno~&&~Ms. The research by I. Zwker et al. was supported
by Grants WI3 44595 and HD 02982 frc& the NIH. The research by
G-D. Block et al. w as supported by NER I’4515264 and the NSF
Center for Biological Timing. The research by A, E&in md CL
~~m&~ was supported by Grants from the N’SMH and the Ait
E&w. office of Scientific Research. The research by R. Silver and J.
LeSauter was supported by Grants NS 24292 and NS 08783 from
NIH and from the AFOSR. The research of J. Dunlap was sup-
ported by Cm 34985 and mH 44651, and of J. Loros by NSF mcB
List of participants
National Institute of Mental Health
Laboratory of Cell Biology
Building 36, Room 3A-15
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Tel.: (1) (301) 4962639
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Block, Gene D.
Center for Biological Timing, Gilmer Hall
University of Virginia
Charlottesville, VA 22901
Tel.: (1) (804) 982-5225
Fax: (1) (804) 982-5626/5221
Bloom, Floyd E.
The Scripps Research Institute
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Bolis, C. Liana
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Dunlap, Jay C.
Dartmouth Medical School
7200 Vail Building, Room 413
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Tel.: (1) (603) 650-1108
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University of Houston
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of Biochemicat and Biophysical Sciences
Montreal Neurological Institute
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Tel.: (1) (514) 398-1938
Fax: (1) (5 14) 3988540
Gadusek, Carleton D.
National Institutes of Health
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Institut de Physiologie
FacultC de Medecine
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Netherlands Institute for Brain Research
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Tel,: (31) (20) 5665500
Fax: (31) (20) 6918466
Moore, Robert Y.
Center For Neuroscience
University of Pittsburgh
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Fax: (1) (412) 648-8376
New York, NY 10027-6598
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Tosteson, Daniel C.
Harvard Medical School
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Turek, Fred W.
2153 N. Campus Drive
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of Neurobiology and Physiology
Wiesel, Torsten N.
Laboratory of Neurobiology
The Rockefeller University
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Section on Biochemical Pharmacology
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