Period determination in the food-entrainable and
methamphetamine-sensitive circadian oscillator(s)
Julie S. Pendergasta, Gisele A. Odab, Kevin D. Niswenderc,d, and Shin Yamazakia,1
aDepartment of Biological Sciences, Vanderbilt University, Nashville, TN 37235-1634;bInstituto de Biociências, Departamento de Fisiologia, Universidade de
São Paulo, São Paulo, Brazil;cMedical Service, Tennessee Valley Healthcare System, Nashville, TN 37212-2637; anddDivision of Diabetes, Endocrinology and
Metabolism, Department of Medicine, Vanderbilt University School of Medicine, Nashville, TN 37232-2358
Edited by Joseph S. Takahashi, Howard Hughes Medical Institute, University of Texas Southwestern Medical Center, Dallas, TX, and approved July 20,
2012 (received for review April 13, 2012)
Daily rhythmic processes are coordinated by circadian clocks, which
are present in numerous central and peripheral tissues. In mam-
mals, two circadian clocks, the food-entrainable oscillator (FEO)
and methamphetamine-sensitive circadian oscillator (MASCO), are
“black box” mysteries because their anatomical loci are unknown
and their outputs are not expressed under normal physiological
conditions. In the current study, the investigation of the timekeep-
three paralogs of the canonical clock gene, Period, revealed unique
and convergent findings. We found that both the MASCO and FEO
in Per1−/−/Per2−/−/Per3−/−mice are circadian oscillators with unusu-
ally short (∼21 h) periods. These data demonstrate that the canon-
ical Period genes are involved in period determination in the FEO
and MASCO, and computational modeling supports the hypothesis
that the FEO and MASCO use the same timekeeping mechanism or
are the same circadian oscillator. Finally, these studies identify
Per1−/−/Per2−/−/Per3−/−mice as a unique tool critical to the search
for the elusive anatomical location(s) of the FEO and MASCO.
food anticipatory activity|restricted feeding|suprachiasmatic nuclei|
with endogenous periods of ∼24 h that can be synchronized to
environmental cues such as the light/dark cycle and food avail-
ability. Circadian studies have traditionally focused on the mas-
ter clock in the suprachiasmatic nuclei (SCN), and more recently
on peripheral oscillators such as those in liver and muscle. Two
extra-SCN oscillators, the food-entrainable oscillator (FEO) and
methamphetamine-sensitive circadian oscillator (MASCO), have
been defined on the basis of circadian behavioral rhythms in-
duced by food and methamphetamine, respectively, but these
remain “black box” mysteries because their anatomical loci are
unknown and their outputs are not expressed under normal
physiological conditions (1–4).
Recent studies of the FEO and MASCO have suggested that
they use a molecular timekeeping mechanism that is distinct
from other circadian oscillators because they function when the
canonical circadian genes are disrupted (5–11; see also refs. 12,
13). Because only two paralogs of the Period gene, Per1 and Per2,
are necessary to generate circadian rhythms in the SCN, previous
studies concluded that the FEO and MASCO are “non-
canonical” clocks in part because they are functional in Per1−/−/
Per2−/−mice (5, 6). Although the third Period paralog, Per3, is
not usually considered a functional component of circadian
clocks, we recently demonstrated that Per3 participates in period
determination in certain peripheral circadian oscillators (14, 15).
We hypothesized that Per3 may also be a constituent of the FEO
and MASCO and/or could be compensating for the loss of
functional PER1 and PER2, therefore necessitating analyses of
the FEO and MASCO in Per1−/−/Per2−/−/Per3−/−mice.
The current study of the FEO and MASCO in Per1−/−/Per2−/−/
Per3−/−mice revealed unique and convergent findings. Though we
confirmed the previous finding that the MASCO is rhythmic when
emporal processes are controlled by circadian clocks, which
produce self-sustained oscillations in physiology and behavior
the canonical clock genes are disrupted, we found that the Period
genes are, in fact, involved in determining the periods of the FEO
and MASCO. Furthermore, both the FEO and MASCO had
same oscillator or that they use the same timekeeping mechanism.
Per1−/−/Per2−/−/Per3−/−MASCO Is a Circadian Oscillator with a Short
Period. We first generated Per1−/−/Per2−/−/Per3−/−mice congenic
with the C57BL/6J strain and found that their wheel-running
activity appeared rhythmic in the light/dark cycle, with activity
beginning ∼2 h before lights off in 12 h light/12 h dark (12L:12D;
Fig. 1 and Fig. S1) and ∼5 h before lights off in 18L:6D (Fig. 2
and Fig. S4), but their daily rhythms of activity were abolished in
constant darkness [similar to other circadian mutant mice (Fig.
1B and Fig. S1); the wild-type wheel-running activity rhythm is
shown in Fig. 1A for reference]. These data suggest that the light/
dark cycle can drive rhythmicity in the SCN (alternatively, the
light/dark input could drive rhythmicity in another brain region
associated with masking, but we will refer to it as the SCN
rhythm herein for ease of discussion) in Per1−/−/Per2−/−/Per3−/−
mice, but the SCN rhythm is abrogated in constant darkness.
To determine if the MASCO is functional in the absence of all
functional PERIOD, we administered methamphetamine (0.005%
in drinking water) to Per1−/−/Per2−/−/Per3−/−mice in constant
darkness (Fig. 1D; the SCN rhythm is dampened in constant
darkness as shown in Fig. 1B and Fig. S1). After several days of
methamphetamine treatment (1–12 d), all Per1−/−/Per2−/−/Per3−/−
mice examined exhibited short (∼21.5 h) methamphetamine-in-
duced wheel-running activity rhythms (Fig. 1D and Fig. S2B). The
Per1−/−/Per2−/−/Per3−/−MASCO rhythm (n = 6) was significantly
shorter than the >24-h wheel-running activity rhythms observed in
wild-type mice (n = 3) administered methamphetamine (t = 5.9,
P < 0.001; Fig. 1C and Fig. S2 A and C). These data demonstrate
that although the MASCO isrhythmicwhen all Period paralogs are
disrupted, functional PERIOD nonetheless participates in the pe-
riod determination of MASCO.
The locomotor activity rhythm in wild-type rodents during
short-term treatment with methamphetamine represents the in-
tegrated outputs of the SCN and MASCO, suggesting that the
two oscillators are coupled (3, 16, 17) (Fig. 1C and Fig. S2A).
However, during long-term exposure to methamphetamine in
a 24-h light/dark cycle, the MASCO rhythm dissociates from the
SCN-controlled activity rhythm in wild-type mice (1, 17, 18) (Fig.
1E and Fig. S3A; the MASCO rhythm dissociates from the SCN-
Author contributions: J.S.P., G.A.O., K.D.N., and S.Y. designed research; J.S.P., G.A.O., and
S.Y. performed research; J.S.P. and G.A.O. analyzed data; and J.S.P. and S.Y. wrote
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
1To whom correspondence should be addressed. E-mail: email@example.com.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
| August 28, 2012
| vol. 109
| no. 35www.pnas.org/cgi/doi/10.1073/pnas.1206213109
controlled rhythm in three of four wild-type mice). To examine
coupling between the light-driven SCN and the MASCO in
Per1−/−/Per2−/−/Per3−/−mice, we administered methamphet-
amine (0.005%) to mice maintained in the light/dark cycle
(18L:6D; Fig. 1F and Fig. S3B). We found that the MASCO
rhythm did not dissociate from the light-driven SCN-controlled
nocturnal activity rhythm during >75 d of methamphetamine
treatment (compared with wild-type mice in the same condition
in Fig. 1E and Fig. S3A), but was relatively coordinated to the
light-driven SCN rhythm, as evidenced by the irregularity in the
onset of activity (Fig. 1F). The 21-h period of the Per1−/−/Per2−/−/
Per3−/−MASCO rhythm was observed upon release of the mice
into constant darkness (Fig. 1F; as in Fig. 1D and Fig. S2B). Fur-
thermore, the MASCO rhythm appeared to free-run from the
phase of activity onset in the light/dark cycle, suggesting that the
Per1−/−/Per2−/−/Per3−/−MASCO was entrained to the light/dark
Variable Patterns of Daily Food Anticipatory Activity in Per1−/−/Per2−/−/
Per3−/−Mice. Food anticipatory activity, which is an output of the
FEO, occurs before food availability only under conditions of
timed food restriction. Food anticipatory activity can be distin-
guished from the locomotor activity rhythm controlled by the SCN
(which occurs during the night in nocturnal mice) by restricting
food availability to the daytime. To investigate timekeeping by the
FEO in the absence of functional PERIOD, we first provided food
only during the day to Per1−/−/Per2−/−/Per3−/−mice in light/dark
conditions (18L:6D; n = 6; Fig. 2 and Fig. S4 D–F: 6 h/day re-
stricted feeding; Fig. S4 A–C: 4 h/d restricted feeding). Per1−/−/
Per2−/−/Per3−/−mice maintained in light/dark conditions (18L:6D)
exhibited daytime food anticipatory activity before food availabil-
conditions, we assessed food anticipatory activity during fasting
after restricted feeding. We found that anticipatory activity
Per2−/−/Per3−/−mice in the light/dark cycle. These data suggest that
in the light/dark cycle (in the presence of light-driven SCN rhyth-
micity), the FEO in Per1−/−/Per2−/−/Per3−/−mice entrains to the
timing of food availability.
mice. Representative double-plotted actograms (5-min bins) from wild-type
(A, C, and E) and Per1−/−/Per2−/−/Per3−/−(B, D, and F) mice. (A and B) Mice
(no methamphetamine treatment) were maintained in 12L:12D (LD; in-
dicated by white and black bars above actograms; the time of darkness is
outlined on the left halves of the actograms) for 6 d and then released into
constant darkness (DD) for 6 d. (C and D) Mice were maintained in constant
darkness (black bar above actograms) and administered 0.005% metham-
phetamine in their drinking water (days 1–33 of methamphetamine treat-
ment shown in the actograms). The days used for χ2periodogram are
indicated by solid vertical lines. (E and F) Mice were administered 0.005%
methamphetamine and maintained in 18L:6D for 78 d and then released
into DD (days 45–95 of methamphetamine treatment are shown here). The
untreated wild-type data (shown in A) was taken from our previously pub-
lished dataset (14). (B–F) Representative actograms from a total of n = 10, 3,
6, 4, and 3 observations, and all individual actograms are presented in Fig. S1
(B), Fig. S2 A and B (C and D), and Fig. S3 A and B (E and F), respectively.
The period of the MASCO rhythm is short in Per1−/−/Per2−/−/Per3−/−
Per1−/−/Per2−/−/Per3−/−mice in the light/dark cycle. (A) Representative dou-
ble-plotted actogram of wheel-running activity (5-min bins) of a Per1−/−/
Per2−/−/Per3−/−mouse maintained in the light/dark cycle (18L:6D, light/dark
conditions indicated by white and black bars, respectively, above the acto-
gram, and the dark phase is outlined with a black box on the left half of the
actogram). The time when food was available is shown by gray shading on
the left half of the actogram. Activity onset occurred ∼5 h before lights off
and ended at lights on during ad libitum feeding. The mouse was fed ad
libitum for 5 d, then fasted for 48 h to characterize activity during fasting
before restricted feeding. The mouse was returned to ad libitum feeding for
3 d, then fed 8 h/d for 2 d and then for 6 h/d for 9 d. On the 10th day of
restricted feeding, food was left in the cage, and the mouse ate ad libitum
for 3 d. The mouse was fasted for 48 h and then returned to ad libitum
feeding. (B) χ2periodogram analysis performed on days 1–9 of restricted
feeding (indicated on the y axis of the actogram in A). The dotted line in the
periodogram shows the significance level P = 0.001. A total of six individual
actograms and periodograms are shown in Fig. S4.
Food anticipatory activity during daily (24-h) restricted feeding of
Pendergast et al. PNAS
| August 28, 2012
| vol. 109
| no. 35
FEO, wenext restricted food availability to Per1−/−/Per2−/−/Per3−/−
mice (n = 15) in constant darkness (the mutant SCN is
not rhythmic in constant darkness; Fig. 3A and Figs. S5 and S6:
6 h/d restricted feeding; Fig. 3C and Fig. S7: 4 h/d restricted
feeding). During food deprivation before restricted feeding,
most Per1−/−/Per2−/−/Per3−/−mice exhibited ultradian bouts of
activity as well as a long (12–24 h) bout of wheel-running activity.
During restricted feeding, locomotor activity was typically con-
solidated before food presentation, but activity sometimes per-
sisted until food was presented, whereas sometimes activity bouts
occurredat various timesbefore food was available (as seen in the
group average activity profiles; Fig. S8 A–C). During food depri-
vation in the majority of Per1−/−/Per2−/−/Per3−/−mice, activity
duringfasting did not occur at the predicted phase of entrainment
to food availability and did not differ markedly from activity ob-
served during food deprivation before restricted feeding (Fig. 3 A
and C and Figs. S5–S7 and S8 A–C). From these data, we con-
24-h cycle of restricted feeding in constant darkness.
Per1−/−/Per2−/−/Per3−/−FEO Is a Circadian Oscillator with a ∼21-h
Period. Our observation that the MASCO period is short in
Per1−/−/Per2−/−/Per3−/−mice drew our attention to a similar
pattern of activity during restricted feeding in these mice—
namely, some components of the food anticipatory activity of
Per1−/−/Per2−/−/Per3−/−mice displayed a short (∼21 h) period
similar to the period of the MASCO rhythm (Fig. 3 and Figs. S5–
S7). χ2periodogram analyses of locomotor activity during re-
stricted feeding sometimes detected this ∼21-h period (in
addition to the 24-h period; Figs. S5B and S6A), especially in
mice fed 4 h/d (Fig. 3D and Fig. S7A).
This observation led us to hypothesize that the Per1−/−/Per2−/−/
Per3−/−FEO is a circadian oscillator, but because it has a short
(21 h) period, it is unable to entrain to the 24-h cycle of restricted
feeding inconstant darkness.Totest this hypothesis, weperformed
restricted feeding ofPer1−/−/Per2−/−/Per3−/−mice on a 21-h cycle in
a 21-h periodicity; group average activity profiles shown in Fig.
S8D). Two Per1−/−/Per2−/−/Per3−/−mice entrained to the 21-h cycle
of restricted feeding and food anticipatory activity also persisted
during food deprivation following an intervening day of ad libitum
feeding (Fig. 4 A and B). Two other Per1−/−/Per2−/−/Per3−/−mice
took several days to entrain to the 21-h cycle of restricted feeding
to the 21-h cycle of restricted feeding (Fig. 4D). χ2periodogram
analyses detected only a 21-h period in the mice that entrained to
demonstrate that the Per1−/−/Per2−/−/Per3−/−FEO is a circadian
oscillator with a ∼21-h period.
Computational Modeling of the FEO/MASCO and SCN as Coupled
Limit-Cycle Oscillators. Our experiments showed that the Per1−/−/
Per2−/−/Per3−/−MASCO was coupled to the light-driven SCN
oscillator in the light/dark cycle, but free-ran with a short period
when the SCN rhythm was dampened in constant darkness. Sim-
ilarly, the Per1−/−/Per2−/−/Per3−/−FEO entrained to a 24-h cycle of
restricted feeding when the mice were maintained in the light/dark
cycle. However, in constant darkness, the Per1−/−/Per2−/−/Per3−/−
FEO entrained to a 21-h cycle, but not a 24-h cycle, of restricted
double-plotted actograms (5-min bins) of wheel-running activity of Per1−/−/Per2−/−/Per3−/−mice maintained in constant darkness (indicated by black bars above
for 2 d (A) or 4 d (C). Mice were then fasted for 48 h and returned to ad libitum feeding. The χ2periodograms (B and D) correspond to the actograms shown in A
and C, respectively. χ2periodogram analysis was performed on days 1–10 of restricted feeding, as indicated on y axes of the actograms. The dotted lines in the
periodograms show the significance level P = 0.001. All individual actograms and periodograms (n = 15 in total) are shown in Figs. S5, S6 and S7.
Variablepatterns offoodanticipatoryactivityduringdaily(24-h) restricted feeding ofPer1−/−/Per2−/−/Per3−/−mice inconstantdarkness. Representative
| www.pnas.org/cgi/doi/10.1073/pnas.1206213109Pendergast et al.
feeding. We next determined if these experimental observations
were consistent with a model where the SCN and FEO/MASCO
are coupled oscillators with distinct periods. We analyzed the
output of a simple mathematical model, where the SCN and FEO/
MASCO are limit-cycle oscillators forced by the light/dark cycle or
by restricted feeding, respectively. For simplicity, coupling between
oscillators was set in only one direction, from the SCN to the FEO/
MASCO, disregarding feedback from the latter to the master
Based on our experimental results in Per1−/−/Per2−/−/Per3−/−
mice, the FEO/MASCO was simulated by an oscillator with
a free-running period of 21 h, and the SCN was simulated by
a damped limit-cycle oscillator. The computer simulation dem-
onstrated that, in constant darkness, the FEO/MASCO free-runs
thus indirectly affecting the dynamics of the FEO/MASCO with
a 24-hperiodicity (Fig. 5B).When thisindirect driveis outside the
range of entrainment of the FEO/MASCO, it becomes relatively
combined, as when daytime restricted feeding (24-h cycle) is per-
5C), as this two-zeitgeber system configuration allows stronger
entrainment, even when they are in antiphase (19). If, however,
24-h restricted feeding is the only input, as in constant darkness,
relative coordination of the FEO/MASCO is observed (Fig. 5D).
is approximated to that of the free-running period of the FEO/
MASCO (21 h), entrainment of this oscillator is achieved in this
Studies of the FEO and MASCO are challenging because their
rhythms are not expressed under normal physiological con-
ditions. Behavioral experiments have demonstrated that both the
ability. (A–E) Double-plotted actograms (5-min bins) of wheel-running ac-
tivity of Per1−/−/Per2−/−/Per3−/−mice maintained in constant darkness
(indicated by black bars above actograms). Gray shading on left halves of
the actograms indicates when food was available. Mice were fed ad libitum
for nine cycles and then fed 6 h per 21-h cycle for 12 cycles. Following one
cycle of ad libitum feeding, mice were food-deprived for 36 h and then fed
ad libitum. The data are plotted on a 21-h cycle (shown on x axes). All in-
dividual actograms (n = 5) are shown. Corresponding periodograms are
shown in Fig. S8.
Per1−/−/Per2−/−/Per3−/−mice entrain to a 21-h cycle of food avail-
FEO limit-cycle oscillators with distinct periods. (A–E) The FEO/MASCO (solid
circle; indicates self-sustained oscillator) and SCN (dashed circle; indicates
damped or passively driven oscillator) are modeled as limit-cycle oscillators
(Left) forced by the light/dark cycle (LD) or by restricted feeding (RF), re-
spectively. Relative circle sizes are proportional to oscillator periods and
amplitudes. Simulated double-plotted actograms (Right) of the locomotor
activity rhythm controlled by the FEO/MASCO (black lines) or SCN (dashed
gray lines) in Per1−/−/Per2−/−/Per3−/−mice are shown for each condition. In
the model, the period of the FEO/MASCO is 21 h. In constant darkness (A),
the SCN is damped and the FEO/MASCO free-runs with a 21-h period. In the
24-h light/dark cycle (B), the SCN is driven and the FEO/MASCO is in relative
coordination. When food is restricted to the daytime on a 24-h cycle in the
light/dark cycle (C), the SCN is driven and the FEO/MASCO is entrained.
When food is restricted on a 24-h cycle in constant darkness (D), the SCN is
damped and the FEO/MASCO is in relative coordination. When food is re-
stricted on a 21-h cycle (deviation from 24-h period indicated by angle of RF
arrow) in constant darkness (E), the SCN is damped and the FEO/MASCO is
entrained. Parameters: damped oscillator SCN: aL= 0.32, bL= 0.3, cL= 0.8,
dL= 0.5; FEO/MASCO: aF= 0.85, bF= 0.52, cF= 0.8, dF= 0.5; coupling CLF=
0.14, CFL= 0. L = 1 (light/dark condition) or L = 0 (constant darkness); Ldur= 1;
F = 0.3 (RF) or F = 0 (ad libitum food); Fdur= 1, phase relationship between LD
and RF ΦLF= 12 h.
Computational simulations of a model of coupled SCN and MASCO/
Pendergast et al.PNAS
| August 28, 2012
| vol. 109
| no. 35
MASCO and FEO rhythms persist in SCN-lesioned rodents and
display the properties of circadian oscillators (reviewed in ref. 4;
see also refs. 1 and 2). However, the anatomical loci of these
oscillators are unknown (despite exhaustive searches for the
FEO; reviewed in ref. 20). Recent studies investigating the FEO
and MASCO in circadian mutant mice have raised the possibility
that these oscillators do not require canonical circadian genes to
maintain their rhythmicities (5–11). In this study, we initially
sought to investigate the putative noncanonical nature of the
MASCO and FEO, and, surprisingly, found that the canonical
Period genes participate in period determination in the MASCO
Relative to the FEO, studies of the circadian properties of the
MASCO are technically more feasible. The free-running MASCO
rhythm is readily expressed within several days of methamphet-
amine administration. In mice with disrupted canonical circadian
genes, the SCN rhythm is disabled by releasing the animals into
constant darkness, thus circumventing the need for SCN lesion.
Using this approach, we found that the MASCO period in Per1−/−/
of wild-type and circadian mutant mice have reported MASCO
periods >24 h (3, 6, 7). Thus, Per1−/−/Per2−/−/Per3−/−mice are
unique in that they have a short-period MASCO rhythm.
In contrast to the MASCO, observing the free-running rhythm
of the FEO has been nearly impossible. By definition, a free-
running circadian rhythm can only be observed in constant con-
ditions—that is, ad libitum feeding or food deprivation (in SCN-
lesioned mice in constant darkness) in the case of the FEO.
However,theoutputoftheFEOis notexpressed underad libitum
feeding conditions and mice cannot survive more than ∼48 h of
food deprivation, thus leading to seemingly insurmountable
technical difficulties in measuring the free-running rhythm of the
FEO. To our knowledge, only one study has reported the obser-
on a 23-h cycle (21). In the current study, we observed the non-
entrained FEO rhythm in Per1−/−/Per2−/−/Per3−/−mice during
restricted feeding on a 24-h cycle in constant darkness. Consistent
with the simulations of the mathematical model presented herein,
we found that when the light-driven SCN rhythm is damped by
release into constant darkness, the Per1−/−/Per2−/−/Per3−/−FEO,
which has a 21-h period, cannot entrain to the 24-h cycle of re-
stricted feeding, thus allowing the expression of the free-running
(or relatively coordinated) FEO rhythm. This finding represents
of the FEO, and this technique can be applied to investigation of
other circadian mutants as well as wild-type rodents.
The cyclic input that we refer to as the light-driven SCN
rhythm could also be conferred if the light/dark cycle drives
rhythmicity in a different brain region (other than the SCN). In
this sense, the damped oscillator in the model could be replaced
by the light/dark cycle zeitgeber and yield the same results.
Therefore, though we believe the SCN is a strong candidate for
the anatomical locus receiving light input and driving rhythmic
behavior in Per1−/−/Per2−/−/Per3−/−mice, this is not necessarily
the case. However, our conclusions about the MASCO and FEO
in Per1−/−/Per2−/−/Per3−/−mice hold true regardless of whether
the SCN or another brain region is receiving light/dark input.
Previous studies of the FEO in mice with disrupted canonical
circadian genes have been inconclusive, in part due to the absence
of clear data showing that food anticipatory behavior persists
during food deprivation (i.e., in constant conditions) in constant
darkness (when the SCN rhythm is disabled) (5, 8, 9, 11). The
finding that food anticipatory activity in Per1−/−/Per2−/−/Per3−/−
mice entrains to a 21-h cycle of restricted feeding and persists
during food deprivation after an intervening day of ad libitum
feeding is definitive evidencethat theFEOis a circadian oscillator
in the absence of all PERIOD. Despite our finding that both the
MASCO and FEO are rhythmic in the absence of functional
PERIOD, the periods of both oscillators were markedly short-
ened. These data demonstrate that the canonical Period genes are
involved in period determination in the MASCO and FEO.
The MASCO appears to be coupled to the light-driven SCN
damped oscillator in light/dark conditions in Per1−/−/Per2−/−/
Per3−/−mice, because the MASCO rhythm did not dissociate from
the nocturnal activity rhythm even after long-term (>75 d) meth-
amphetamine treatment in the light/dark cycle (although the
MASCO may be relatively coordinated to the SCN). Similarly, our
data show that the food anticipatory activity of Per1−/−/Per2−/−/
Per3−/−mice entrains to a 24-h cycle of restricted feeding in the
light/dark cycle, but not in constant darkness, suggesting that the
FEO is coupled to the light-driven SCN. These experimental data
are consistent with our mathematical model of coupled SCN and
FEO/MASCO limit-cycle oscillators, where the SCN oscillator is
light-driven, but damps in constant darkness.
be the same circadian oscillator (2, 4, 22–24). The current study
provides additional evidence supporting this hypothesis. First, the
MASCO and FEO in Per1−/−/Per2−/−/Per3−/−mice have similar
and unique short periods. Second, the Per1−/−/Per2−/−/Per3−/−
MASCO and FEO behave distinctly in light/dark and constant
darkness conditions, but the distinct outputs are similar between
the MASCO and FEO, and are consistent with the computer
simulations of the model.
an invaluable tool in the search for the anatomical location(s) of
the MASCO and FEO. Per1−/−/Per2−/−/Per3−/−mice are unique
because their FEO and MASCO have distinctive short periods,
whereas classical circadian oscillators requiring PERIOD (such as
the SCN) are disabled in constant darkness. Thus, the search for
the MASCO and FEO can be revitalized by surveying tissues to
identify loci with ∼21-h periods of rhythmicity in Per1−/−/Per2−/−/
In summary, the work presented here demonstrates that the
canonical Period genes are involved in timekeeping in the FEO
and MASCO, and we have identified Per1−/−/Per2−/−/Per3−/−
mice as a tool for solving the enigmas of the MASCO and FEO.
Materials and Methods
Animals. We obtained mPer1−/−, mPer2−/−, and mPer3−/−mice (congenic with
the 129/Sv genetic background, and provided by David Weaver, University of
Massachusetts, Worcester, MA) (25, 26) and backcrossed them with wild-type
C57BL/6J mice (Jackson Laboratory) for at least 15 generations (14, 27)
(C57BL/6J Per1−/−, Per2−/−, and Per3−/−mice were deposited at Jackson
Laboratory, stock nos. 10491, 10492, and 10493, respectively). Period mutant
mice were crossed with each other until we generated several Per1+/−/Per2−/−/
Per3−/−breeding pairs. Per1−/−/Per2−/−/Per3−/−mice used for experiments
(males and females) were generated from these breeders. Genotyping for
the Period genes was performed as previously described (25, 26). C57BL/6J
wild-type mice were from our breeding colony at Vanderbilt University. The
wild-type mouse shown in Fig. 1A is a Per2+/+from our previous study (14).
The mice were bred and group housed in the Vanderbilt University animal
facility in a 12L:12D cycle [light intensity ∼350 lux (lx)] and provided food
and water ad libitum. The mean (± SD) ages of the mice at the beginning of
the experiments were as follows: wild type, 93 ± 35 d; Per1+/−/Per2−/−/Per3−/−,
105 ± 39 d). All experiments were conducted in accordance with the guide-
lines of the Institutional Animal Care and Use Committee at Vanderbilt
Recording and Analyses of Circadian Behavior. Animals were singly housed in
cages (length × height × width: 29.5 × 11.5 × 12 cm) with unlimited access to
a running wheel (diameter 11 cm), food (unless otherwise indicated), and
water. The cages were placed in light-tight, ventilated boxes in light/dark
conditions (light intensity: 200–300 lx) or in constant darkness (as indicated
for each experiment). Cages were changed every 3 wk. Wheel-running ac-
tivity (recorded every minute by computer) was monitored and analyzed
using ClockLab (Actimetrics). Normalized activity data were double-plotted
in actograms in 5-min bins using ClockLab. Periods were determined by χ2
periodogram with α = 0.001 (the days used for analyses are indicated for
| www.pnas.org/cgi/doi/10.1073/pnas.1206213109Pendergast et al.
each experiment). When multiple periods were detected by χ2, fast Fourier Download full-text
transform was used to determine the dominant period.
Methamphetamine Treatment and Analyses. The mice were provided with
0.005% methamphetamine (Sigma) in their drinking (tap) water. χ2perio-
dogram analyses were used to determine the periods of the methamphet-
amine-induced wheel-running rhythms on days 12–33 of methamphetamine
treatment (it often took several days for a stable methamphetamine-in-
duced rhythm to be observed). Changes in phases and/or periods of meth-
amphetamine-induced rhythms occurred frequently (both spontaneously
and after cage changes), and the days used for χ2analyses were altered in
these instances (as indicated in figures and legends).
Restricted Feeding and Analyses. Mice were fed LabDiet 5L0D (Purina). Be-
cause we previously found that Bmal1−/−mice must be offered food on the
bottom of the cage during restricted feeding to prevent death (11), we
placed food on the bottom of the cage and in the hopper during restricted
feeding of Per1−/−/Per2−/−/Per3−/−mice. Mice were allowed to eat as much as
they desired during the time when food was available. When food was re-
moved, the light-tight box was opened and all food was removed from the
wire top and from the bottom of the cage. During restricted feeding
experiments performed in constant darkness, an infrared viewer (FIND-R-
SCOPE Infrared Viewer; FJW Optical Systems, Inc.) was used to add and
remove food from cages so that mice were not exposed to visible light.
During ad libitum feeding and food deprivation, the light-tight boxes were
not opened to avoid any external cues associated with food availability.
During this time, the well-being of the mouse was monitored by assessing
wheel-running data collected by computer.
Restricted feeding in the light/dark cycle was performed in 18L:6D because
we found in our previous studies that food anticipatory activity was more
robust in long photoperiods, and we wanted to provide ideal conditions for
observing food anticipatory in our current experiments (11). Restricted
feeding protocols differed slightly for each experiment (as indicated in
figures and legends), but the typical protocol was as follows: After a brief
period (5–14 d) of acclimation to the wheel-running cage and light-tight
box, mice were food-deprived for 48 h to assess activity during fasting be-
fore restricted feeding. Following 1–3 d of ad libitum feeding, mice were
food-deprived for 24 h and then fed 6 h/d (Figs. 2 and 3A and Figs. S4 D and
E, S5, and S6) or 4 h/d (Fig. 3C and Figs. S4 A–C and S7) for 10 d. After this
period of restricted feeding, mice were fed ad libitum for 0–4 d [Fig. 3A and
Fig. S5: 2 d ad libitum; Fig. S6: 0 d ad libitum; Fig. 2 and Fig. S4: 3 d ad
libitum; Fig. 3C and Fig. S7: 4 d ad libitum; Fig. 4 and Fig. S9: one cycle (21 h)
ad libitum] and then food-deprived for 48 h.
Statistical Analysis. Statistical analysis was performed using SigmaStat (Systat
Software, Inc.). Independent t tests (two-tailed) were used to compare two
groups. Significance was ascribed at P < 0.05.
Computer Simulations. SCN and FEO/MASCO oscillators were simulated by
coupled Pittendrigh-Pavlidis equations, forced by light/dark (L) or restricted
feeding (F), respectively (SI Materials and Methods). In all simulations, L had
a 1-h duration and amplitude 1, whereas F had amplitude 0.3 and 1-h du-
ration. A fixed 12-h phase relationship was set between L and F. To attain
a damped SCN oscillator, the parameter set values were chosen so as to leave
the oscillator out of the self-sustainment domain. Simulations were per-
formed using the CircadianDynamix software (www.neurodynamix.net),
which is an extension of Neurodynamix II (28).
ACKNOWLEDGMENTS. We thank Dr. W. Otto Friesen for Neurodynamix
software. This research was supported by National Science Foundation Grant
IOS-1146908 and the National Mouse Metabolic Phenotyping Centers
MICROMouse Program U24DK076169 (to S.Y.; www.mmpc.org). Support
was also provided by the Tennessee Valley Healthcare System; National Insti-
tutes of Health Grant DK085712; and Diabetes Research and Training Center
Grant DK20593 (to K.D.N.).
1. Honma K, Honma S, Hiroshige T (1986) Disorganization of the rat activity rhythm by
chronic treatment with methamphetamine. Physiol Behav 38:687–695.
2. Honma K, Honma S, Hiroshige T (1987) Activity rhythms in the circadian domain
appear in suprachiasmatic nuclei lesioned rats given methamphetamine. Physiol Be-
3. Tataroglu O, Davidson AJ, Benvenuto LJ, Menaker M (2006) The methamphetamine-
sensitive circadian oscillator (MASCO) in mice. J Biol Rhythms 21:185–194.
4. Mistlberger RE (1994) Circadian food-anticipatory activity: Formal models and physi-
ological mechanisms. Neurosci Biobehav Rev 18:171–195.
5. Storch KF, Weitz CJ (2009) Daily rhythms of food-anticipatory behavioral activity do
not require the known circadian clock. Proc Natl Acad Sci USA 106:6808–6813.
6. Mohawk JA, Baer ML, Menaker M (2009) The methamphetamine-sensitive circadian
oscillator does not employ canonical clock genes. Proc Natl Acad Sci USA 106:
7. Honma S, Yasuda T, Yasui A, van der Horst GT, Honma K (2008) Circadian behavioral
rhythms in Cry1/Cry2 double-deficient mice induced by methamphetamine. J Biol
8. Pitts S, Perone E, Silver R (2003) Food-entrained circadian rhythms are sustained in
arrhythmic Clk/Clk mutant mice. Am J Physiol Regul Integr Comp Physiol 285:R57–R67.
9. Iijima M, et al. (2005) Altered food-anticipatory activity rhythm in Cryptochrome-
deficient mice. Neurosci Res 52:166–173.
10. Masubuchi S, Honma S, Abe H, Nakamura W, Honma K (2001) Circadian activity
rhythm in methamphetamine-treated Clock mutant mice. Eur J Neurosci 14:
11. Pendergast JS, et al. (2009) Robust food anticipatory activity in BMAL1-deficient mice.
PLoS ONE 4:e4860.
12. Feillet CA, et al. (2006) Lack of food anticipation in Per2 mutant mice. Curr Biol 16:
13. Mendoza J, Albrecht U, Challet E (2010) Behavioural food anticipation in clock genes
deficient mice: Confirming old phenotypes, describing new phenotypes. Genes Brain
14. Pendergast JS, Friday RC, Yamazaki S (2010) Distinct functions of Period2 and Period3
in the mouse circadian system revealed by in vitro analysis. PLoS ONE 5:e8552.
15. Pendergast JS, Niswender KD, Yamazaki S (2012) Tissue-specific function of Period3 in
circadian rhythmicity. PLoS ONE 7:e30254.
16. Honma S, Honma K, Hiroshige T (1991) Methamphetamine effects on rat circadian
clock depend on actograph. Physiol Behav 49:787–795.
17. Masubuchi S, et al. (2000) Clock genes outside the suprachiasmatic nucleus involved in
manifestation of locomotor activity rhythm in rats. Eur J Neurosci 12:4206–4214.
18. Cuesta M, Aungier J, Morton AJ (2012) The methamphetamine-sensitive circadian
oscillator is dysfunctional in a transgenic mouse model of Huntington’s disease.
Neurobiol Dis 45:145–155.
19. Oda GA, Friesen WO (2011) Modeling two-oscillator circadian systems entrained by
two environmental cycles. PLoS ONE 6:e23895.
20. Davidson AJ (2009) Lesion studies targeting food-anticipatory activity. Eur J Neurosci
21. Stephan FK (1992) Resetting of a feeding-entrainable circadian clock in the rat.
Physiol Behav 52:985–995.
22. Honma S, Kanematsu N, Honma K (1992) Entrainment of methamphetamine-induced
locomotor activity rhythm to feeding cycles in SCN-lesioned rats. Physiol Behav 52:
23. Honma S, Honma K, Hiroshige T (1989) Methamphetamine induced locomotor
rhythm entrains to restricted daily feeding in SCN lesioned rats. Physiol Behav 45:
24. Mistlberger RE (2011) Neurobiology of food anticipatory circadian rhythms. Physiol
25. Shearman LP, Jin X, Lee C, Reppert SM, Weaver DR (2000) Targeted disruption of the
mPer3 gene: Subtle effects on circadian clock function. Mol Cell Biol 20:6269–6275.
26. Bae K, et al. (2001) Differential functions of mPer1, mPer2, and mPer3 in the SCN
circadian clock. Neuron 30:525–536.
27. Pendergast JS, Friday RC, Yamazaki S (2009) Endogenous rhythms in Period1 mutant
suprachiasmatic nuclei in vitro do not represent circadian behavior. J Neurosci 29:
28. Friesen WO, Friesen JA (2009) Neurodynamix II. Concepts in Neurophysiology Illus-
trated by Computer Simulations (Oxford Univ Press, New York).
Pendergast et al.PNAS
| August 28, 2012
| vol. 109
| no. 35