2147
J Physiol 596.11 (2018) pp 2147–2157
Functional decoupling of melatonin suppression and
circadian phase resetting in humans
Shadab A. Rahman1,2 , Melissa A. St Hilaire1,2 , Claude Gronfier1,2,3 , Anne-Marie Chang1,2 ,
Nayantara Santhi1,2,4 , Charles A. Czeisler1,2 , Elizabeth B. Klerman1,2 and Steven W. Lockley1,2
1
Division of Sleep and Circadian Disorders, Departments of Medicine and Neurology, Brigham & Women’s Hospital, Boston, MA, USA
Division of Sleep Medicine, Harvard Medical School, Boston, MA, USA
3
Lyon Neuroscience Research Center, Integrative Physiology of the Brain Arousal Systems (WAKING) team, INSERM UMRS 1028, CNRS UMR 5292,
Université Claude Bernard Lyon 1, Université de Lyon, F-69000, Lyon, France
4
Surrey Sleep Research Centre, Faculty of Health and Medical Sciences, University of Surrey, Guildford, Surrey, UK
2
The Journal of Physiology
Edited by: Ole Paulsen & Kim Barrett
Key points
r There is assumed to be a monotonic association between melatonin suppression and circadian
phase resetting induced by light exposure.
r We tested the association between melatonin suppression and phase resetting in humans.
r Sixteen young healthy participants received nocturnal bright light (9500 lux) exposure of
continuous or intermittent patterns, and different durations ranging from 12 min to 6.5 h.
r Intermittent exposure patterns showed significant phase shifts with disproportionately less
melatonin suppression.
r Each and every bright light stimulus in an intermittent exposure pattern induced a similar
r
degree of melatonin suppression, but did not appear to cause an equal magnitude of phase
shift.
These results suggest that phase shifts and melatonin suppression are functionally independent
such that one cannot be used as a proxy measure of the other.
Abstract Continuous experimental light exposures show that, in general, the conditions that
produce greater melatonin suppression also produce greater phase shift, leading to the assumption
that one can be used as a proxy for the other. We tested this association in 16 healthy individuals
who participated in a 9-day inpatient protocol by assessing melatonin suppression and phase
resetting in response to a nocturnal light exposure (LE) of different patterns: (i) dim-light control
(<3 lux; n = 6) or (ii) two 12-min intermittent bright light pulses (IBL) separated by 36 min of
darkness (9500 lux; n = 10). We compared these results with historical data from additional
LE patterns: (i) dim-light control (<3 lux; n = 11); (ii) single continuous bright light exposure
of 12 min (n = 9), 1.0 h (n = 10) or 6.5 h (n = 6); or (iii) an IBL light pattern consisting of
six 15-min pulses with 1.0 h dim-light recovery intervals between them during a total of 6.5 h
Shadab Rahman’s research interests are in sleep and circadian neurophysiology. His primary research interest is in basic and applied
circadian photobiology. He has been studying how the spectral, duration and pattern characteristics of light modulate behavioural,
endocrine and molecular outputs that are under the control of the biological clock. His secondary interest is in peripheral rhythms
including metabolic, immune and reproductive rhythms. He is exploring the endogenous circadian regulation of these rhythms
and the effects of light exposure on these rhythms. His goal is to combine the findings from these two research streams to develop
effective photobiological countermeasures for circadian disruption.
C 2018 The Authors. The Journal of Physiology
C 2018 The Physiological Society
DOI: 10.1113/JP275501
2148
S. A. Rahman and others
J Physiol 596.11
(n = 7). All light exposure groups had significantly greater phase-delay shifts than the dim-light
control condition (P < 0.0001). While a monotonic association between melatonin suppression
and circadian phase shift was observed, intermittent exposure patterns showed significant phase
shifts with disproportionately less melatonin suppression. Each and every IBL stimulus induced a
similar degree of melatonin suppression, but did not appear to cause an equal magnitude of phase
shift. These results suggest unique specificities in how light-induced phase shifts and melatonin
suppression are mediated such that one cannot be used as a proxy measure of the other.
(Received 30 October 2017; accepted after revision 2 February 2018)
Corresponding author S. A. Rahman: Division of Sleep and Circadian Disorders, Department of Medicine, Brigham
and Women’s Hospital, 221 Longwood Ave., Boston, MA 02115, USA. Email: sarahman@rics.bwh.harvard.edu
Introduction
Light is the strongest environmental time cue that resets
the mammalian circadian pacemaker, located in the suprachiasmatic nucleus (SCN) (Czeisler et al. 1989; Meijer
et al. 1992, 1998), with maximal phase shifts during
the biological night (St Hilaire et al. 2012). Ocular
light exposure during the biological night also acutely
suppresses release of the pineal hormone melatonin (Lewy
et al. 1980; Zeitzer et al. 2000). The light information
required to entrain the SCN or suppress melatonin is
detected by specialized retinal photoreceptors and transduced to the SCN via the retinohypothalamic tract (Klein
& Moore, 1979; Johnson et al. 1998; Gooley et al. 2001).
Subsequently, the SCN sends light information to the
pineal via the retina–SCN–superior cervical ganglion
(SCG)–pineal pathway, the canonical pathway by which
light can suppress melatonin (Klein et al. 1971; Moore,
1978).
Light-induced melatonin suppression shows sensitivities seemingly similar to those for phase resetting with
respect to the duration (Chang et al. 2012), intensity
(Boivin et al. 1996; Zeitzer et al. 2000) and spectral
characteristics of the light exposure (Wright & Lack,
2001; Lockley et al. 2003; Gooley et al. 2010). The degree
of melatonin suppression and the magnitude of phase
resetting have been reported to be linearly correlated
(Lockley et al. 2003) and, at least at the photoreceptor
input level, have been proposed to be predictive of the
strength of the photic stimulus on the SCN and therefore
broader circadian function (Brainard et al. 2001; Rea et al.
2005, 2010). Given that melatonin suppression can only
occur during the biological night (defined from the onset
(SynOn) of melatonin until synthesis offset (SynOff)) (St
Hilaire et al. 2007) and light can cause phase shifts at times
when melatonin is not secreted (Jewett et al. 1997; Khalsa
et al. 2003), the two responses can clearly be functionally
independent.
In the present study we compared the magnitude
and pattern of melatonin suppression with circadian
phase resetting in humans during exposure to a range
of continuous and intermittent light pulses in order to
examine the extent to which melatonin suppression and
phase resetting are functionally coupled.
Methods
Ethical approval
The study was approved by the Partners Human Research
Committee, and participants provided written informed
consent prior to study. The study conformed to the
standards set by the latest revision of the Declaration of
Helsinki and the study was registered as a clinical trial on
www.clinicaltrials.gov (NCT no. 01330992).
The protocol for the 16 participants studied under the
dim-light control condition and the 1.0 h intermittent
bright light (IBL) condition is reported here. The protocols
for the 0.2, 1.0, 6.5 and 6.5 h IBL conditions have been
reported previously (Gronfier et al. 2004; Chang et al. 2012;
St Hilaire et al. 2012). Differences between the current and
previous protocols are detailed below.
Participants and prestudy conditions
Sixteen young healthy individuals (9 females; mean age
± SD: 23.1 ± 2.8 years; range 19–29 years) participated
in the current inpatient study at the Intensive Physiological Monitoring Unit in the Centre for Clinical
Investigation (CCI) at Brigham and Women’s Hospital.
All were healthy as determined by physical, psychological
and ophthalmologic exam. For at least 3 weeks prior
to admission, participants maintained a self-selected,
constant 8 h sleep/rest/dark schedule confirmed with calls
to a time- and date-stamped voicemail at bedtime and
wake time. Throughout screening, participants were asked
to refrain from use of any prescription or non-prescription
medications, supplements, recreational drugs, caffeine,
alcohol or nicotine. Compliance was verified by urine and
blood toxicology during screening and urine toxicology
upon entry to the unit. For at least 7 days prior to
entering the unit, subjects’ schedules were monitored
C 2018 The Authors. The Journal of Physiology
C 2018 The Physiological Society
J Physiol 596.11
Decoupled melatonin suppression and phase resetting
with actigraphy (Actiwatch-L, Minimitter, Inc., Bend, OR,
USA).
Historical data
Forty-three individuals (13 females; mean age ± SD:
22.7 ± 3.5 years; range 18–31 years) participated in the
previously reported protocols (Gronfier et al. 2004; Chang
et al. 2012; St Hilaire et al. 2012). All prestudy conditions
were identical.
Study protocols
Participants were studied for 9 or 10 days in an
environment free of time cues (i.e. no access to windows,
clocks, watches, live TV, radio, internet, telephones and
current newspapers and continually supervised by staff
trained not to reveal information about the time of day)
(Fig. 1A and B). The 9-day schedule consisted of a 3-day
baseline (8 h:16 h sleep:wake cycle based on average sleep
times in the 7 days prior to study entry), an initial 50 h
constant routine (CR1, described below), a 16 h light
exposure day, followed by 8 h of sleep. Following the
subsequent 8 h sleep episode, participants completed a
30 h constant routine (CR2) followed by a final 8 h sleep
episode, after which they were discharged (Fig. 1A). The
10-day protocol (Gronfier et al. 2004) differed in including
a 26.2 h CR1 and a 64.0 h CR2. During the constant routine
procedures, participants were asked to remain awake while
supervised in constant dim light in a semi-recumbent
posture, with daily nutritional intake divided into hourly
portions (150 mEq Na+ /100 mEq K+ (±20%) controlled
nutrient, isocaloric (basal energy expenditure × 1.3) diet,
2000–2500 mL fluids/24 h; Mills et al. 1978; Duffy & Dijk,
2002).
Lighting conditions
During the first 2.5 baseline days, maximum ambient light
during scheduled wake was 48 µW cm−2 (190 lux)
maximum when measured in the horizontal plane at
a height of 187 cm and 23 µW cm−2 or (88 lux)
when measured in the vertical plane (137 cm). In the
9-day study, midway through day 3, maximum ambient
light was decreased to <0.4 µW cm−2 [approximately
0.1 µW cm−2 (<3 lux; 1.5 lux)] when measured in
the vertical plane at 137 cm and maintained at that
level for the remainder of the study. In the 10-day
study, lighting conditions for day 3 were maintained
at 90 lux until bedtime. Ambient room lighting was
generated using ceiling-mounted 4100K fluorescent lamps
(F96T12/41U/HO/EW, 95W; F32T8/ADV841/A, 32W;
F25T8/TL841, 25 W; Philips Lighting, The Netherlands)
with digital ballasts (Hi-Lume 1% and Eco-10 ballasts,
Lutron Electronics Co., Inc., Coopersburg, PA, USA)
C 2018 The Authors. The Journal of Physiology
C 2018 The Physiological Society
2149
transmitted through a UV-stable filter (Lexan 9030
with prismatic lens, GE Plastics, Pittsfield, MA, USA).
Routine illuminance and irradiance measures were
conducted using an IL1400 radiometer/powermeter with
an SEL-033/Y/W or SEL-033/F/W detector, respectively
(International Light, Inc., Newburyport, MA, USA).
Study groups and experimental light exposure
There were six light exposure (LE) conditions: (i)
dim-light control, n = 17; (ii) 1.0 h bright continuous
light, n = 10 (Fig. 1C); (iii) 0.2 h bright continuous light,
n = 9 (Fig. 1D); (iv) 1.0 h intermittent bright light (IBL),
n = 10 (Fig. 1E); (v) 6.5 h bright continuous light, n = 6
(Fig. 1F); and (vi) 6.5 h bright IBL, n = 7; (Fig. 1G). All
LE occurred in constant posture conditions. The different
LE conditions were aligned relative to the midpoints of LE
timing, except for the 0.2 h LE, which occurred slightly
earlier, with the midpoint 12 min earlier than the other
groups. The 1.0 h IBL condition included two 12 min
bright light pulses with a 36 min dark (lights off) interval in between. The 1.0 h IBL condition was timed so
that the first pulse corresponded to the start of the single
0.2 h LE condition. The 6.5 h IBL condition included
six 15 min bright light pulses with five 60 min dim light
(<1 lux) intervals in between. The dim-light control group
received dim (<3 lux) ambient light during the constant
posture. All participants in the 0.2 continuous, 1.0 h IBL,
1.0 h continuous and 10 participants in the control group
completed the 9-day inpatient study (Chang et al. 2012).
All participants in the 6.5 h continuous, 6.5 h IBL and seven
participants in the control group completed the 10-day
inpatient study (Gronfier et al. 2004). The phase shift
and melatonin suppression results have been previously
reported for the 0.2 h continuous (Chang et al. 2012),
1.0 h continuous (Chang et al. 2012), 6.5 h continuous
(Gronfier et al. 2004; Chang et al. 2012), 6.5 h IBL (Gronfier
et al. 2004) and 11 participants from the control group
(Gronfier et al. 2004; St Hilaire et al. 2012). The LE
conditions were centred within the 16 h wake episode and
timed to occur at a circadian phase expected to induce
melatonin suppression and phase delay shifts.
The target corneal light illuminance during the
experimental light exposure (LE) was 9500 lux
(2900 µW cm−2 , 6700 melanopic lux; Lucas et al.
2014) and 1.5 lux (0.1 µW cm−2 , 1.0 melanopic lux).
The LE and dim-light control conditions were generated
using the same overhead fluorescent lamps as described
previously. In the 9-day inpatient study the constant posture for the light exposure session began 6 h after wake
time on the LE day for 4.5 h. In the 10-day inpatient study
the constant posture for the LE session began 3 h after
wake time on the LE day for 8 h. Participants remained
seated in a specific location of the room for the entire
constant posture. Participants wore clear unshaded UVEX
2150
S. A. Rahman and others
glasses (UVEX Winter Optical, Smithfield, RI, USA) and
were asked to maintain a ‘fixed gaze’ on a target on the
wall directly in front of them for at least 4 min alternating
with an equal interval ‘free gaze’ during which they were
allowed to look elsewhere, as long as they did not close their
eyes or shield them from the light. The 0.2 h continuous
and 1.0 h continuous conditions used a 6 min fixed/free
gaze interval; the 1.0 h IBL used a 4 min fixed/free gaze
interval during the 1.0 h exposure interval and a 5 min
fixed/free gaze interval during the remainder of the 4.5 h
constant posture; the 6.5 h continuous and 6.5 h IBL
conditions used a 5 min fixed/free gaze interval throughout
the exposure interval. Light illuminance was measured at
every change of gaze with the sensor placed next to the
participant’s eye and pointed at the target during ‘fixed
gaze’ and in the angle of gaze during ‘free gaze’. A research
technician was present during the LE session to administer
the LE, measure and record light readings, and monitor
adherence to study procedures.
Outcome measures of melatonin phase and
melatonin suppression
In the current study (n = 16), blood samples were collected
every 5–60 min throughout the protocol and assayed by
A
1
J Physiol 596.11
radioimmunoassay for melatonin concentration. Blood
collection frequency varied between protocols. Melatonin
concentration was determined by double-antibody radioimmunoassay (Vaughan, 1993) by a laboratory unaware
of experimental conditions (Specialty Assay Research Core
Laboratory, Brigham and Women’s Hospital, Boston, MA,
USA). The plasma melatonin intra-assay coefficient of
variation (%CV) was 8.5% at 5.85 pg mL−1 and 8.0%
at 25.46 pg mL−1 , and the interassay %CV was 10.8%
at 5.98 pg mL−1 ; 14.5% at 23.07 pg mL−1 . The previous
studies had comparable %CV coefficients. In the 10-day
study (Gronfier et al. 2004), which included the 6.5 h
continuous, 6.5 h IBL and dim-light control conditions
(n = 20), samples were assayed for melatonin by use of
radioimmunoassay techniques (Diagnostech/Pharmasan,
Osceola, WI, USA). The assay sensitivity was 2.5 pg mL−1 .
The average intra-assay coefficients of variation were 6.4%
below 50 pmol L−1 and 4.9% above. In the previous
9-day studies (Chang et al. 2012; St Hilaire et al. 2012),
which included the 0.2 h continuous, 1.0 h continuous
conditions (n = 19) and the dim-light control condition
(n = 4), melatonin was assayed by radioimmunoassay
(Pharmasan). Sensitivity of the assay was 2.5 pg mL−1 ;
inter-assay and intra-assay coefficients of variance were
9.2–13.2% and 9.8–12.1%, respectively.
Clock Time
ADM
22.00
3
C
D
Study Day
7
04.00
06.00 h
6
8
10
12
1.0 h
0.2 h
(Continuous)
9
D/C
E
ADM
1.0 h IBL
(0.2 h x
2 pulses)
2
F
4
6.5 h
(Continuous)
6
G
8
10
24.00
02.00
(Continuous)
5
B
24.00
(0.25 h x
6 pulses)
D/C
04.00
08.00
12.00
6.5 h IBL
16.00
Clock Time
20.00 24.00 h
4
Time from Wake (h)
Figure 1. Study protocol to assess the functional coupling between melatonin suppression and phase
resetting
Participants were enrolled in a 9-day (n = 39; A) or 10-day (n = 20; B) inpatient protocol in an environment free of
time cues. White bars indicate exposure to ambient room light (90 lux) and grey bars indicate exposure to dim
ambient light (<3 lux). Black bars show scheduled sleep episodes in darkness that, for the first 3 baseline days,
was scheduled the same time as the self-selected sleep schedule maintained for 3 weeks prior to admission. The
schedule consisted of a 3-day baseline following admission (ADM), with 8:16 h sleep:wake cycle; an initial 50 h
(A) or 26 h (B) constant routine, an 8 h sleep (sleep delayed by 10 h from pre-admission self-selected sleep
schedule) and a 16 h wake episode in which the light exposure was centred; followed by 8 h sleep (sleep delayed
by 10 h from pre-admission self-selected sleep schedule) and then a second 30 h (A) or 64 h (B) constant
routine followed by an 8 h recovery sleep at the same time as the baseline days and then discharge (D/C), the 8 h
interval during which the light exposure of different patterns and durations occurred is shown as a hatched bar.
Participants received bright light exposure of different patterns and durations (C–G).
C 2018 The Authors. The Journal of Physiology
C 2018 The Physiological Society
J Physiol 596.11
Decoupled melatonin suppression and phase resetting
The dim light melatonin onset (DLMOn) was used as
the endogenous phase marker and calculated as the time at
which levels of melatonin went above the 25% threshold
of the three harmonic fitted peak-to-trough amplitude
of melatonin during CR1 (Lockley et al. 2003; Gronfier
et al. 2004). Phase shifts were calculated as the difference
in clock time between CR1 and CR2 phases. Due to the
length of CR1 (50 h) in the 9-day study, there were two
DLMO phase measures determined for each participant
during this constant routine; phase shifts were calculated
using the DLMOn that occurred temporally closer (24 h
prior) to the LE.
Melatonin suppression was determined in three ways:
(i) the area under the curve (AUC) during the
light exposure was compared to the AUC during the
corresponding time window 24 h earlier during CR1,
and the resulting percentage melatonin suppression due
to the bright light was calculated using the formula
[1 − (AUCLE /AUCCR1 )] × 100 (Zeitzer et al. 2000; Lockley
et al. 2003); (ii) the difference in melatonin levels at the
start and end of each light pulse (interpolated linearly),
expressed as a percentage; and (iii) AUC during the interval between DLMO and DLMOff (downward crossing of
the 25% threshold) flanking the light-exposure stimulus
and the interval between DLMO and DLMOff 24 h earlier
during CR1. Percentage melatonin suppression due to the
bright light was calculated as described earlier.
Data analysis
Data from two participants in the current study were
excluded from further analysis. One participant (1.0 h IBL
condition) was excluded from analysis because post hoc
analysis showed that the LE occurred 6.2 h after DLMOn,
which is outside the interval (−3 to + 6 h around
DLMOn) for inducing a phase delay (St Hilaire et al. 2012).
The second individual (dim-light control condition)
showed considerable daytime melatonin secretion, which
is unusual, and a reliable DLMO could not be determined.
Due to differences in blood sampling frequency, melatonin
values were linearly interpolated at the start and end of
each light pulse and also at the start and end of the entire
exposure window.
Melatonin phase shift and suppression data were
compared among the six LE conditions using a general
linear model one-way ANOVA with LE condition as the
main factor. If a significant main effect was observed, then
data were subjected to Student–Newman–Keuls (SNK)
post hoc analysis. The percentage change between the onset
and offset of the light pulses was compared between groups
using a general linear model with LE type as the main
factor. If a significant main effect was observed then data
were subjected to SNK post hoc analysis. All analyses were
performed using SAS 9.3 (SAS Institute Inc., Cary, NC,
USA).
C 2018 The Authors. The Journal of Physiology
C 2018 The Physiological Society
2151
Table 1. Correlation between phase shift and melatonin
suppression within each light exposure condition
Light exposure condition
Dim-light control
1.0 h continuous
0.2 h continuous
1.0 h IBL
6.5 h continuous
6.5 h IBL
Pearson’s r
P
0.26
0.41
0.47
−0.37
0.18
−0.42
0.34
0.24
0.20
0.32
0.74
0.35
Results
Melatonin suppression and phase resetting
Phase resetting increased with longer duration exposures,
proportional to both the sum of the light exposure
duration (Fig. 2A) and the entire exposure window
(start of the first pulse to the end of the last pulse in
the IBL conditions; data not shown). All light exposure
groups had significantly greater delay shifts than the
dim-light control condition. The phase shift in the 0.2 h
continuous, 1.0 h IBL (0.4 h total light exposure) and 1.0 h
continuous groups were not significantly different. The
6.5 h continuous and 6.5 h IBL (1.5 h total light exposure)
groups were both significantly greater than the other three
LE groups (P = 0.01) but not significantly different from
each other (Fig. 2A).
In contrast to phase resetting, melatonin suppression
did not increase monotonically with summed LE duration
(Fig. 2B). All LE groups had significantly greater
suppression than the control group but the 6.5 h IBL
group (1.5 h total light exposure) had relatively less
suppression than the 6.5 h continuous condition, and less
than what could be expected from a 90 min exposure
interval. The 0.2 h continuous and 6.5 h IBL (1.5 h total
light exposure) conditions had similarly low melatonin
suppression (25%) whereas the 1 h continuous and 6.5 h
continuous conditions had similar higher suppression
(60–80%). The rank order of the amount of melatonin
suppression by condition did not correspond to the order
observed for phase resetting magnitude. Therefore, the IBL
conditions induced disproportionally larger phase shifts
than melatonin suppression (Fig. 2C).
To assess the relationship further, individually matched
phase shift and melatonin suppression values were plotted
for all individuals to examine whether the magnitudes of
both responses were parallel. Figure 2C shows that the
phase-shift magnitudes are highly variable and are not
associated with the suppression response. Correlational
analyses between melatonin suppression and phase shifts
were not significant within any of the six different light
exposure conditions (Table 1).
2152
S. A. Rahman and others
Temporal dynamics of melatonin suppression
for suppression is apparent in the plots seen as a lower
melatonin value at the end of the second pulse compared
to the start of that pulse (Fig. 3E), we did not detect a
significant change in melatonin levels between the start
and end of the light pulse for the 0.2 h continuous
condition and the second pulse of the 1.0 h IBL condition,
likely because the 36 min resetting interval was not
sufficiently long to allow for full recovery of the melatonin
levels, which would have allowed for observation of greater
suppression.
Melatonin levels decreased during each light pulse and
started to recover shortly after the end of the light pulse,
as expected. Melatonin levels remained low throughout
the 1.0 h and 6.5 h continuous conditions (Fig. 3A–G).
Between the start and end of the 1.0 h and 6.5 h light
pulses, melatonin changed 59.3 ± 5.8% and 78.0 ± 7.3%,
respectively (Fig. 3B–H). In the 6.5 h IBL condition,
melatonin levels decreased during each 15 min pulse and
recovered to pre-pulse levels or higher by the end of
each 60 min recovery interval (Fig. 3I). Each of the six
pulses reduced melatonin levels similarly (range 25–43%)
without any evidence of desensitization in subsequent
responses (Fig. 3J).
The first 12 min pulse of the 1.0 h IBL condition
decreased melatonin levels by 15.7 ± 5.8%, which was
not significantly different from the mean reduction in
melatonin levels induced by the six 15 min pulses of
the 6.5 h IBL condition (27.1 ± 6.3%). While the trend
Discussion
A positive relationship between melatonin suppression
and phase delay resetting in response to nocturnal light
exposure has been suggested (Boivin et al. 1996; Zeitzer
et al. 2000; Lockley et al. 2003; Gooley et al. 2010; Chang
et al. 2012), which may lead to the assumption that a
light pulse that induces melatonin suppression may also
B 100
−1
0
60
40
20
0
CTRL 0.2 h 1.0 h 1.0 h 6.5 h 6.5 h
IBL
IBL
Group 1
Suppression (%)
C
CTRL 0.2 h 1.0 h 1.0 h 6.5 h 6.5 h
IBL
IBL
Group 2
Group 1
Group 2
100
−4
75
−3
50
−2
25
−1
0
0
−25
Suppression
Phase Shift
−50
CTRL
Group 1
1
2
Delay
−2
80
Phase shift (h)
−3
Advance
−4
Suppression (%)
Phase delay (h)
A
J Physiol 596.11
Group 2
RANK ORDER OF PHASE RESETTING
Figure 2. Relationship between melatonin suppression and circadian phase resetting
A and B, relationship between light pattern and (A) phase shift, assessed as the difference in circadian phase over
3 circadian cycles between the initial and final constant routines, and (B) melatonin suppression, assessed as the
change in AUC over the interval within which the light exposure took place (0.2, 1.0 or 6.5 h, as appropriate) and
the corresponding interval which occurred 24 h earlier. For IBL exposures, the entire exposure interval, including
the intervening intervals of dim light, was used (e.g. for the 0.2 h condition, AUC was calculated over the entire
1 h interval). Three groups are apparent based on the rank order of the magnitude of phase shift: CTRL < Group
1 (0.2 h, 1.0 h IBL and 1.0 h continuous) < Group 2 (6.5 h IBL and 6.5 h continuous). C, individually matched
phase shift and melatonin suppression values plotted for all individuals across the three groups as generated in A;
data are shown as means ± SEM.
C 2018 The Authors. The Journal of Physiology
C 2018 The Physiological Society
Decoupled melatonin suppression and phase resetting
100
C
80
60
40
20
0
140
0
B
25
50
75
100
0
D
25
120
50
G
I
% of Mean plasma melatonin
100
E
Less melatonin
A
2153
75
*
80
100
0
F
140
25
120
50
100
75
*
80
500
400
100
0
H
25
300
200
100
0
200
160
120
% Change in plasma melatonin
J Physiol 596.11
50
75
100
0
J
25
50
80
40
0
75
*
4
6
8
10
12
100
RR1 2 3 45 6
Time from wake (h)
Pulse no.
Melatonin - % of Mean
Melatonin - % Change
Light Pulse
Figure 3. Melatonin suppression and circadian phase resetting are not functionally coupled
Melatonin levels are expressed as a percentage of the mean during the 8 h interval from 22.00 to 06.00 h shown
by hatched bars in Fig. 1A and B, which contains the light exposure for each individual, and then compared across
individuals in the 1.0 h continuous (A), 0.2 h continuous (C), 1.0 h IBL (E), 6.5 h continuous (G) and 6.5 h IBL (I)
conditions (mean ± SEM). Values were interpolated at the start and end of the light pulse and in 0.5 intervals
starting 1 h before the pulse and ending 1.0 h after the pulse. The light pulse is shown as the grey line. The
corresponding change in melatonin levels from each pulse calculated as the percentage change between the start
and end of the light pulse is shown as black bars in B, D, F, H and J (mean ± SEM; ∗ signifies P < 0.05). This method
of calculating melatonin suppression is different from the AUC-based method used for calculating suppression
in Fig. 2, and allows for calculating the change in melatonin from the start to the end of each intermittent and
continuous light pulse. The corresponding continuous conditions used as a comparative reference (denoted as
Pulse no. R) are re-plotted as grey bars in the grey hatched area. For the 1.0 h IBL condition, the comparator
conditions include the 1.0 h and the 12 min continuous conditions. For the 6.5 h IBL condition, the comparator
condition includes the 6.5 h continuous condition.
C 2018 The Authors. The Journal of Physiology
C 2018 The Physiological Society
2154
S. A. Rahman and others
induce circadian phase shifts, and that the responses are
proportional such that melatonin suppression can be
used as a proxy measure for phase resetting (Brainard
et al. 2001; Thapan et al. 2001; Najjar et al. 2014; Rea
et al. 2005, 2010). Poor association between melatonin
suppression and circadian phase resetting has been previously reported, but a functional decoupling between
the two responses was not elucidated (Zeitzer et al. 1997,
2011). Results from the current study show, however, that
such close functional coupling does not exist, suggesting
that any relationship is associative and inconsistent rather
than causative. We present evidence that a similar degree
of melatonin suppression occurs each and every time a
light pulse is applied during the biological night (Fig. 3J),
which is not matched by a similar phase shifting response
to each pulse, based on the overall phase shifting response
(Fig. 2A). The findings suggest that the phase shifting
capacity of the SCN is diminished after an initial exposure
and that this decreased response lasts for a specific interval
during which additional photic stimuli have marginally
additional phase resetting effects. Importantly, however,
light continues to induce melatonin suppression during
that interval. We hypothesize that the SCN appears to be
able to relay the timing and ‘strength’ of the intermittent
light exposures to the pineal while being insensitive to
cumulative phase shifting effects of all pulses of light, most
likely due to the topographical division of retinorecipient
and circadian rhythm generation areas within the SCN.
Notwithstanding the different temporal patterns of these
two responses to light, these results further caution
against using melatonin suppression as an indirect
measure of the circadian resetting strength of a photic
stimulus.
If melatonin suppression and phase resetting were
functionally coupled, then the 0.2 h continuous, 1.0 h
IBL and the 6.5 h IBL conditions could be expected to
cause similar phase shifts since they induce similar levels of
melatonin suppression. In contrast, the incongruent phase
shift and melatonin suppression responses observed in the
current study support the lack of a functional coupling
between melatonin suppression and phase resetting. The
results, therefore, show a divergence from the expected
relationship between melatonin suppression and phase
resetting, suggesting that melatonin suppression is not a
reliable proxy for phase resetting.
The IBL data could be interpreted as indicating that
most of the phase resetting effect is induced by the first
few light pulses with subsequent light pulses adding less.
The data do not support this hypothesis, however. First,
the single 12 min (0.2 h) pulse induced the same phase shift
of 1 h as the 60 min pulse. Second, when two 12 min
pulses were administered within a 1 h exposure window,
the same phase shift of 1 h was observed, suggesting
that the second 12 min pulse did not induce additional
phase resetting and that only the first pulse actively shifted
J Physiol 596.11
the clock, despite both pulses inducing similar melatonin
suppression.
Since melatonin levels returned to pre-light exposure
levels after 60 min, it could be hypothesized that SCN
sensitivity to the phase resetting effects of light recovers
within 60 min. If this were the case, the 6.5 h IBL exposure
should induce a 6 h phase shift from the sum of six
15 min pulses administered with 60 min of recovery interval between pulses, given that one 15 min pulse induces
a 1 h shift. The data show that only half of this phase
resetting response is achieved following six 15 min pulses
and therefore the pulses are not interpreted as six equal
exposures by the SCN in terms of phase shifting. The
3 h shifts obtained are equivalent to approximately three
individual 12 min shifts, consistent with the hypothesis
that every other pulse induces a phase shift (pulses 1,
3 and 5) with the ‘even’ pulses contributing minimally
to phase shifting, despite causing an equal amount of
melatonin suppression each time. If this is the case, then
the time interval during which there is reduced SCN phase
resetting capacity starts as early as 15 min after the pulse,
and remains low for 60 to 135 min after the pulse.
An alternate hypothesis is that the first three of the six
pulses induced the entire phase resetting response and
the latter three pulses were ineffective. In this case the
interval of reduced sensitivity would be 36–120 min.
Data from mechanistic studies in animal models do
not support this conclusion, however. Multiple lines of
evidence in animal models show that the SCN resolves
individual pulses and returns to baseline between pulses.
Light exposure-induced c-Fos expression in the SCN peaks
30 min after the pulse and is undetectable 60 min after
the pulse (Butcher et al. 2003). Phosphorylated cAMP
response element-binding protein (pCREB) induction
peaks 20 min after a pulse and returns to baseline within
60 min (Butcher et al. 2003). Light pulses delivered 60 and
120 min after the first 15 min pulse induce a second and
third round of c-Fos and pCREB expression in the SCN
(Butcher et al. 2003). Importantly, even though the SCN
can resolve individual pulses in 60 min intervals, additive
phase resetting by multiple pulses may occur in 120 min
intervals. In mice, two 15 min pulses administered 60 min
apart did not induce significantly greater phase shift than
the first pulse alone whereas when delivered 120 min apart,
the two pulses cause significantly greater phase shift than
the shift by one pulse alone (Best et al. 1999), consistent
with our data.
The molecular mechanisms of phase resetting explored
in animal models, in conjunction with the current results,
support longer duration (>15 min) pulses being resolved
into discrete pulses. For example, exposure to a 120 min
continuous pulse leads to extracellular-signal-regulated
kinase (ERK) activation in the SCN, which is necessary
and influences the magnitude of phase resetting (Butcher
et al. 2002, 2003; Dziema et al. 2003) within 15 min of the
C 2018 The Authors. The Journal of Physiology
C 2018 The Physiological Society
J Physiol 596.11
Decoupled melatonin suppression and phase resetting
pulse. ERK activation then decreases to near basal levels
(30% of the initial induction) by 60 min and remains at
basal levels until the end of the light pulse (Butcher et al.
2003). In contrast, the G-protein Ras, which is upstream
of ERK in the mitogen-activated protein kinase pathway,
remains in an activated state throughout the 120 min
light pulse. Taken together, these results suggest that the
SCN is responsive to a continuous light pulse but that
the resetting capacity becomes minimal 15 min after the
start of the pulse. This hypothesis is further supported
by the sustained firing activity of SCN neurons to light
pulses longer than 15 min and the discrete sustained
response of SCN neuronal firing to a train of intermittent
15 min pulses but a lack of an additive increase in phase
resetting in response to multiple pulses (Meijer et al.
1992). This ability of the SCN to continue receiving the
photic stimulus likely explains the sustained melatonin
suppression observed with the 1.0 h and 6.5 h continuous
conditions. In addition, all six pulses within the 6.5 h IBL
induced the same level of melatonin suppression.
Additionally, exogenous melatonin inhibits SCN firing
(Mason & Brooks, 1988; Zhou et al. 2000), a marker of
SCN activation in response to light and monotonically
related to the magnitude of phase resetting (Meijer et al.
1992). Therefore, there is a likely impact of melatonin on
the differential phase resetting observed under continuous
and intermittent light exposure patterns. Robust and
sustained melatonin suppression during continuous
bright light exposure may have allowed the SCN to fully
phase shift in response to light, whereas high melatonin
levels (low melatonin suppression) during intermittent
bright light exposure may have partially inhibited SCN
phase shifting in response to light. This may explain the
slightly reduced phase resetting, albeit not significantly,
observed in the 6.5 h IBL condition compared to the
6.5 h continuous light exposure patterns, but requires
additional studies to be confirmed.
Overall, our data suggest that melatonin suppression
and phase resetting are sometimes correlated, but
ultimately are generated by distinct processes downstream of retinal signal transduction (Paul et al. 2003).
A limitation of the current study was the limited
sample size in each light exposure condition, however.
Additionally, the blood sampling frequency was different
between protocols, which was accounted for post hoc by
linearly interpolating melatonin values. Future studies
with additional participants in the IBL conditions, and
the same sampling frequency for melatonin are necessary
to confirm the decoupled phase resetting and melatonin
suppression under intermittent light exposure conditions.
We hypothesize that the photic signal received at the
retinorecipient core of the SCN is transmitted to the
pineal, given that the light pulse induces immediate and
continuous melatonin suppression for the entire light
exposure duration, each and every time a light pulse is
C 2018 The Authors. The Journal of Physiology
C 2018 The Physiological Society
2155
given between SynOn and SynOff. In contrast, the same
photic stimulus induces an initial phase resetting response
within the SCN, although if the following pulse occurs
within 36–135 min, the SCN appears to be considerably
less sensitive to additional phase resetting. After the interval of reduced phase shifting capacity ends, the system
appears to respond fully to a second phase shifting pulse.
The ability to reliably predict and estimate phase
resetting capacity of a given photic stimulus from the
melatonin suppression induced by the same stimulus
appears attractive in applied settings primarily due to
less resource requirements for assessing melatonin suppression. Our work shows clearly, however, that melatonin
suppression is not a reliable surrogate for phase
resetting, often the major chronobiotic and chronotherapeutic outcome of interest. Therefore, melatonin
suppression is a reliable marker of the SCN’s ability to
detect light and transduce that information to the pineal
gland but cannot be used as a surrogate measure of the
stimulus strength that induces circadian phase resetting in
humans.
References
Best JD, Maywood ES, Smith KL & Hastings MH (1999). Rapid
resetting of the mammalian circadian clock. J Neurosci 19,
828–835.
Boivin DB, Duffy JF, Kronauer RE & Czeisler CA (1996).
Dose-response relationships for resetting of human
circadian clock by light. Nature 379, 540–542.
Brainard GC, Hanifin JP, Greeson JM, Byrne B, Glickman G,
Gerner E & Rollag MD (2001). Action spectrum for
melatonin regulation in humans: Evidence for a novel
circadian photoreceptor. J Neurosci 21, 6405–6412.
Butcher GQ, Doner J, Dziema H, Collamore M, Burgoon PW
& Obrietan K (2002). The p42/44 mitogen-activated protein
kinase pathway couples photic input to circadian clock
entrainment. J Biol Chem 277, 29519–29525.
Butcher GQ, Lee B & Obrietan K (2003). Temporal regulation
of light-induced extracellular signal-regulated kinase
activation in the suprachiasmatic nucleus. J Neurophysiol 90,
3854–3863.
Chang AM, Santhi N, St Hilaire M, Gronfier C, Bradstreet DS,
Duffy JF, Lockley SW, Kronauer RE & Czeisler CA (2012).
Human responses to bright light of different durations.
J Physiol 590, 3103–3112.
Czeisler CA, Kronauer RE, Allan JS, Duffy JF, Jewett ME,
Brown EN & Ronda JM (1989). Bright light induction of
strong (type 0) resetting of the human circadian pacemaker.
Science 244, 1328–1333.
Duffy JF & Dijk DJ (2002). Getting through to circadian
oscillators: Why use constant routines? J Biol Rhythms 17,
4–13.
Dziema H, Oatis B, Butcher GQ, Yates R, Hoyt KR & Obrietan
K (2003). The ERK/MAP kinase pathway couples light to
immediate-early gene expression in the suprachiasmatic
nucleus. Eur J Neurosci 17, 1617–1627.
2156
S. A. Rahman and others
Gooley JJ, Lu J, Chou TC, Scammell TE & Saper CB (2001).
Melanopsin in cells of origin of the retinohypothalamic
tract. Nat Neurosci 4, 1165.
Gooley JJ, Rajaratnam SMW, Brainard GC, Kronauer RE,
Czeisler CA & Lockley SW (2010). Spectral responses of the
human circadian system depend on the irradiance and
duration of exposure to light. Sci Transl Med 2, 31ra33.
Gronfier C, Wright KP Jr, Kronauer RE, Jewett ME & Czeisler
CA (2004). Efficacy of a single sequence of intermittent
bright light pulses for delaying circadian phase in humans.
Am J Physiol Endocrinol Metab 287, E174–E181.
Jewett ME, Rimmer DW, Duffy JF, Klerman EB, Kronauer RE
& Czeisler CA (1997). Human circadian pacemaker is
sensitive to light throughout subjective day without evidence
of transients. Am J Physiol Regul Integr Comp Physiol 273,
R1800–R1809.
Johnson RF, Moore RY & Morin LP (1998). Loss of
entrainment and anatomical plasticity after lesions
of the hamster retinohypothalamic tract. Brain Res 460,
293–313.
Khalsa SBS, Jewett ME, Cajochen C & Czeisler CA (2003). A
phase response curve to single bright light pulses in human
subjects. J Physiol 549, 945–952.
Klein DC & Moore RY (1979). Pineal N-acetyltransferase and
hydroxyindole-O-methyltransferase: Control by the
retinohypothalamic tract and the suprachiasmatic nucleus.
Brain Res 174, 245–262.
Klein DC, Weller JL & Moore RY (1971). Melatonin
metabolism: neural regulation of pineal serotonin: acetyl
coenzyme A N-acetyltransferase activity. Proc Natl Acad Sci
USA 68, 3107–3110.
Lewy AJ, Wehr TA, Goodwin FK, Newsome DA & Markey SP
(1980). Light suppresses melatonin secretion in humans.
Science 210, 1267–1269.
Lockley SW, Brainard GC & Czeisler CA (2003). High
sensitivity of the human circadian melatonin rhythm to
resetting by short wavelength light. J Clin Endocrinol Metab
88, 4502–4505.
Lucas RJ, Peirson SN, Berson DM, Brown TM, Cooper HM,
Czeisler CA, Figueiro MG, Gamlin PD, Lockley SW,
O’Hagan JB, Price LL, Provencio I, Skene DJ & Brainard GC
(2014). Measuring and using light in the melanopsin age.
Trends Neurosci 37, 1–9.
Mason R & Brooks A (1988). The electrophysiological effects of
melatonin and a putative melatonin antagonist
(N-acetyltryptamine) on rat suprachiasmatic neurones in
vitro. Neurosci Lett 95, 296–301.
Meijer JH, Rusak B & Ganshirt G (1992). The relation between
light-induced discharge in the suprachiasmatic nucleus and
phase shifts of hamster circadian rhythms. Brain Res 598,
257–263.
Meijer JH, Watanabe K, Schaap J, Albus H & Detari L (1998).
Light responsiveness of the suprachiasmatic nucleus:
long-term multiunit and single-unit recordings in freely
moving rats. J Neurosci 18, 9078–9087.
Mills JN, Minors DS & Waterhouse JM (1978). Adaptation to
abrupt time shifts of the oscillator[s] controlling human
circadian rhythms. J Physiol 285, 455–470.
Moore RY (1978). The innervation of the mammalian pineal
gland. Biology 4, 1–29.
J Physiol 596.11
Najjar RP, Chiquet C, Teikari P, Cornut PL, Claustrat B, Denis
P, Cooper HM & Gronfier C (2014). Aging of non-visual
spectral sensitivity to light in humans: compensatory
mechanisms? PLoS One 23, e85837.
Paul KN, Fukuhara C, Tosini G & Albers HE (2003).
Transduction of light in the suprachiasmatic nucleus:
evidence for two different neurochemical cascades regulating
the levels of Per1 mRNA and pineal melatonin. Neuroscience
119, 137–144.
Rea MS, Figueiro MG, Bierman A & Bullough JD (2010).
Circadian light. J Circadian Rhythms 8, 2.
Rea MS, Figueiro MG, Bullough JD & Bierman A (2005). A
model of phototransduction by the human circadian system.
Brain Res Brain Res Rev 50, 213–228.
St Hilaire MA, Gooley JJ, Khalsa SB, Kronauer RE, Czeisler CA
& Lockley SW (2012). Human phase response curve to a 1 h
pulse of bright white light. J Physiol 590, 3035–3045.
St Hilaire MA, Gronfier C, Zeitzer JM & Klerman EB (2007). A
physiologically-based mathematical model of melatonin
including ocular light suppression and interactions with the
circadian pacemaker. J Pineal Res 43, 294–304.
Thapan K, Arendt J & Skene DJ (2001). An action spectrum for
melatonin suppression: Evidence for a novel non-rod, noncone photoreceptor system in humans. J Physiol 535,
261–267.
Vaughan GM (1993). New sensitive serum melatonin
radioimmunoassay employing the Kennaway G280
antibody: Syrian hamster morning adrenergic response.
J Pineal Res 15, 88–103.
Wright HR & Lack LC (2001). Effect of light wavelength on
suppression and phase delay of the melatonin rhythm.
Chronobiol Int 18, 801–808.
Zeitzer JM, Kronauer RE & Czeisler CA (1997). Photopic
transduction implicated in human circadian entrainment.
Neurosci Lett 5, 135–138.
Zeitzer JM, Dijk DJ, Kronauer RE, Brown EN & Czeisler CA
(2000). Sensitivity of the human circadian pacemaker to
nocturnal light: Melatonin phase resetting and suppression.
J Physiol 526, 695–702.
Zeitzer JM, Ruby NF, Fisicaro RA & Heller HC (2011).
Response of the human circadian system to millisecond
flashes of light. PLoS One 6, e22078.
Zhou XJ, Jiang XH, Yu GD & Yin QZ (2000). Modulation of
circadian rhythm of discharges of suprachiasmatic nucleus
neurons in rat hypothalamic slices by melatonin. Sheng Li
Xue Bao 52, 215–219.
Additional information
Competing interests
Competing interests are reported for the last two years. A.M.C.,
N.S. and C.G. have no conflicts of interest to declare.
S.A.R. holds a patent for prevention of circadian rhythm
disruption by using optical filters and improving sleep
performance in subject exposed to light at night; he owns equity
in Melcort Inc.; he is a co-investigator on studies sponsored by
Biological Illuminations, LLC and Vanda Pharmaceuticals Inc.
He has provided paid consulting services to Sultan & Knight Ltd
t/a Circadia.
C 2018 The Authors. The Journal of Physiology
C 2018 The Physiological Society
J Physiol 596.11
Decoupled melatonin suppression and phase resetting
M.S.H. has provided paid consulting services to The
MathWorks, Inc. (Natick, MA, USA) and the CRC for Alertness,
Safety, and Productivity (Melbourne, Australia).
C.A.C. reports having received consulting fees from or
served as a paid member of scientific advisory boards for:
Bose Corporation; Boston Celtics; Boston Red Sox; Columbia
River Bar Pilots; Institute of Digital Media and Child
Development; Klarman Family Foundation; Novartis; Samsung
Electronics; Quest Diagnostics, Inc.; Vanda Pharmaceuticals
and V-Watch/PPRS. He has also received education/research
support from Cephalon Inc., Mary Ann & Stanley Snider via
Combined Jewish Philanthropies, Optum, Philips Respironics,
Inc., ResMed Foundation, San Francisco Bar Pilots, Schneider
Inc., and Sysco. He has received lecture fees from American
Academy of Sleep Medicine (AADSM), CurtCo Media Labs
LLC, Global Council on Brain Health/AARP, Hawaii Sleep
Health and Wellness Foundation, Harvard School of Public
Health, Maryland Sleep Society, National Sleep Foundation,
University of Michigan, University of Washington, and Zurich
Insurance Company, Ltd. The Sleep and Health Education
Program of the Harvard Medical School Division of Sleep
Medicine (which C.A.C. directs) has received Educational
Grant funding from Cephalon, Inc., Jazz Pharmaceuticals,
Takeda Pharmaceuticals, Teva Pharmaceuticals Industries Ltd,
Sanofi-Aventis, Inc., Sepracor, Inc. and Wake Up Narcolepsy.
He is the incumbent of an endowed professorship provided
to Harvard University by Cephalon, Inc. and holds a number
of process patents in the field of sleep/circadian rhythms (e.g.
photic resetting of the human circadian pacemaker). Since 1985,
he has also served as an expert on various legal and technical
cases related to sleep and/or circadian rhythms including those
involving the following commercial entities: Bombardier, Inc.;
Continental Airlines; FedEx; Greyhound; and United Parcel
Service (UPS). He owns or owned an equity interest in Somnus
Therapeutics, Inc., and Vanda Pharmaceuticals. He received
royalties from McGraw Hill, Houghton Mifflin Harcourt and
Koninklijke Philips Electronics, N.V. for the Actiwatch-2 and
Actiwatch-Spectrum devices. His interests were reviewed and
managed by Brigham and Women’s Hospital and Partners
HealthCare in accordance with their conflict of interest policies.
E.B.K. reports non-university income from consulting for a
law firm on a transportation-related case.
S.W.L. reports receiving consulting fees from Atlanta
Falcons, Atlanta Hawks, Carbon Limiting Technologies Ltd
on behalf of PhotoStar LED, Perceptive Advisors, PlanLED,
Serrado Capital, Slingshot Insights, and has ongoing consulting
contracts with Akili Interactive, Consumer Sleep Solutions,
Delos Living LLC, Environmental Light Sciences LLC, Focal
Point LLC, Headwaters Inc., Hintsa Performance AG, Light
Cognitive, Mental Workout, OpTerra Energy Services Inc.,
Pegasus Capital Advisors LP, Wyle Integrated Science and
Engineering. He has received unrestricted equipment gifts from
Biological Illuminations LLC; Bionetics Corporation; and F.
Lux Software LLC; royalties from Oxford University Press;
honoraria plus travel, accommodation or meals for invited seminars, conference presentations or teaching from Estee Lauder,
Informa Exhibitions, and Teague; travel, accommodation and/or
C 2018 The Authors. The Journal of Physiology
C 2018 The Physiological Society
2157
meals only (no honoraria) for invited seminars, conference
presentations or teaching from Hintsa Performance AG,
Lightfair and USGBC; ongoing investigator-initiated research
grants from Biological Illumination LLC and F. Lux Software
LLC; has completed service agreements with Rio Tinto Iron
Ore and Vanda Pharmaceuticals Inc.; and has completed
three sponsor-initiated clinical research contracts with Vanda
Pharmaceuticals Inc. He also holds a process patent for the
use of short-wavelength light for resetting the human circadian
pacemaker and improving alertness and performance which is
assigned to the Brigham and Women’s Hospital per Hospital
policy. He has also served as a paid expert witness in two cases
related to sleep, circadian rhythms, work hours and/or light. He
also serves as a Program Leader in the Cooperative Research
Centre for Alertness, Safety and Productivity.
Author contributions
This work was completed at the Intensive Physiological
Monitoring Unit at the Centre for Clinical Investigation at
Brigham and Women’s Hospital, Boston, MA, USA. Authors
responsible for conception or design of the work: S.A.R., M.S.H.,
C.G., A.M.C., N.S., C.A.C., E.B.K. and S.W.L.; acquisition,
analysis or interpretation of data for the work: S.A.R., M.S.H.,
C.G., A.M.C., N.S., C.A.C., E.B.K. and S.W.L.; drafting the
work or revising it critically for important intellectual content:
S.A.R., M.S.H., C.G., A.M.C., N.S., C.A.C., E.B.K. and S.W.L. All
authors approved the final version of the manuscript, agree to be
accountable for all aspects of the work in ensuring that questions
related to the accuracy or integrity of any part of the work are
appropriately investigated and resolved. All persons designated
as authors qualify for authorship, and all those who qualify for
authorship are listed.
Funding
This work was supported by NASA NAG 5-3952,
NSBRI HFP02801, HFP02802, and NIH P01-AG09975,
R01-HL114088 (E.B.K.), RC2-HL101340-0 (E.B.K., S.W.L.,
S.A.R.), K02-HD045459 (E.B.K.), K24-HL105664 (E.B.K.),
T32-HL07901 (M.S.H., S.A.R.), HL094654 (C.A.C.). The project
described was supported by grants from the National Centre
for Research Resources to the General Clinical Research Centre
GCRC-M01-RR02635, NIH Grant Number 1UL1 TR001102-01,
8UL1TR000170-05, UL1 RR 025758, Harvard Clinical and
Translational Science Centre, from the National Centre for
Advancing Translational Science. The content is solely the
responsibility of the authors and does not necessarily represent
the official views of the National Centre for Research Resources,
the National Centre for Advancing Translational Science or the
National Institutes of Health.
Acknowledgements
We thank the research staff and research participants at the
Division of Sleep Medicine, BWH; and the technical, dietary,
nursing and medical staff at the Centre for Clinical Investigation
at the Brigham and Women’s Hospital.