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Sleep Science 2010 v.3, n. 1, p.1-62, Jan/Mar 2010
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©2010 – Sleep Science
Contents
Quarterly
Sleep Science 2010 v.3, n. 1, p.1-62, Jan/Mar 2010
V
EDITORIALS
ORIGINAL ARTICLES
1
Work start time and chronotype of indoor and outdoor daytime workers
Horários de início de trabalho e cronotipo de trabalhadores diurnos de ambientes internos e
externos
Flávio Back, Claudia Roberta de Castro Moreno, Fernando Mazzilli Louzada, Luis Menna-Barreto
7
Morningness/eveningness chronobiological assessment of Dental students: chronotype
Avaliação de matutinidade/vespertinidade dos estudantes de Odontologia: cronótipo
Milva Maria Figueiredo De Martino, Karla Figueiredo De Martino Pasetti, César Bataglion, Maria Filomena Ceolim
11
Environmental and organizational conditions for napping during night work:
a qualitative study among Nursing professionals
Condições ambientais e organizacionais dos cochilos durante o trabalho noturno: um estudo
qualitativo entre profissionais de Enfermagem
Aline Silva-Costa, Milena Maria de Araújo, Roberta Nagai, Frida Marina Fischer
16
Onset and stability of melatonin treatment effect in childhood sleep onset insomnia
Início e estabilidade dos efeitos do tratamento com melatonina na insônia de início de sono
infantil
Ingeborg M. van Geijlswijk, Robert Didden, Kristiaan B. van der Heijden, Marcel G. Smits, Jan F. van Leeuwe
22
Human period-3 gene involvement in diurnal preference among Argentinean bipolar
disorders patients
O envolvimento do gene humano período-3 na preferência diurna de uma população argentina
de pacientes com transtornos bipolares
Leandro Pablo Casiraghi, Diego Martino, Eliana Marengo, Ana Igoa, Ezequiel Ais, Sergio Strejilevich, Diego Andrés Golombek
27
Anticipatory behavioral rhythm to scheduled glucose availability in rats
Ritmo comportamental antecipatório à disponibilidade programada de glicose em ratos
Breno Tercio Santos Carneiro, Fabiano Santos Fortes, John Fontenele Araujo
32
Effects of chronobiology on prostate cancer cells growth in vivo
Efeitos da cronobiologia no crescimento de células cancerígenas da próstata in vivo
Abraham Haim, Adina Yukler, Orna Harel, Hagit Schwimmer, Fuad Fares
36
The presence of neuronal-specific nuclear protein (NeuN) in the circadian timing
system of the capuchin monkey (Cebus apella)
A presença da proteína nuclear específica neuronal (NeuN) no sistema de temporização
circadiano do macaco capuchinho (Cebus apella)
Rayane Bartira Silva do Nascimento, Janaína Siqueira Borda, Rovena Clara Galvão Januário Engelberth, Raysa Oliveira de
Medeiros, Renata Frazão, Luciana Pinato, André Luiz Bezerra de Pontes, Expedito Silva do Nascimento Jr, Maria Inês Nogueira,
Roelf Justino Cruz-Rizzolo, Miriam Stela Maris de Oliveira Costa, Jeferson de Souza Cavalcante
40
Mathematical model of the interaction between the dorsal and ventral regions of the
suprachiasmatic nucleus of rats
Modelo matemático da interação das regiões dorsal e ventral do núcleo supraquiasmático de ratos
Bruno da Silva Brandão Gonçalves, Breno Tercio Santos Carneiro, Crhistiane Andressa da Silva, Diego Alexandre da Cunha
Fernandes, Fabiano Santos Fortes, João Miguel Gonçalves Ribeiro, Rafaela Cobuci Cerqueira, Sergio Arthuro Mota Rolim,
John Fontenele Araújo
45
Evening chronotypes experience poor sleep quality when taking classes with early
starting times
Estudantes com cronótipo vespertino apresentam pior qualidade do sono quando as aulas
iniciam mais cedo
Ádison Mitre Alves de Lima, Gabriela Carminhola Gomes Varela, Hosana Aparecida da Costa Silveira,
Renata Davin Gomes Parente, John Fontenele Araujo
49
Masking effect in rats bearing partial lesions of the suprachiasmatic nucleus
Efeito máscara em ratos apresentando lesões parciais do núcleo supraquiasmático
Manuel Ángeles-Castellanos, Jorge M. Amaya, Ruud M. Buijs, Carolina Escobar
SHORT COMMUNICATION
53
Effect of brief constant darkness and illumination on mitochondrial respiratory
control of the pineal gland, Harderian gland, spleen and thymus of adult rat
Efeitos da escuridão e da luminosidade breve e constante no controle da respiração
mitocondrial das glândulas pineal, Harderiana, baço e timo
Oscar Kurt Bitzer-Quintero, Airam Jenny Dávalos Marín, Fermín Paul Pacheco-Moises, Erandis Dheni Torres-Sánchez and
Genaro Gabriel Ortíz
CASE REPORT
56
Circadian variations in the capacity to adjust behavior to environmental changes
Variações circadianas na capacidade de ajuste comportamental em face de alterações
ambientais
Aída García, Candelaria Ramírez, Pablo Valdéz
61
AUTHORS INSTRUCTIONS
Sleep
Science
EDITORIAL
São Paulo, February 4th, 2010.
In 2010 the Sleep Science Journal enters the third year of scientific divulging in the fields
of Chronobiology and Sleep.
In the last year, four issues were published, totalizing 21 articles and a special issue for
the International Congress of Chronobiology that took place in Natal (RN, Brazil) on October 2009.
These issues were distributed for free in Brazil and abroad for the members of Brazilian
Sleep Society, representatives of the Latin American Federation of Sleep Societies, researchers
and professionals of the field.
We have been receiving articles on unpublished subjects of significant relevance from authors from Brazil and other parts of the world, including Argentine, USA, Israel and Mexico.
Our goal for 2010 is indexing the Sleep Science Journal on an international database,
and we count on all readers’ collaboration through the submission of papers and mention of
published articles.
Sleep Science is the only journal with a representative number of scientific articles in the
field of Chronobiology and Sleep of Latin America, and it has been adapting to editorial patterns in compliance with international standards.
All articles are published in English, which increases its reach and visibility.
We appreciate the support of our editorial staff and of our readers in making real this
project that was idealized by all professionals of the Sleep Science journal.
Best regards,
Dr. Lia Rita Azeredo Bittencourt
Editor in chief
Sleep Sci. 2010;3(1):v
Sleep
Science
EDITORIAL
Nowadays, there has been an evident increase in research related to the basic Biology and
health fields, which include chronobiologic tools and knowledge. Treatments for seasonal
affective disorders, eating disorders, nonseasonal depression, among other health problems,
have been delivered by means of phototherapy, prescription of melatonin and many other
medications, all based on chronobiologic concepts.
Though it is known in many countries, only in the 1980s the Chronobiology became
subject of study in Brazil. Since then, this new range of knowledge has been theme of
research on Biology academic ambient. Researchers of Health have started to use chronobiologic means to comprehend and interpret physiological events that vary regularly
throughout time.
Besides that, the advances of science in this area have led to the recognition that there is
a control of circadian rhythm in each cell, concerning the molecular level. This knowledge
allows us to better understand the synchronizing processes of biological rhythms. However,
if on the one hand there is a relevant contribution for the understanding of circadian timing
system mechanisms, on the other hand, the complexity of this system is evidenced, revealing
that there is much to do on this subject.
In this special issue, the Sleep Science Journal assumes with competence its role of divulging Chronobiology and publishes the main articles presented at the X Latin American
Symposium on Chronobiology, which happened on 2009, in Natal (RN, Brazil).
Claudia Moreno
Associate Editor
Sleep Sci. 2010;3(1):��
vi
Sleep
Science
ORIGINAL ARTICLE
Work start time and chronotype of indoor
and outdoor daytime workers
Horários de início de trabalho e cronotipo de trabalhadores
diurnos de ambientes internos e externos
Flávio Back1, Claudia Roberta de Castro Moreno2, Fernando Mazzilli Louzada3,
Luis Menna-Barreto4
ABSTRACT
Background and objective: Natural light exposure has important
effects on the biological timing systems. One could suppose that
this exposure might promote a better adjustment between biological
rhythms and early working times among outdoor diurnal workers.
The aim of this study was to compare the morningness/eveningness
preferences and the relationship between actual and ideal timing
to work on diurnal workers exposed to different light conditions.
Methods: The study was conducted with two groups of workers
(n=49) living in a rural area and exposed to similar social conditions.
One group worked indoor (n=20, mean age 30.8 years (21-50);
standard deviation=9.8), and the other group worked outdoor
(n=29, mean age 30.8 years (17-50); standard deviation=10.0). The
workers filled out a morningness-eveningness questionnaire (MEQ).
A one-way ANOVA was carried out in order to compare MEQ
scores between the two groups of workers. Results: As expected,
Outdoor Environment Group (OEG) showed a higher average when
compared to Indoor Environment Group (IEG), which means a trend
to a morning preference (OEG: 58.4±7.9; IEG: 47.4±6.4), with a
significant difference (F=26.22; p<0.001). According to the reported
data related to working times, the OEG would like to postpone the
working time by 31 minutes, while the IEG would postpone by 96
minutes their actual working time (F=7.71; p<0.01). Conclusions:
The results of this study suggested that natural light exposure may
promote better adjustment to early working hours.
Keywords: Chronobiology phenomena; Work hours; Night work;
Sleep deprivation; Rural workers
RESUMO
Introdução e objetivo: A exposição à luz natural tem efeitos relevantes no sistema de temporização biológica. Pode-se supor que essa
exposição poderia promover um ajuste melhor entre os ritmos biológicos e os horários de início de trabalho entre trabalhadores diurnos
de ambientes externos. O objetivo deste estudo foi comparar a matutinidade/vespertinidade e a relação entre o horário de trabalho real e
o ideal em trabalhadores diurnos expostos a condições de iluminação
distintas. Métodos: O estudo foi conduzido com dois grupos de trabalhadores (n=49) que residiam em uma área rural e tinham condições
sociais similares. Um grupo trabalhava em ambiente interno (n=20,
idade média 30,8 anos (21-50); desvio padrão=9,8) e o outro grupo
trabalhava em ambiente externo (n=29, idade média 30,8 anos (1750); desvio padrão=10,0). Os trabalhadores preencheram um questionário de matutinidade/vespertinidade (MEQ). Foi realizada uma
ANOVA de um fator com o intuito de comparar os escores do MEQ
entre os dois grupos de trabalhadores. Resultados: Como esperado,
o Grupo do Ambiente Externo (GAE) apresentou média de escores
mais elevada que o Grupo do Ambiente Interno (GAI), o que significa uma tendência à matutinidade (GAE: 58,4±7,9; GAI; 47,4±6,4),
com significância estatística (F=26,22; p<0,001). De acordo com os
dados relatados em relação aos horários de trabalho, o GAE gostaria de
atrasar seu horário de trabalho em 31 minutos, em média, enquanto
que o GAI gostaria de atrasar em 96 minutos seu horário de trabalho
(F=7,71; p<0,01). Conclusões: Os resultados desse estudo sugerem
que a exposição à luz natural pode promover um ajuste melhor aos
horários de início de trabalho matutinos.
Descritores: Fenômenos cronobiológicos; Jornada de trabalho; Trabalho noturno; Privação do sono; Trabalhadores rurais
INTRODUCTION
The solar clock, that is, the alternation between day and night
has been considered as the main environmental event which
is able to synchronize the organism oscillators (1-3). Synchronization starts with the action of light on the ganglion cells of
the retina. The processing of such information by the central
nervous system allows entrainment, triggering internal syn-
Study carried out at Instituto de Ciências Biomédicas, Universidade de São Paulo – USP, São Paulo (SP), Brazil.
1
Instituto de Ciências Biomédicas, Universidade de São Paulo – USP, São Paulo (SP), Brazil.
2
Faculdade de Saúde Pública, Universidade de São Paulo – USP, São Paulo (SP), Brazil.
3
Setor de Ciências Biológicas, Universidade Federal do Paraná – UFPR, Curitiba (PR), Brazil.
4
Instituto de Ciências Biomédicas, Universidade de São Paulo – USP, São Paulo (SP), Brazil; Escola de Artes, Ciências e Humanidades, Universidade de São Paulo – USP,
São Paulo (SP), Brazil.
Corresponding author: Fernando Mazzilli Louzada – Department of Physiology, Universidade Federal do Paraná – Caixa Postal 19031 – CEP 81531-990 – Curitiba (PR),
Brazil – Tel.: (41) 3361-1552 – E-mail: [email protected]
Received: February 20, 2009; Accepted: March 31, 2010
Sleep Sci. 2010;3(1):1–6
2
Work start time and chronotype
chronization processes. In order to trigger such processes, the
central nervous system uses different output signals to spread
the circadian message throughout the brain and the body (4).
This process involves direct and indirect neuronal projections
and secretion of polypeptides (5,6). The internal synchronization process is based on multiple feedback mechanisms, resulting in a coordinated rhythmicity of the organism’s physiology and behavior. Detailing the mechanisms that involve
the synchronizing processes allows for the understanding of
their plasticity, as well as their limits.
Human species is diurnal. However, there are marked
individual differences concerning preferences for sleep and
wakefulness as well as other activities, which is a characteristic called “chronotype” originally described by Horne and
Östberg (7) in 1976. In the last decade, diurnal preferences
have been associated to clock-genes polymorphisms (8-10) and
to the endogenous circadian period. Morning-type individuals show a shorter intrinsic period when compared to
evening-type individuals (11). Differences between the phase
angles of entrainment have been reported, with the shorter
phase angles being related to evening-types (12,13). Wright
et al. (14) showed that the phase angle of entrainment may
be affected by the intrinsic period and by the duration and
intensity of light exposure.
There is room for the supposition that the reduction of
time exposure to natural light in contemporary society may
modify the phase angle of entrainment and affect morningness/eveningness preferences. Roenneberg et al. (15) evaluated
the chronotype distribution of 500 individuals and found a
positive correlation between time exposure to natural light
and advanced sleep phase. Goulet et al. (16) described patterns
of light exposure along 24 consecutive hours in individuals
with different diurnal preferences in a situation with minimal external constraints on their sleep schedules and found
that morning-type individuals are exposed to higher light
intensities in the morning when compared to evening-type
individuals. Recently, Roenneberg et al. (17) showed an association of longitude to sleep phase preferences. People living
in cities with early sunrise times showed a tendency for more
pronounced morningness. The authors suggest that the human circadian-timing system is predominantly entrained by
sun time rather than social time. Although the availability
of food can be stronger than light among most of animals,
with a genetic regulation as well, there is a need for further
studies to discuss it in the case of human beings (18).
Exposure to light has been used as a means to promote
adjustment of the worker’s biological rhythms (19,20). It has
also been demonstrated that short periods of exposure to
light may produce the same effects as long periods of exposure to light. For instance, there is a study that demonstrates that exposure to intermittent light (six 40-minSleep Sci. 2010;3(1):1–6
ute light pulses) produces an adjustment of the circadian
rhythmicity (21).
On an extended review article about the subject, Burgess et
al. (22) recommend exposure to medium intensity light and/or to
intermittent light during night work as a strategy to promote
adaptation to the work schedule. On the other hand, exposure
to solar light on the way back home after night work may prevent adaptation. Wearing sunglasses on the way back home has
also been recommended by many researchers (22,23).
Based on these findings, one could suppose that the natural
light exposure is capable of promoting a better adjustment
between biological rhythms and early working times among
outdoor diurnal workers, when compared to indoor workers.
The aim of this study was to compare the morningness/
eveningness preferences and the relationship between actual
and ideal timing to work on diurnal workers exposed to different light conditions.
METHODS
Subjects
The study was conducted with two groups of workers (n=49)
in a rural area in Paraná, Brazil, between June and October
2007 (average of annual temperature=22.1ºC; average of relative humidity=69.3%). One group worked indoor (n=20,
mean age 30.8 years (21-50); standard deviation (SD)=9.8)
and the other group worked outdoor (n=29, mean age 30.8
years (17-50); SD=10.0).
The Indoor Environment Group (IEG) was comprised by
bench lathe workers, electrical machine operators and one
desk-bound office worker. The working time of this group
was from 8 to 18h, from Monday to Friday, and Saturday
from 8 to 16h. Their commuting time ranged from 5 to
20 minutes walking or riding a motorcycle. The Outdoor
Environment Group (OEG) was comprised by agricultural
workers. This group worked from Monday to Friday (7 to
17h); their commuting time ranged from 30 to 60 minutes.
They used to walk for around 15 minutes to get a ride in
an opened truck body. These workers had been submitted
to natural bright light during working time for 11.7±10.6
years, while IEG had been exposed to artificial light during
working time for 1.8±1.2 (four months to five years).
Both groups lived at the same city (latitude: S 23º 5’;
longitude: W 52º 36’), with 2,000 inhabitants, and they had
similar sociocultural background. Workers taking drugs
that interfered in the sleep-wake cycle and those who reported sleep disturbances were excluded from the sample.
Light intensity measures
The light intensity in both workplaces was measured in the
morning (8h) and in the afternoon (15h) using a light meter
with a maximum of sensitivity of at 20,000 lux (THDL-
Back F, Moreno CRC, Louzada FM, Menna-Barreto L
Morningness-eveningness identification and questions
about working times
The workers filled out a Portuguese version of the Morningness-Eveningness Questionnaire (MEQ) (7) and questionnaires about their working times. A one-way ANOVA was
carried out in order to compare MEQ scores between the two
groups of workers.
A questionnaire on working times was answered by the
workers as well as questions about work span, commuting
time, time of starting and ending work. A question related to
workers’ preference for the ideal time to start working taking
into account their sleep quality was also included. Difference
between the ideal working time (according to their preferences)
and the actual working time was calculated.
Differences between the actual and ideal work time
were calculated and the averages were compared through
ANOVA.
For each group, linear regression analysis between age and
MEQ scores was carried out and a correlation coefficient was
obtained.
The study was approved by the ethical committee of
Universidade de São Paulo (USP).
According to the data obtained, the OEG would like to
postpone their working time by 31 minutes, while the IEG
Light intensities (lx)
Light exposure intensities
Time (hours)
Figure 1: Light exposure pattern of one subject per group within 24h
(measured each 4 min). Black circles represent an Outdoor Envirnoment
Group individual and open squares represent an Indoor Environment
Group individual. On the Y axis are shown the light intensity level, in lux,
logaritimic scale. Axis X represents time in hours.
Diurnal preference distribution
Number of observations
400®). A photodiode sensor was held at the researcher’s eyes
level to measure the light intensity at the workplace.
One subject of each group wore a wrist actigraph with
light sensor (Ambulatory Monitoring®) during 24 hours
of a workday in order to measure duration and intensity of
light exposure. The device had a light sensitivity ranging
from 0 to 3,995 lx. These results were used to illustrate the
light exposure pattern of the groups. Workers did not wear
goggles during the study period.
There was a gradual increase of the light intensity during
sunrise, which used to take around 70 minutes to reach the
limit of sensivity at 20,000 lux. During data collection, the
photoperiod varied: sunrise ocurred between 5h58 and 7h06
and sunset between 17h52 and 18h35.
Score
RESULTS
The IEG was exposed to a range of 50 to 500 lx during
most part of the day and the OEG was exposed up to at least
20,000 lx, measured by a portable light sensor, although the
detection limit of the wrist light sensor was 3,995 lx.
Examples of light exposure intensities of every hour on
two workers from each group have been plotted on Figure 1.
As expected, OEG showed a higher average of MEQ than
the IEG (OEG: 58.4±7.9; IEG: 47.4±6.4). A significant difference between groups was detected (F=26.22; p<0.001).
It was also observed that the chronotype distribution was
quite wide, with the extremes being far apart. The distribution of MEQ scores for each group is shown in Figure 2.
Figure 2: Distribution of morningness-eveningness questionnaire (MEQ)
scores for the two groups overlapped. Gray columns represent the Outdoor Environment Group (OEG), while black columns represent the Indoor Environment Group (IEG). On the axis Y are shown the number of subjects. On the
axis X are shown the MEQ scores. Higher morningness-eveningness scores are
associated with morning-like preferences, whereas lower scores are associated
with evening-like preferences.
would like to have it postponed by 96 minutes. In other
words, the OEG seems to be more satisfied with their working hours. Figure 3 shows the differences between actual and
ideal work time. A significant difference was detected between groups (F=7.71; p<0.01).
Sleep Sci. 2010;3(1):1–6
3
Work start time and chronotype
Time difference (min.)
Difference between actual and ideal work time
Group
Figure 3: Columns represent Outdoor Environment Group (OEG) and Indoor Environment Group (IEG). On the Y axis are displayed time difference
between actual and ideal working times. Open squares represent means and
bar error refers to the standard error of the mean.
Differences according to age were detected as well. Figure 4 shows the correlations between age and MEQ score for
both groups. Older workers showed higher MEQ score. A
positive significant correlation was found only on the OEG
(r=0.61; p=0.0004), IEG (r=0.28; p=0.21).
Correlations between age and Horne & Österg scores
Scores
4
Age
Figure 4: Correlation between age and morningness-eveningness questionnaire (MEQ) scores. Black circles represent Outdoor Environment Group
(OEG); open squares represent the Indoor Environment Group (IEG). On
the Y axis are shown the morningness-evemimgmess questionnaire scores.
On the X axis are plotted the age of the subjects.
DISCUSSION
The discussion about the deleterious effects upon alertness
due to early working times is not new in the literature. According to Tucker et al. (24), an early work starting time,
Sleep Sci. 2010;3(1):1–6
around 6h, for instance, might lead to reduced alertness
and is also associated with poor psychological and physical
health. The results of this study indicate that natural light
exposure may promote better adjustment to early working
hours. The workers exposed to natural light during working
time (OEG) were earlier types when compared to workers
from the indoor group. This fact could explain why the differences between ideal and actual working times are smaller
in this group, even taking into account that they started
work one hour earlier than those working indoor (IEG).
Several factors are related to the expression of morningness-eveningness preference. According to some studies,
morning type individuals are exposed to natural light earlier
when compared to evening types (15,16). Diurnal preference
changes with development. Adolescents tend to delay and
older adults advance their circadian rhythms (25-27). Genetic influences on these tendencies have been proposed (8-10).
Studies in rodents and humans suggest that differences in
duration and intensity of light exposure since birth also play
a significant role (28-32). In addition, recent studies show that
recent photic history modifies the responses of the circadian
system; melatonin secretion depends on previous exposure
to light (33-35). OEG subjects were submitted to natural light
during the day with levels up to 20,000 lx. It is reasonable
to suppose that their circadian system response to artificial
light at home is quite different when compared to the responses of IEG subjects.
The significant correlation between age and chronotype
in the OEG suggests that environmental factors may have a
role of increasing or modifying ontogenetic trends.
Morning tendencies in OEG subjects might be explained exclusively by the fact that they were exposed
to natural bright light predominantly during advanced
portions of the phase response curve. However, recently,
Scheer et al. (36) compared the intrinsic period of subjects submitted to different light/dark regimens. The results represent the first demonstration of the plasticity
of the human intrinsic period, which had already been
described in rodents by Pittendrigh and Daan (37). In the
interpretation of our data, effects of chronic exposure to
bright light on the intrinsic period plasticity should not
be ruled out.
The effects of artificial light exposure in worksites have
been documented as a means of promoting alertness during
night work or for promoting adjustment of workers’ biological rhythms (22,38). However, few studies have aimed to
investigate the effect of natural light exposure on workers.
Kaida et al. (39) showed, in a lab study, the positive effects
of natural light exposure at lunchtime on workers’ performance and alertness levels. Our results reinforce the idea
of natural light exposure as a means of promoting alertness
Back F, Moreno CRC, Louzada FM, Menna-Barreto L
and facilitating early awakening among day workers with an
early start to the day shift.
Limitations and future studies
The present study was conducted under limitations. The
limited number of workers who have used the actigraphs
with light sensor and the limitations of the light meter
sensitivities are some examples. Another limitation is the
fact that the two groups studied had different work starting
times. Also, data regarding sleep habits and diurnal sleepiness were not gathered. Thus, further investigation involving larger numbers of subjects is necessary to provide better insight into the discussion of the effects of natural light
exposure on morningness/eveningness characteristics as well
as on sleepiness and sleep, safety, health and working time
satisfaction.
ACKNOWLEDGMENTS
We acknowledge the support of Fundação de Amparo à
Pesquisa do Estado de São Paulo (Fapesp), grant number
05/57597-2.
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2. Khalsa SB, Jewett ME, Cajochen C, Czeisler CA. A phase response
curve to single bright light pulses in human subjects. J Physiol.
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3. Klerman EF, Duffy JF, Dijk DJ, Czeisler CA. Circadian phase resetting in older people by ocular bright light exposure. J Investig
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4. Brandstaetter R. Circadian lessons from peripheral clocks: is
the time of the mammalian pacemaker up? Proc Natl Acad Sci.
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5. Albrecht U. The mammalian circadian clock: a network of gene
expression. Front Biosci. 2004;9:48-55.
6. Saper CB, Scammel TE, Lu J. Hypothalamic regulation of sleep
and circadian rhythms. Nature. 2005;437(7063):1257-63.
7. Horne J, Östberg O. A self-assessment questionnaire to determine
morningess-eveningness in human circadian rhythms. Int J Chronobiol. 1976;4(2):97-110.
8. Katzenberg D, Young T, Finn L, Lin L, King DP, Takahashi JS,
et al. A CLOCK Polymorphism associated with human Diurnal
preference. Sleep. 1998;21(6):569-76.
9. Archer SN, Robilliard DL, Skene DJ, Smits M, Williams A, Arendt J, et al. A length Polymorphism in the circadian clock gene
Per3 is linked to delayed sleep phase syndrome and extreme diurnal preference. Sleep. 2003;26(4):413-5.
10.Pereira DS, Tufik S, Louzada FM, Benedito-Silva AA, Lopez AR,
Lemos NA, et al. Association of the length polymorphism in the
human Per3 gene with the delayed sleep-phase syndrome: does
latitude have an influence upon it? Sleep. 2005;28(1):29-32.
11.Duffy JF, Rimmer DW, Czeisler CA. Association of intrinsic circadian period with morningness-eveningness, usual wake time,
and circadian phase. Behav Neurosci. 2001;115(4):895- 9.
12.Duffy JF, Dijk DJ, Hall EF, Czeisler CA. Relationship of endogenous circadian melatonin and temperature rhythms to self-reported preference for morning or evening activity in young and
older people. J Investig Med. 1999;47(3):141-50.
13.Liu X, Uchiyama M, Shibui K, Kim K, Kudo Y, Tagaya H, et
al. Diurnal preference, sleep habits, circadian sleep propensity
and melatonin rhythm in healthy human subjects. Neurosci Lett.
2000;280(3):199-202.
14.Wright KPJr, Gronfier C, Duffy JF, Czeisler CA. Intrinsic period
and light intensity determine the phase relationship between melatonin and sleep in humans. J Biol Rhythms. 2005;20(2):168-77.
15.Roenneberg T, Wirz-Justice A, Merrow M. Life between clocks –
daily temporal patterns of human chronotypes. J Biol Rhythms.
2003;18(1):80-90.
16.Goulet G, Mongrain V, Desrosiers C, Paquet J, Dumont M. Daily
light exposure in morning-type and evening-type individuals. J
Biol Rhythms. 2007;22(2):151-8.
17.Roenneberg T, Kumar CJ, Merrow M. The human circadian clock
entrains to sun time. Curr Biol. 2007;17(2):44-5.
18.Fuller PM, Lu J, Saper CB. Differential rescue of light- and foodentrainable circadian rhythms. Science. 2008;320(5879):1074-7.
19.Czeisler CA, Dijk DJ. Use of bright light to treat maladaptation
to night shift work and circadian rhythm sleep disorders. J Sleep
Res. 1995;4(S2):70-3.
20.Crowley SJ, Lee C, Tseng CY, Fogg LF, Eastman CI. Combinations
of bright light, scheduled dark, sunglasses, and melatonin to facilitate circadian entrainment to night shift work. J Biol Rhythms.
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Am J Physiol. 1999;277(6 Pt2):R1598-604.
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Sleep
Science
ORIGINAL ARTICLE
Morningness/eveningness chronobiological
assessment of Dental students: chronotype
Avaliação de matutinidade/vespertinidade dos estudantes de Odontologia: cronótipo
Milva Maria Figueiredo De Martino1, Karla Figueiredo De Martino Pasetti2, César Bataglion3, Maria Filomena Ceolim1
ABSTRACT
Background and objective: Considering that chronotype is an
individual differential characteristic reflecting the time of day when
individuals have their best performances, some people prefer to
wake up early in the morning and are more alert in the first part
of the day, others prefer to wake up later, as their peak time of the
day is in the evening, and they prefer to go to bed late at night.
The purpose of this study was to analyze the chronotype of undergraduate dental students and to identity the relationship between
chronotype and periods of both physical activity and study, as well
as to verify the correlation to age. Methods: Seventy-five first-year
students, who participated voluntarily in the study, both genders,
whose mean age was 19 years old (±1.5), answered the Horne and
Ostberg Questionnaire (1976), which was modified and validated by
Benedito-Silva et al. or Brazilian population and was applied at the
Dental School of the University of São Paulo, Ribeirão Preto (USP).
Results: The results revealed a predominance of “Indifferent” chronotype, followed by “Moderately eveningness”, “Moderately morningness” and “Definitely eveningness” chronotypes. A significant
statistical correlation was not observed between chronotypes and
individual characteristics. Conclusions: There was a predominance
of indifferent chronotype among dental students and we did not find
any correlation between chronotype, time of study, physical activity,
gender and age.
Keywords: Circadian rhythm; Exercise; Students, dental
RESUMO
Introdução e objetivo: Considerando-se que o cronótipo é uma
característica individual que reflete o período do dia em que os indivíduos tem o seu melhor desempenho, algumas pessoas preferem
acordar cedo e estão mais alertas nas primeiras horas da manhã, enquanto outras preferem acordar mais tarde, pois seu pico de desempenho é vespertino e elas preferem ir para a cama tarde da noite.
A proposta deste estudo foi analisar o cronótipo de estudantes de
odontologia e identificar a relação entre cronótipo, período de atividade física e de estudo, como também verificar a correlação com
idade. Métodos: Participaram 75 estudantes voluntários do primeiro ano do curso de Odontologia, de ambos os sexos, com média de
idade de 19 anos (±1,5). Responderam ao questionário de Horne &
Ostberg (1976) modificado e validado por Benedito-Silva et al. para
a população brasileira. Resultados: Os resultados mostraram a predominância do “cronótipo indiferente”, seguido pelo “moderamente
vespertino”, “moderamente matutino” e “definitivamente vespertino”. Não foi verificada correlação significativa entre o cronótipo e
as características individuais dos sujeitos. Conclusão: Houve uma
predominância do cronótipo indiferente entre estudantes de odontologia e não foi encontrada correlação entre cronótipo, tempo de
estudo, atividade física, sexo e idade.
Descritores: Ritmo circadiano; Exercício; Estudantes de Odontologia
INTRODUCTION
Chronobiology is a branch of science that studies biological
rhythms in living organisms and systematizes this knowledge. It may be defined as “a study of mechanisms and alterations of each organism’s temporal structure under various situations (1). From this point of view, time is seen as “a
fundamental dimension of living structures” (2).
Biological rhythms are endogenous and cannot be manipulated by the subject. A person who studies at schedule
times that conflict with his/her biological rhythm may pay
a price in terms of health, quality of life and performance (3).
An individual’s chronotype, with morningness/eveningness characteristics, is important to determine periods of
best performances and well-being. This information can be
used to optimize the quality of study and to minimize any
associated disturbance.
Study carried out at Dental School of the Universidade de São Paulo – USP, Ribeirão Preto (SP), Brazil.
1
Department of Nursing, Faculty of Medical Sciences, Universidade Estadual de Campinas – UNICAMP, Campinas (SP), Brazil.
2
Dental Surgeon, Pontifícia Universidade Católica de Campinas – PUCCAMP, Campinas (SP), Brazil.
3
Universidade de São Paulo – USP, Ribeirão Preto (SP), Brazil.
Corresponding author: Milva Maria Figueiredo De Martino – Rua Renato Reis, 56 – CEP 13084445 – Campinas (SP), Brazil – Phone: (19) 3289-4907 – E-mail:
[email protected]
Received: December 10, 2009; Accepted: March 18, 2010
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8
Morningness/eveningness chronobiological assessment
According to Horne and Ostberg (4), individuals may
be classified into three chronotypes, taking into account
individual differences in temporal allocation of biological
rhythms. The chronotypes are the following: morningness
(subdivided into extreme and moderate), eveningness (subdivided into extreme and moderate) and indifferent. Morningness individuals are those who prefer going to bed early
(around 22 or 23h) and consequently get up early (around
6h) without any difficulty, resulting in good physical and
mental performances. Eveningness individuals, on the other
hand, go to bed late (around 1h) and consequently wake up
late (around 10h), presenting better performance in the afternoon and in the beginning of the evening. The indifferent
chronotypes, however, are more flexible and choose intermediary times according to their routine needs (5).
Zubioli (6) studied the chronotypes of nursing assistants
who work in a hospital in the State of Parana and observed
that individual characteristics are not always respected
when placing workers to the different shifts. For example,
the morning shift may be tiring for workers who have eveningness chronotype due to the fact that it demands that
they wake up earlier than it is adequate for their organism.
Campos and De Martino (7) verified the chronotype of a
group of nursing professors. They concluded that individual
preferences were in accordance to work regimen of the study
population who had chosen the work period according to
their chronotypes.
Studies on circadian rhythm of the sleep-wake cycle revealed that nurses who worked on night shifts had a peculiar characteristic regarding sleep duration. The data showed
that individuals who worked during the night slept up to
14 hours soon after their work shift, suggesting the need
to replace hours of sleeping more than the number of hours
that were in fact lost. Some of these nurses presented characteristics that did not correspond with their best ability,
which suggests that their chronotypes should be analyzed (8).
Zavada et al. (9) performed a comparative study using two
questionnaires: Munich Chronotype Questionnaire (MEQ)
and Horne–Ostberg (HO). Time of mid-sleep on free days and
on workdays was assessed using MEQ. It was concluded that
MEQ was more strongly correlated to midpoint of sleeping
on free days. Sleep onset was correlated to MEQ score and it
was usually slightly better than sleep-end times, especially on
workdays. It was also observed that mid-sleep on free days is a
good predictor of chronotype (as judged by sleep preferences).
In this study, the individual characteristics and chronotypes of dental university students were analyzed in order to
suggest more adequate hours for their academic performance
and to contribute to an improvement on their quality of life,
in harmony with their biological rhythms.
Sleep Sci. 2010;3(1):����
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OBJECTIVES
The purpose of this study was to analyze the chronotype of
undergraduate dental students and to identity the relationship between chronotype and periods of both physical activity and study, as well as correlation to age.
METHODS
Subjects
First-term dental university students from Universidade de
São Paulo (Ribeirão Preto, Brazil) were invited to participate
in this study. From a total of 100 students, some were absent
at the time of the class on the day of the research and others
refused to participate. Seventy-five students took part voluntarily in the research and signed the informed consent term.
The study was approved by the Ethics Research Committee
from Universidade de São Paulo, Brazil.
Data collection instruments
Horne and Ostberg (4) Questionnaire (HO) with a Portuguese version, adapted and validated by Benedito-Silva et
al. (10) was employed. These authors applied the questionnaire on several different areas of Brazil, adapting the score
according to habits and characteristics of the Brazilian
population. The questionnaire consists of questions about
habitual situations of daily life and the individuals should
register their favorite time periods for each activity.
The questionnaire results were shown in a numeric
score between 16 and 86, according to which the individual could be classified into five chronotypes: eveningness (16 to 30), moderately eveningness (31 to 41), indifferent (42 to 58), moderately morningness (59 to 69)
and extreme morningness (70 to 86). The students also
answered to items regarding social-demographic data
(gender, age, nationality, level of physical activities and
hours of study).
Procedures
Data were collected in the classroom before classes began
and the subjects were asked to answer the questionnaire and
to return it when it was finished.
Data analysis
Tables and graphs were used to organize the data in absolute
numbers and percentages and to submit to a descriptive statistics analysis. Chronotype classification was performed using a program developed by De Martino and Pirola*. A nonparametric test was used to verify the correlation between
the score of the HO questionnaire and age (Spearman’s rank
correlation) for all subjects. Kruskal-Walllis test (11) was used
*
De
�������������������������������������������������������������������������������
Martino MMF, Pirola D. Software sobre questionário de identificação de indivíduos matutinos e vespertinos. Programa datilografado, 1999.
De Martino MMF, Pasetti KFDM, Bataglion C, Ceolim MF
to score the physical activity and study times, as well as to
compare the HO score to age.
RESULTS
The students’ ages ranged from 17 to 27 years old, mean age
of 19 (±1.5) years. The mean age of females was 18. 9 (±1.2)
years, while the mean age of males was 19.1 (±1.7) years.
The “indifferent” chronotype was found to be predominant (IN, 76%), followed by the “moderately evening” (ME,
13%), “moderately morning” (MM, 8%) and “definitely
evening” (DE,3%), as shown in Figure 1.
30
30
Age 25
Age
20
20
15
15
16 23 30 37 44 51 58 65 72 79 86
physical activity performed, followed by gymnastics, soccer,
volleyball and walking. Other categories such as basketball,
tennis and swimming were cited only once (Figure 4).
ME
14
IN
12
MM
10
DE
76%
DE: definitely evening; ME: moderate evening; IN: indifferent;
MM: moderately morning
Figure 1: Distribution of 75 students classified according to chronotype.
The score obtained by the participants ranged from 29 to
66. No significant statistical correlation was observed between
the age of the students and the score obtained in the HO questionnaire (Spearman R=0.0271), as shown in Figure 2.
29
DE
27
Age
ME
IN
MM
25
23
21
19
17
15
20
30
40
50
60
70
HO Score
DE: definitely evening; ME: moderate evening; IN: indifferent;
MM: moderately morning
Figure 2: Distribution of subjects according to the HO questionnaire
score and age.
No correlation was also observed between the HO questionnaire score and age of subjects according to gender, as
observed in Figure 3.
An analysis of the physical activities practiced by the students showed that 41.9% of all students performed different categories of physical exercises, as demonstrated by figure 4. Body building was observed to be the most frequent
HO score
Figure 3: Distribution of subjects according to the HO score for age and
gender, dark spots for male and vacant circles for female.
Frequency
8%
16 23 30 37 44 51 58 65 72 79 86
HO score
13%
3%
25
13
8
6
6
4
4
3
2
2
0
1
1
1
1
Types of physical activities
Body building
Walking
Gymnastics
Basketball
Soccer
Tennis
Volley ball
Swimming
Figure 4: Frequency of physical activities performed by the university
students.
No correlation was observed between the time of the day
in which the physical activity was practiced and the score
obtained in the HO questionnaire (Spearman’s R=0.2101).
It should be observed that only 22 students informed the
time when physical activity was performed. Most of these
activities probably took place in the evenings, since the dental course is full-time and evenings are the only time available. It is interesting to note that 33% of the morningness
chronotype students referred practicing physical activities
while 42% of the eveningness chronotype and 44% of the
indifferent type answered positively.
It was also verified that regarding time of study, the subjects reported that they studied an average of four hours per
day, at least two hours, a question answered by 53 students.
No correlation was observed between the time of study and
the HO questionnaire score (Spearman R=0.1051). It was
verified that 33% of the morningness chronotype, 75% of
the eveningness chronotype (moderate and definitely) and
Sleep Sci. 2010;3(1):����
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Morningness/eveningness chronobiological assessment
74% of the indifferent participants indicated their time of
study.
DISCUSSION
Another study on the chronotype of undergraduate nursing
students (3) revealed the predominance of the indifferent, followed by the moderate morningness. It is well known that
eveningness is an inherent characteristic of younger individuals who demonstrate it as they reach adolescence. The
fact that the subjects were full-time, and also their ages were
homogenous, may have contributed to the inexistence of statistical significant differences in this study.
Recently, another study (12) described the prepubertal
children’s chronotypes in order to examine the validity between children’s chronotype measures and sleep-wake cycles, as well as to assess the associations between these measures. In this study, the authors used actigraphy to assess
the children’s sleep and also the sleeping diaries reported by
the children’s parents. The authors concluded that using the
measures CCTQ (MSF, M/E and CT) it is possible to evaluate the chronotype in prepurbertal children between 4 and
11 years old.
We concluded that among the researched students, there
has been the predominance of “Indifferent” chronotype, followed by “Moderately Eveningness”, “Moderately Morningness” and “Definitely Eveningness” chronotypes.
The students alternated study periods with physical activities that were practiced in the evenings by 41.89% of
the subjects. Time of study as well as physical activity did
not show any relationship with the HO questionnaire score.
No significant statistical correlation was verified between
the chronotype score obtained in the HO questionnaire with
regard to gender and age.
It is suggested that more longitudinal studies, that accompany the students during their entire course, as well as
cross-sectional studies that compare students who are in different years of the course should be done, as performed by
De Martino and Ling (3).
There was a predominance of indifferent cronotype
among these students and we did not find any correlation
between cronotype, time of study, physical activity, gender
and age.
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Sleep
Science
ORIGINAL ARTICLE
Environmental and organizational
conditions for napping during night work:
a qualitative study among Nursing professionals
Condições ambientais e organizacionais dos cochilos durante o trabalho
noturno: um estudo qualitativo entre profissionais de Enfermagem
Aline Silva-Costa1, Milena Maria de Araújo1, Roberta Nagai1, Frida Marina Fischer1
ABSTRACT
Background and Objective: Permission to take a nap during the
night shift may compensate for shorter daytime sleep duration at
home and improve alertness on the job. This study aimed to describe
the importance of napping and characterize the environmental
and organizational conditions of taking a nap during a night shift.
Methods: This is a qualitative study in which 20 registered nurses and
nurse assistants of a hospital in the city of São Paulo were interviewed.
Results: The results showed that, despite the permission to nap, the
occurrence of napping depended on the workload. Although there was
a location designated for napping, there was insufficient space and
couches for everyone. In this event, many workers improvised other
locations to nap. Although the conditions were not always adequate,
participants reported napping/rest as an important factor for physical
and mental rest and for maintaining alertness during the night
shift. Conclusions: The statements of the participants emphasize
that, in order to obtain satisfactory benefits from napping, adequate
conditions for this purpose should be implemented.
Keywords: Night work; Work schedule tolerance; Nursing staff;
Sleep deprivation; Arousal; Shift work; Working environment; Workload; Sleep disorders; Circadian rhythm
RESUMO
Objetivo: A permissão para cochilar durante o trabalho noturno pode
contribuir para compensar a reduzida duração de sono em casa e melhorar o estado de alerta durante o trabalho. Os objetivos deste estudo
foram conhecer as condições ambientais e organizacionais do cochilo
e a sua importância durante o trabalho noturno entre profissionais de
Enfermagem. Métodos: Trata-se de um estudo qualitativo, no qual 20
entrevistas foram realizadas com enfermeiras e auxiliares de enfermagem de um hospital da cidade de São Paulo. Resultados: Os resultados mostraram que, apesar da permissão para o cochilo, a sua ocorrência dependia da demanda de trabalho. Havia uma sala destinada para
cochilar, mas não havia espaço e poltronas disponíveis para todos.
Neste caso, muito trabalhadoras improvisavam outro local. Embora
nem sempre em condições adequadas, as participantes relataram que
cochilar/descansar é importante para o descanso físico e mental e para
manter o alerta ao longo da noite de trabalho. Conclusões: Os relatos
das participantes reforçam que, para que os benefícios dos cochilos
sejam obtidos satisfatoriamente, também é importante que sejam implementadas condições adequadas para a sua ocorrência.
Descritores: Trabalho noturno; Tolerância ao trabalho programado;
Recursos humanos de Enfermagem; Privação do sono; Nível de alerta;
Trabalho em turnos; Ambiente de trabalho; Carga de trabalho; Transtornos do sono; Ritmo circadiano
INTRODUCTION
Health services are available to the population 24 hours a
day. For this reason, hospitals have adopted continuous shift
work schedules in which employees work in shifts, covering
the day and night periods, seven days a week throughout
the year. Professionals who are assigned to night shifts suffer several consequences to their health and well-being due
to the maladjustment of their biological rhythms (1). Individuals who work during night shifts are clearly affected by
nighttime sleep deprivation (2).
Night shift nurses often experience a significant lack of
sleep hours each week, which leads to increased sleepiness
and, at long term, contributes to increased symptoms of fatigue (3). Among nurses and Nursing assistants who work
during night shifts, the quality of nighttime sleep on their
days off is better than the quality of daytime sleep on work
days (4). This night sleep deprivation may compromise not
only the health of workers, but also the quality of patient
care. The accumulation of multiple jobs is another characteristic of Nursing, adding to the duration of the work day
(5)
, with possible effects on sleep.
In this context, several intervention methods have been
proposed in the literature with the aim of raising the alert-
Study carried out at Faculdade de Saúde Pública, Universidade de São Paulo – USP, São Paulo (SP), Brazil.
1
Department of Environmental Health, Faculdade de Saúde Pública, Universidade de São Paulo – USP, São Paulo (SP), Brazil.
Corresponding author: Aline Silva-Costa – Avenida Doutor Arnaldo, 715 – CEP 01246-904 – São Paulo (SP), Brazil – Phone: (11) 3061-7115 – E-mail: [email protected]
There is no conflict of interest in this study.
Received: December 14, 2009; Accepted: March 18, 2010
Sleep Sci. 2010;3(1):�����
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12
Environmental and organizational conditions for napping during night work
ness of night shift employees, including interventions with
bright light (6) and the use of caffeinated beverages (7). Permission to nap during the night shift (8) is a strategy that
seeks to minimize sleep complaints.
A study (9) with nurses and physicians showed that napping during the night shift improved their performance and
decreased fatigue at the end of the work day. Napping during the night shift was also associated with reduced sleepiness during the shift among nurses (10). Another study (11)
showed that the nurses who took naps during the night shift
had the same total amount of sleep as the day shift workers.
Thus, given the benefits of naps during night shifts and
the lack of qualitative studies in this area, the current study
aimed to understand the importance of napping and to characterize the environmental and organizational conditions that
are favourable or unfavourable to napping during the night
shift among the Nursing staff at a hospital in São Paulo.
METHODS
Sample population
This study included 20 female Nursing professionals (assistants, technicians and registered nurses) who worked during
night shifts (19h to 7h) in a hospital in São Paulo that allows
napping during the night shift.
The mean age of study participants was 37 years (SD=7
years old). Most of them (60.0%) had College degrees, 55.0%
were married, 35.0% were single and 10.0% divorced. Half
of the interviewed individuals were nurses and the other half
were assistants or nurse technicians; 85.0% reported having
only one job. The working schedule consisted of 12 hours
of night work followed by 36 hours off. There were 6 or 7
nights of work every 14 days.
The studied sectors were: emergency rooms, adult intensive care unit, maternity, medical-surgical clinical unit and
pediatrics wards.
Interviews
When aiming to understand and interpret a particular phenomenon in terms of the meanings that people attach to it,
it is vital to perform qualitative research (12).
In this study, semi-structured interviews were recorded
with permission and transcribed for later analysis.
The developed interview script was previously tested in
a population similar to that of the current study. The questions used were: 1) Is it important for you to nap? Why? 2)
How is napping time organized in your sector? What do you
think about this napping time organization? 3) Could you
describe the place where you usually take the nap? I would
like you to describe this location exactly in terms of lighting, noise, ventilation, temperature, privacy and comfort in
general.
Sleep Sci. 2010;3(1):�����
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The study sample was selected according to convenience,
depending on the availability of the professionals to participate in interviews, because they were held at the hospital
during night shifts.
Data analysis
Interviews were analyzed according to the method of “Collective Subject Discourse” (CSD). Using this technique, it
was possible to select the individuals’ key expressions and
central ideas from each answer (13). CSD is a methodological
tool that is capable of depicting the social representations of
a particular group (13).
The key words are verbatim transcripts or excerpts of the
speech of each individual. They reveal the essence of the testimony or of the discursive content of the segments in which
the statement is divided (14).
The central idea can be understood as a summary of
the individual’s statement. Therefore, the central idea of a
speech is not an interpretation, but rather a description of
the meaning of a statement or a set of statements (14).
For the development of collective subject discourse, all of
the key expressions comprised by the same central idea were
grouped together.
Ethical principles
This study was approved by the Research Ethics Committees of the School of Public Health of Universidade de São
Paulo (USP) and of the University Hospital where the research was carried out. The workers were informed about (i)
the research objectives, (ii) their freedom to refuse to participate in the study without prejudice toward their professional activity and (iii) the guarantee of the confidentiality
of the results. The Nursing professionals who were invited
to participate signed a consent form that was drafted in accordance with the ethical principles described in Resolution
196/96 from the Brazilian Ministry of Health.
RESULTS
Collective Subject Discourse (CSD)
Below, we present the results of the three analyzed questions
and their central ideas (CI):
Question 1 (Is it important for you to nap? Why?) generated three central ideas, here defined as CI-A, CI-B and CI-C.
CI-A: Napping is important because it provides rest,
increases attention, and reduces fatigue and the risk of
errors.
CSD - Central Idea A: “It is important because it somewhat decreases fatigue. There are moments when I’m very
sleepy and, when I rest a bit, I get better. Sometimes, when
you cannot nap due to the workload in the sector or because
Silva-Costa A, Araújo MM, Nagai R, Fischer FM
we work with a reduced number of employees, I realize that
I’m more distracted. The risk of a mistake due to sleep is very
great because of distraction. There comes a time when fatigue
is really intense. Sometimes I read something and I cannot
understand what I’m reading. So you have to stop in order to
have a mind-rest as well. When I work twelve hours straight,
sometimes I cannot even assimilate what I have been doing.”
CI-B: Napping is important because it is a physiological need.
CSD - Central Idea B: “Napping is important because
we are human beings; we need to rest even if just for half
an hour, because the night was made for sleeping. There are
people who work every night and sleep during the day. But
sometimes, (these people) cannot sleep because during the
day the sleep-wake cycle changes a lot, so it is important to
rest during the night.”
CI -C: Napping is not important, but resting is.
CSD - Central Idea C: “For me, it is not important to
nap because napping will not solve much. But it is important to have a peaceful environment where you can restore
some of the energy lost during the shift. It is important to
close your eyes, rest, stretch your legs, even if you don’t sleep.
This helps to reinvigorate, even if not for long. On very busy
nights when there is no rest, your day is very cumbersome.”
Question 2 (How is napping time organized in your sector? What do you think about this napping time organization?) generated four central ideas, here defined as CI-A,
CI-B, CI-C and CI-D.
CI-A: I like the organization of napping schedules
because we share in accordance with each person’s fatigue and we do not leave anyone alone on duty.
CSD - Central Idea A: “I think it’s good because we
share the time of the nap in accordance with each person’s
fatigue. It is usually organized into two periods: half of the
staff rests in the first half, and the remainder in the second.
If there are a large number of employees, the rest is divided
into three periods. We divide the napping time according to
the needs of the employee. This is good because not everyone
will nap at the same time, so the sector is not empty. Because
if suddenly there is a complication, being alone is complicated, it is important to have another colleague to help. It is
a commitment among all.”
CI-B: I like the organization of the napping schedule, but I think they should increase resting time.
CSD of Central Idea B: “I think the organization is
good, although I think the hospital should allow a little ex-
tra resting time for those who work at night because we get
very tired. One hour is too little.”
CI-C: I like the organization of napping schedules,
but I think it should be something more formal.
CSD of Central Idea C: “We even have the time to rest,
but it seems as if it is always furtive. Sometimes we even get
insecure about the boss coming in. He knows that we take
time to rest, but we still feel terribly ashamed. It is embarrassing; everyone knows that it happens in all sectors across
the hospital. It seems like we’re doing something wrong,
even though we know it is not. If someone is resting, it is
because they actually can do it.”
CI-D: I don’t like the napping schedules because
there is no predefined time and there is not always time
to rest.
CSD of Central Idea D: “It’s disorganized because there
is not an exact time and we cannot always rest. The schedule
is defined by the head nurse. Since it depends on the shift, the
type of patient, the team and the sector, it is not always possible
to rest. There are shifts when you cannot even sit down. The
priority is to work. There is no strict organization, where you
would rest in a particular period of time. If one is scheduled for
a time, but had to give medication or the patient called, then
we cannot rest in our schedule. So we will not rest at all. I think
we should really have a standard schedule, ‘on the clock’ for
everyone. However, we do work, many times, understaffed.”
Question 3 (Could you describe the place where you usually take the nap? I would like you to describe this location
exactly in terms of lighting, noise, ventilation, temperature, privacy and comfort in general) generated three central
ideas, here defined as CI-A, CI-B and CI-C.
CI-A: The place where I rest is comfortable.
CSD of Central Idea A: “We, here at the hospital, have
a rest area where there are reclining sofas, a private restroom
and two seats. It is very quiet, comfortable and darker than
the other places. In the rest room, the light is very soft and
it has two areas: one with a television, and others only with
sofas for those who like to sleep in the dark. The room has
air conditioning, and we can warm up when it is very cold,
it feels good for resting. I think it’s a very quiet place, with
comfort, appropriate to sleep quietly.”
CI-B: The place where I rest is not comfortable.
CSD of Central Idea B: “It’s not a comfortable place.
The hospital even has a proper place to rest, but it has few
sofas for the number of employees. There is no privacy. It is
mixed, males and females, from all sectors. It has an elevator
Sleep Sci. 2010;3(1):�����
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13
14
Environmental and organizational conditions for napping during night work
nearby, so you have a lot of noise of people coming in and
out. Often, there is no seat available, so we rest in any unoccupied office or room. In these places, you can be called back
to work at any time. Also, there’s no bed, we stretch a little
on the couch or reclining seats; that is, we do not lie down.
Sometimes we lay on stretchers, mattresses, or even on the
floor, where we lie on a sheet. It’s all improvised. There are
rooms that do not have adequate ventilation, no windows.
Then it gets hot, stuffy. And there is a lot of noise, people
coming in all the time. We end up not resting, but instead,
as we usually say, just stretching our legs.”
CI-C: I don’t know because it has been a while since
I’ve rested.
CSD of Central Idea C: “I don’t know because it has
been a long time since I’ve rested.”
DISCUSSION
The analysis of the importance of napping during night jobs
presented the following ideas: napping is important because
it provides rest and alertness, reduces fatigue and the risk of
errors, and is a physiological need. These statements resemble the research report presented at the 2008 annual meeting of Associated Professional Sleep Societies (APSS). In the
report, nurses reported that short naps (of 20-30 minutes)
were sufficient for improving alertness, presenting a restorative function (8).
During night work, sleepiness and fatigue can reduce the
attention of workers and lead them to mistakes. A study
conducted with nurses who worked during the night shift
for longer than a year showed that they were at a higher
risk for work injuries with medical leaves (15). The feelings
of guilt, shame and fear experienced by Nursing staff after
the occurrence of errors at work may, in turn, interfere negatively with work (16).
The data from the current study corroborate other findings stating that napping during night work was shown to
be an important strategy for increasing the level of alertness
among health professionals (9,10), air traffic controllers (17), factory operators (18) and drivers (19), as well as reducing the occurrence of errors and accidents at work.
Naps, or simply rest, during night shift work was also effective in the reduction of fatigue complaints. These results
were also found in studies with physicians and nurses (9) and
computer operators (20), in which naps during night shifts
were associated with reductions in sleepiness and fatigue
complaints.
As described in the literature (21), napping during the
night shift could have the role of “anchor” sleep, which is
also quite beneficial for the chronobiological aspect. This
confirms the importance of this strategy in minimizing the
Sleep Sci. 2010;3(1):�����
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symptoms of sleepiness and fatigue caused by the organization of night work, as reported in the statements of the
interviewees.
In the current study, the statements did not include reports on the preference for a certain time of the night to nap.
Research conducted in a laboratory (22) showed the influence
of the nap duration and the time at which it occurred in the
performance of night shift workers. Naps that occurred after
the middle of the night were considered better in terms of
sleep quality than those that occurred earlier in the night.
However, performance was found to be worse after later naps
due to sleep inertia (22). Results of other experimental research (23) showed that alertness improved with a nap of 30 or
50 minutes at different times during the night shift. These
conflicting results show that there is no consensus in the
literature about the best time and duration for a nap during
the night shift.
Concerning the environment in which the naps occurred,
we observed positive and negative statements. The absence
of beds, lack of privacy, inadequate ventilation, presence of
light and noise were factors considered negative by several
interviewees. However, other workers did not mention these
factors and were satisfied with the designated napping location.
Individual differences in the perception of working conditions and the tolerance to night work (24) may be one explanation for these seemingly contradictory discourses. For
this reason, rest areas of only average quality may not meet
everyone’s needs. It is important to support nurses and provide comfortable environments in order to provide them the
necessary rest during the night shift (25).
Regarding the scheduling of naps, as noted in a previous
study conducted in São Paulo (10), we found that Nursing
teams take turns in groups such that their work sector is not
understaffed. Several interviewees complained about the informality of naps despite being allowed. Similar statements
were also reported in a Canadian study (25) in which several
nurses reported that they felt tired, but were afraid to take
a nap.
Permission for Nursing teams to nap during night work
remains controversial, mainly because this group of workers
deals with continued assistance to patients. As the demands
of night jobs are often considered to be lesser than those of
day jobs, the number of employees is typically reduced in
the night shift (26). This reduction complicates the organization of napping during working hours. Therefore, aspects
of the organization of work must be considered so that they
may be used in Nursing teams’ discussions about the regulation of nap during night work.
The statements suggest that several aspects of night time
rest/naps should be improved. Environmental factors (venti-
Silva-Costa A, Araújo MM, Nagai R, Fischer FM
lation, lighting, and noise), location of the room (away from
the sector area), and lack of time (depending on the demands
that prevent workers from leaving the workplace) were some
of the impediments to achieving satisfactory naps.
In view of these reports, it is important that aspects of
the physical and organizational environment are taken into
account when planning the nighttime naps in hospitals.
Simply offering a place to rest is not sufficient for obtaining
the benefits of the nighttime nap on the work environment.
Study limitations
The interviews in this study were not analyzed according to
sector and job function. Because these variables could have
an important role, especially in regard to the perception of
the site and schedule of naps, future studies should evaluate
the influence of function and sector.
ACKNOWLEDGMENTS
We thank Professor Fernando Lefèvre for his assistance during the initial phase of this study, suggesting the script of
the interview.
The author Aline Silva-Costa is sponsored by FAPESP
(2008/51616-3); Roberta Nagai was sponsored by CAPES
during her Doctoral studies; Milena Maria de Araújo was
sponsored by CNPq/PIBIC; Frida Marina Fischer receives
productivity Grant IB from CNPq.
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Sleep
Science
ORIGINAL ARTICLE
Onset and stability of melatonin treatment
effect in childhood sleep onset insomnia
Início e estabilidade dos efeitos do tratamento com
melatonina na insônia de início de sono infantil
Ingeborg M. van Geijlswijk1, Robert Didden2, Kristiaan B. van der Heijden3, Marcel G. Smits4, Jan F. van Leeuwe5
ABSTRACT
Backgroud and objective: To evaluate onset and stability of therapeutic effect of 4-week melatonin treatment for chronic sleep onset insomnia in elementary school-aged children. Methods: Retrospective
analysis of unpublished data obtained from two previously published
randomized, double-blind and placebo-controlled trials on melatonin
treatment efficacy for childhood insomnia in children with chronic
sleep onset insomnia, age 6-12 years (n=49). Intervention consisted
of placebo (n=25) or melatonin 5 mg (n=24) administered at 6 (n=9)
or 19h (n=40) during four weeks. Collected data were “light-out
time”, “sleep onset latency”, “sleep onset”, “total sleep time”, “wakeup time”, and subjective sleep measures recorded in a diary. Results:
Melatonin treatment showed a phase-advance of lights out of 21h15
(1.05) to 20h28 (1.07); sleep onset advanced from 22h05 (0.93) to
20h45 (1.09) and sleep latency decreased from 53 (39) to 18 minutes
(16). After the 4-week trial period, these values were 20h44 (1.27),
21h09 (1.33), 25 minutes (39). Conclusions: Melatonin advances
sleep latency and sleep onset and increases total sleep time starting
right from the first treatment night in children with chronic sleep onset insomnia. �������������������������������������������������������
Evidence is provided that the onset of melatonin treatment effect can be expected within a few days after commencement
and, then, remains stable.
Keywords: Sleep initiation and maintenance disorders/drug therapy;
Melatonin/therapeutic use; Education, primary and secondary; Child
RESUMO
Objetivo: Avaliar o início dos efeitos e a estabilidade do tratamento
para insônia de início do sono com melatonina por quatro semanas
em crianças cursando escola primária. Métodos: Análise retrospectiva de dados inéditos obtidos de dois ensaios randomizados, duplocegos e controlados por placebo sobre a eficácia do tratamento da
melatonina para insônia na infância em crianças com início de insônia crônica com idades entre 6 e 12 anos (n=49). A intervenção
consistiu em placebo (n=25) ou melatonina 5 mg (n=24) ministrados às 18 (n=9) ou 19h (n=40) durante quatro semanas. Os dados
coletados foram “horário em que as luzes foram apagadas”, “latência
até o início do sono”, “início do sono”, “tempo total de sono”, “horário de despertar” e medidas subjetivas do sono registrados em um
diário. Resultados: O tratamento com melatonina evidenciou fase
adiantada quando as luzes estavam apagadas das 21h15 (1.05) às
20h28 (1.07); o início do sono avançou das 22h05 (0.93) às 20h45
(1.09) e latência ao sono diminuiu de 53 (39) para 18 minutos (16).
Ao término das quatro semanas, esses valores passaram a ser 20h44
(1,27), 21h09 (1.33), 25 minutos (39). Conclusões: A melatonina
adianta a latência ao sono e ao início do sono aumentando o tempo
total de sono a partir da primeira noite em crianças que sofrem de
insônia de início de sono crônica. As evidências demonstram que o
efeito pelo tratamento de melatonina para o início do sono pode ser
esperado dentro de alguns dias após o início do tratamento e, então,
se mantém estável.
Descritores: Distúrbios
�������������������������������������������������
do início e da manutenção do sono/quimioterapia; Melatonina/uso terapêutico; Ensino fundamental e médio;
Criança
INTRODUCTION
Sleep onset insomnia is a highly prevalent disorder among
school-age children. A chronic and severe presentation leading to long-term sleep deprivation can seriously affect a
child’s physical and mental development. We have previously shown in two randomized, double-blind and placebo-controlled studies in elementary school children with chronic
sleep onset insomnia suggestive of delayed sleep phase disorder that melatonin (5 mg) advanced sleep-wake rhythm
and lengthened total sleep duration (1,2). However, the data
Study carried out at Pharmacy Department, Faculty of Veterinary Medicine, Utrecht University, The Netherlands.
1
Pharmacy Department, Faculty of Veterinary Medicine, Utrecht University; Utrecht Institute for Pharmaceutical Sciences – UIPS, Department of Pharmacoepidemiology
and Pharmacotherapy, Faculty of Science, Utrecht University, The Netherlands.
2
Department of Special Education, Radboud University Nijmegen, The Netherlands.
3
Department of Clinical Child and Adolescent Studies, Faculty of Social Sciences, University of Leiden, The Netherlands.
4
Department of Sleep-Wake Disorders and Chronobiology, Gelderse Vallei Hospital Ede, The Netherlands.
5
Department of Applied Statistics, Radboud University Nijmegen The Netherlands.
Corresponding author: Ingeborg M. van Geijlswijk – Department of Pharmacy, Faculty of Veterinary Medicine, Utrecht University – Yalelaan 106 – 3584 CM Utrecht
– The Netherlands – Phone: +31-30-2532066 – Fax: +31-30-2532065 – E-mail: [email protected]
Received: December 14, 2009; Accepted: March 18, 2010
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van Geijlswijk IM, Didden R, van der Heijden KB, Smits MG, van Leeuwe JF
published merely included treatment measurements performed within the fourth week. We have further analyzed
these findings by considering all data obtained from Day 1
to Week 4 in the treatment period, instead of focusing on
endpoint data. Knowledge of the time-course and stability
of the treatment effects during therapy initiation is clinically relevant for clinicians and researchers for this can prevent
unnecessary long administration of melatonin, for example,
when the appropriate dose of melatonin needs to be determined (3). If, during the initiation phase, the dose effect is
evaluable after days instead of weeks, the initiation phase of
effective therapy can be shortened from weeks to days.
While long-term treatment efficacy has already been established by several research groups (4,5), and direct soporific
effects of melatonin were recently described in the context of
sedation for electroencephalogram (EEG) recording (6), this
is the first study to report on the time-course and stability of
melatonin treatment effects for insomnia treatment.
This study’s aim was to evaluate the acute, wanted effects
of melatonin administration to children with sleep onset insomnia. The long-term safety, especially addressing the influence on puberty onset, is a still to be sufficiently answered
issue. For this population, long-term effects, including the
adverse events, are described elsewhere (7).
METHODS
Subjects were elementary school children with chronic sleep
onset insomnia, who participated in one of two randomized
placebo-controlled studies published earlier (1,2). They were
aged 6 to 12 years and in otherwise good general health;
their sleep problems were not related to any other pathology
than attention deficit hyperactivity disorder (ADHD) and
suggestive of delayed sleep phase disorder. Other
���������������
sleep pathology was excluded by ambulatory polysomnography with
24-hour cassette electroencephalography at the child’s home
and parental report.
The institutional review board on human research approved both studies and informed consent was obtained from
the parents of all participants. A 1-week baseline phase preceded a 4-week treatment phase. Melatonin immediate release tablets (5 mg) or identical-looking placebo were given
at 18h (melatonin: n=19; placebo: n=19) daily in the first
study (1) and at 19h (melatonin: n=35; placebo: n=36) in the
second (2). Sleep onset insomnia was defined as (a) complaints
of sleep-onset problems expressed by parents and/or child; (b)
occurrence on at least four days/week for longer than one year;
(c) average sleep onset later than 20h30 for children at age 6
years and for older children 15 minutes later per year; and (d)
average sleep onset latency exceeding 30 minutes. Exclusion
criteria were disturbed sleep architecture as measured by ambulatory polysomnography, and sleep maintenance insomnia
(one awakening>30 minutes or two or more awakenings >5
minutes summing up to at least 40 minutes, occurring on one
or more nights a week, for a period of at least 4 weeks preceding the start of the trial). Further exclusion criteria were mental handicap, severe learning disabilities, liver disease, renal
failure, chronic pain, and severe neurological or psychiatric
disorders. Finally, any prior use of melatonin, use of hypnotics, antidepressants, and neuroleptics was exclusion criterion.
Allowed medications were methylphenidate and salbutamol
by inhalation.
In the present paper, a sleep log was considered incomplete when it did not cover the entire four-week treatment
period (which is the period of interest in the present study).
This is in contrast to the previously published articles, when
it was considered incomplete when there were no data of the
fourth treatment week (which was the period of interest in
the previous studies). This complete coverage of the fourweek treatment was accomplished in 49% of the originally
included participants. The 51% missing data during the
first three treatment weeks is due to the fact that the decision to record sleep data over the entire four-week trial period (instead of during the fourth treatment week only) was
made at a time when both trials were already in progress.
Parents were asked to complete the following parameters in sleep logs daily: light-out time (time when the
light was turned off before sleeping), sleep onset time (estimated time when the child fell asleep, assessed by checking on the child through listening or watching every ten
minutes, as noninvasive as possible), wake-up time (estimated time of waking up), get up time (time of getting
out of bed). Means and standard deviations were calculated
over the baseline week and each treatment week, resulting
in five mean (SD) values per parameter. Intra, intergroup
and interaction effects were tested using multivariate analyses of variance (α≤0.05). Differences in means for four
sleep parameters, i.e. sleep onset time , sleep onset latency,
wake-up time and total sleep time were tested for each of
the 34 pairs of consecutive days (1st and 2nd night… 34th
and 35th night) using the Wilcoxon signed-rank test. To
account for multiple comparisons the α-level was set at
0.00147 (0.05 was divided by the number of comparisons:
34). Besides the mean values above mentioned, we tried
to rate therapy success with an individual parameter. We
defined as criterion for successful therapy the number of
patients with a decreased sleep onset latency of at least 25
minutes (being the standard deviation in sleep onset latency of both groups and even more important: a decrease
expected to be experienced as a clinically relevant relief of
sleep onset insomnia). We assessed this parameter for both
groups (melatonin and placebo) after one, two, three and
four weeks of therapy compared to baseline.
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17
18
Onset and stability of melatonin treatment effect
RESULTS
In the first study (1), there were 38 participants, of which
33 sleep logs were available on the fourth treatment week;
however of those 33 only 9 sleep logs were completed during the entire four-week treatment period. In the other paper
(2)
, there were 62 participants, of which 55 sleep logs were
completed during fourth treatment week; however, only 40
sleep logs were completed during the entire four-treatment
weeks – data are used for the present study.
Of the total sample included in the present study (n=49),
25 children (17 male and 8 female; 6 of trial (1) and 19 of
trial (2)) were assigned to melatonin treatment and 24 children (19 male and 5 female; 3 of trial (1) and 21 of trial (2))
to placebo.
Figure 1 depicts the mean values for lights out and
sleep onset time over the course of treatment. As can be
seen, robust phase advances occur within the first week of
melatonin treatment, after which the values remain stable
except for a slight delay within the third treatment week.
The convergent direction of the lights out and sleep onset
lines indicate a decrease of sleep onset latency within the
first treatment week.
Figure 1: Mean light-out time and sleep onset time Day 1-7 (i.e. baseline)
and Day 8-35 (i.e. intervention).
This sleep onset latency is of importance, since this
parameter clearly demonstrates the trouble falling asleep
for children sent to bed. Mean (± SD) sleep onset latency
changed in the Treatment Group from baseline 53 (±39) to
18 minutes (±16) in treatment Week 1 to 16 minutes (±17)
in Week 3, to 19 minutes (±21) in Week 4 and 25 minutes
(±38) in Week 5.
On the contrary, lights out, sleep onset and therefore
sleep onset latency did not significantly change in the Placebo Group, as illustrated by the white gap in Figure 1 (62
(±43), 55 (±48), 58 (±47), 60 (±51) and 49 (±52) minutes
in the last treatment week) (Figure 2).
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Figure 2: Mean sleep onset latency in Week 1 (i.e. baseline) and Week 2
to 5 (i.e. intervention).
Responders, defined as patients with a 25-minute decrease
in sleep onset latency, were compared between weeks and
groups, the group effect tested by type III between-subjects
tests. During the 1st treatment week, in the melatonin group,
14 patients responded with a >25-minute decrease in mean
sleep onset latency, while this was 4 in the Placebo Group
(F=9.379; p=0.004). Responders in the 2nd week were 16
patients in Melatonin and 5 in Placebo Group (F=11.035;
p=0.002); 13 in Melatonin and 4 in Placebo in the 3rd week
(F=7.505; p=0.009), and 13 in Melatonin and 7 in Placebo
in the 4th week (F=2.679; p=0.108). In the Melatonin Group,
11 patients met this criterion for success for each of the 4
weeks, against 1 in Placebo (F=10.177; p=0.003).
It is noteworthy that two patients in the Melatonin
Group had any response and one patient in the Placebo
Group had a consistent high response.
Figure 3 depicts wake-up time over the course of treatment, variation which shows an advanced phase that is most
robust in the first treatment week and gradually stabilizes in
the next treatment weeks. Values in placebo show a continuing irregular pattern.
Figure 3: Mean wake-up time in Days 1 to 7 (i.e. baseline) and Days 8 to
35 (i.e. intervention).
van Geijlswijk IM, Didden R, van der Heijden KB, Smits MG, van Leeuwe JF
Table 1: Results of multivariate analyses of variance for sleep parameters
Parameter/type
‘Exposure’ variablea
df1
df2
F
p-value
Form of the function
Sleep onset time
Main
Week number
4
44
25.226
< 0.001
Advances
Week number*treatment group
4
44
16.000
< 0.001
Advances
Treatment group
1
47
16.511
< 0.001
Advances
Time contrasts
Week 1 versus later*treatment group
1
47
39.311
< 0.001
Advances
Week 1 versus Week 2*treatment group
1
47
30.186
< 0.001
Advances
Sleep latency
Main
Week number
4
44
9.011
< 0.001
Decreases
Week number*treatment group
4
44
7.168
< 0.001
Decreases
Treatment group
1
47
19.240
< 0.001
Decreases
Time contrasts
Week 1 versus later*treatment group
1
47
19.489
< 0.001
Decreases
Week 1 versus Week 2*treatment group
1
47
18.344
< 0.001
Decreases
Total sleep time
Main
Week number
4
44
11.203
< 0.001
Increases
Week number*treatment group
4
44
2.523
0.054
Treatment group
1
47
16.284
< 0.001
Increases
Time contrasts
Week 1 versus later*treatment group
1
47
7.375
0.009
Increases
Week 1 versus Week 2*treatment group
1
47
8.076
0.007
Increases
Difficulty falling asleep
Main
Week number
4
44
20.095
< 0.001
Decreases
Week number*treatment group
4
44
7.168
< 0.001
Decreases
Treatment group
1
47
29.833
< 0.001
Decreases
Time contrasts
Week 1 versus later*treatment group
1
47
18.853
< 0.001
Decreases
Week 1 versus Week 2*treatment group
1
47
18.348
< 0.001
Decreases
Feeling rested (morning)
Main
Week number
4
44
4.925
0.002
Increases
Week number*treatment group
4
44
3.226
0.021
Increases
Treatment group
1
47
7.108
0.010
Increases
Time contrasts
Week 1 versus later*treatment group
1
47
0.849
0.362
Week 1 versus Week 2*treatment group
1
47
5.465
0.024
Increases
a
: week number and week number*treatment group effects tested by Wilks’ lambda; treatment group effect tested by type III between-subjects test.
Table 2: Results of the first treatment night in the melatonin treatment
group
Sleep parameter
z
p-value
-3.92
0.00009
-3.95
0.00008
-3.77
0.00016
Sleep latency
Difference in means of 7th night - 8th night
Sleep onset time
Difference in means of 7th night - 8th night
Total sleep time
Difference in means of 7th night - 8th night
Results of multivariate analyses of variance are summarized in Table 1. The results show that pretreatment to
treatment changes of sleep onset, sleep latency, total sleep
time light-out and wake-up time are significantly different
whilst melatonin treatment as compared to placebo treatment (i.e. Week 1 versus later * treatment group). Further
analysis shows that the difference in pretreatment to treatment changes of these sleep parameters are already significant after one week of treatment (i.e. Week 1 versus Week 2
* treatment group), except for get up time.
Main effects of week number and treatment group on
total sleep time are significant when the interaction is not.
This means that the Melatonin Group exceeds the Placebo
Group on the average, but that the trend over the weeks does
not differ between the two groups. Compared to placebo,
melatonin resulted in a significantly larger decrease in children’s perceived difficulties in falling asleep, from baseline
to later weeks as well as from baseline to the second week.
As compared to placebo, the Melatonin Group showed a
significantly different change in feeling rested in the morning from baseline to the first treatment week. However, this
effect did not sustain during the four-week treatment period. No significant effects were found for mood during the
evening, mood at daytime and perceived sleep quality (not
shown).
Finally, Table 2 shows the change in sleep latency, sleep
onset time, and total sleep time from the final baseline night
to the first treatment night within the Melatonin Treatment
Group. The results reveal significant improvements in all
three parameters during this first treatment night. The
Wilcoxon signed-rank test revealed that for the 34 pairs of
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19
20
Onset and stability of melatonin treatment effect
consecutive daily means for sleep latency, sleep onset time
and total sleep time the difference between consecutive pairs
of nights was only statistically significant (α-level set at
0.00147) for the change over the final baseline night to the
first treatment night in the Melatonin Treatment Group.
Differences between consecutive night pairs of the four
parameters within the Placebo Group were all statistically
non-significant.
DISCUSSION
This paper is a further analysis of data from our two previous
publications, and we have now found that effects of melatonin on sleep latency, sleep onset time and total sleep time
occur from the first night of treatment. These effects were
stable during a four-week intervention period. Melatonin
initially also improved feeling rested in the morning, which
however did not sustain during intervention.
Individual results for sleep onset latency and using this
result as a criterion for successful therapy corroborate the
mean study population results, since the variance of sleep
measures (sleep onset and wake-up time in particular) is
large in a population with varying sleep needs (age 6 to
12 years) and this variance is much smaller in the mean
calculated measure sleep onset latency. What strikes in the
responders results is that most responders in the Melatonin Group are consistent during the four treatment weeks
(11)
, as in the Placebo Group this was only applicable for
one patient.
In contrast to the Melatonin Group (which showed immediate decrease of sleep onset latency starting from Treatment Day 1), the Placebo Group showed decreasing sleep
onset latency values from Week 1 to Week 4, although not
statistically significant. One reason for this finding could be
the conditioning effect of parents checking on the child every ten minutes. �����������������������������������������
If children are anxious, knowing and having a parent come in and check on them in set intervals
may have a curing effect on their insomnia. It is a common
finding in randomized clinical trials studying treatment effect in insomniacs that placebo produces significant changes
on self-reported sleep measures (8). However, in our studies,
sleep measures did not significantly change during placebo
treatment, with one exception. This might be explained by
the fact that the sleep measures were not assessed by the
patients themselves.
There are several limitations of this study; one of which
is that we did not assess sleep objectively by means of polysomnography or actigraphy. However, parents of sleep-disturbed infants have shown to be accurate reporters of actigraphically assessed sleep onset and sleep duration (9).
We used the data of two studies, with different timing of melatonin administration, although the two
Sleep Sci. 2010;3(1):�����
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subsets show very similar characteristics of sleep-wake
rhythm and endogenous melatonin onset. Moreover, the
effect of melatonin on sleep parameters did not differ
between the two studies (1-2). Melatonin treatment for
chronic insomnia in children has been criticized in the
Netherlands. Jenni (10) stressed that cultural variation
and developmental variation and biological variability
of sleep behavior among normal healthy children should
be taken into account. However, in our studies we did
not include healthy children, but children with chronic
complaints of insomnia who showed impaired general
health and quality of life likely due to their insomnia
problems (11).
To conclude, the results presented here provide proof that
onset of melatonin treatment effect can be expected within
a few days and this effect remains stable in the weeks after
that, in addition to the earlier evidence (1,2,12) that melatonin
is an effective treatment for sleep onset insomnia in children
with late melatonin onset.
ACKNOWLEDGEMENTS
We thank Professor ACG Egberts from Utrecht Institute
for Pharmaceutical Sciences (UIPS), Department of Pharmacoepidemiology and Pharmacotherapy, Faculty of Science,
Utrecht University, and
�����������������������������������
Professor H Vaarkamp from �����
Pharmacy Department, Faculty of Veterinary Medicine,,Utrecht
University) for helpful discussions and critically reading the
manuscript.
REFERENCES
1. Smits MG, Nagtegaal EE, van der Heijden J, Coenen AM, Kerkhof GA. Melatonin for chronic sleep onset insomnia in children: a randomized placebo-controlled trial. J Child Neurol.
2001;16(2):86-92.
2. Smits MG, van Stel HF, van der Heijden K, Meijer AM, Coenen
AM, Kerkhof GA. Melatonin improves health status and sleep in
children with idiopathic chronic sleep-onset insomnia: a randomized placebo-controlled trial. J Am Acad Child Adolesc Psychiatry. 2003;42(11):1286-93.
3. Arendt J, Skene DJ. Melatonin as a chronobiotic. Sleep Med Rev.
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4. Carr R, Wasdell MB, Hamilton D, Weiss MD, Freeman RD, Tai
J, et al. Long-term effectiveness outcome of melatonin therapy in
children with treatment-resistant circadian rhythm sleep disorders. J Pineal Res. 2007;43(4):351-9.
5. Wasdell MB, Jan JE, Bomben MM, Freeman RD, Rietveld WJ,
Tai J, et al. A randomized, placebo-controlled trial of controlled
release melatonin treatment of delayed sleep phase syndrome and
impaired sleep maintenance in children with neurodevelopmental
disabilities. J Pineal Res. 2008;44(1):57-64.
6. Ashrafi MR, Mohammadi M, Tafarroji J, Shabanian R, Salamati P, Zamani GR. Melatonin versus chloral hydrate for recording sleep EEG. Eur J Paediatr Neurol. 2009;doi:10.1016/j.
ejpn.2009.06.010. [Epub ahead of print]
van Geijlswijk IM, Didden R, van der Heijden KB, Smits MG, van Leeuwe JF
7. Hoebert M, van der Heijden KB, van Geijlswijk IM, Smits
MG. Long-term follow-up of melatonin treatment in children
with ADHD and chronic sleep onset insomnia. J Pineal Res.
2009;47(1):1-7.
8. Perlis ML, McCall WV, Jungquist CR, Pigeon WR, Matteson SE.
Placebo effects in primary insomnia. Sleep Med Rev. 2005;9(5):
381-9.
9. Sadeh A. Evaluating night wakings in sleep-disturbed infants: a
methodological study of parental reports and actigraphy. Sleep.
1996;19(10):757-62.
10.Jenni OG. Sleep onset insomnia during childhood or poor fit
between biology and culture: comment on van der Heijden et
al. ‘Prediction of melatonin efficacy by pre-treatment dim light
melatonin onset in children with idiopathic chronic sleep onset
insomnia’. J Sleep Res. 2005;14(2):195-7; discussion 197-9.
11.van der Heijden KB, Smits MG, van Someren EJ, Boudewijn Gunning W. Reply to Jenni - Childhood chronic sleep onset insomnia
and late sleep onset: What’s the difference? J Sleep Res. 2005;14(2):
197-9.
12.van der Heijden KB, Smits MG, van Someren EJ, Boudewijn
Gunning W. Prediction of melatonin efficacy by pretreatment
dim light melatonin onset in children with idiopathic chronic
sleep onset insomnia. J Sleep Res. 2005;14(2):187-94.
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21
Sleep
Science
ORIGINAL ARTICLE
Human period-3 gene involvement in diurnal preference
among Argentinean bipolar disorders patients
O envolvimento do gene humano período-3 na preferência diurna de
uma população argentina de pacientes com transtornos bipolares
Leandro Pablo Casiraghi1, Diego Martino2, Eliana Marengo2, Ana Igoa2, Ezequiel Ais2, Sergio Strejilevich2, Diego Andrés
Golombek1
ABSTRACT
Background and objective: Due to circadian disturbances observed
among bipolar disorder (BD) patients, several studies have analyzed a
possible link with genes involved in the molecular generation of biological rhythms. Some of these genes have been previously associated
with diurnal preference (i.e., chronotype) in healthy individuals. In
this study, we aimed to establish the influence of two genetic polymorphisms on chronotypes among two local populations of healthy
subjects and individuals affected by bipolar disorders. Methods: The
polymorphisms analyzed were a variable number of tandem repeats (4
or 5-repeat alleles; 4R or 5R) in exon 18 of hper3, and a T/C single
nucleotide polymorphism in clock. Chronotypes of healthy individuals
(n=39) and patients (n=37) were determined by the Horne and Ostberg Questionnaire, and the control population was divided into three
groups according to results. Results: No difference in allele or genotype
frequencies was detected between control and bipolar disorder populations. In controls, the 5R allele in hper3 and the C allele in clock were
sub-represented among evening-type individuals (p<0.05). Moreover,
subjects homozygous for the 4R allele of hper3 had significantly lower
scores (evening preference) than 5R allele carriers among both controls
(p<0.05) and patients (p<0.001).Conclusions: We have confirmed the
strong correlation of hper3 variable number of tandem repeats (VNTR)
polymorphism with diurnal preference on a local population, even
when not taking into account chronotype classification. This correlation was replicated with an even stronger robustness, on patients with
bipolar disorders. These findings add to the growing body of evidence
supporting an important role for hper3 on human circadian physiology.
Keywords: Chronobiology disorders; Bipolar disorder; ��������
Polymorphism, genetic; Sleep/physiology; Phenotype; Minisatellite repeats
RESUMO
Introdução e objetivo: Devido a distúrbios circadianos observados
entre pacientes portadores de transtornos bipolares (TBP), vários estudos têm se focado em uma possível associação genética na geração
molecular de ritmos biológicos. Alguns genes já foram associados anteriormente à preferência diurna (por exemplo, o cronótipo) em indi-
víduos saudáveis. O enfoque neste estudo foi estabelecer a influência
que dois polimorfismos genéticos exercem sob os cronótipos pertencentes a duas populações locais saudáveis e a indivíduos portadores de
transtornos bipolares. Métodos: Os polimorfismos analisados consistiram em um número variado de repetições em pares (alelos de 4 ou 5
repetições; 4R ou 5R) em exon 18 do hper 3, e um T/C polimorfismo
de nucleotídeo simples em relógio. Os cronótipos de indivíduos saudáveis (n=39) e de pacientes com transtornos bipolares (n=37) foram
determinados pelo questionário Home-Ostberg, e a população controle foi dividida em três grupos de acordo com os resultados colhidos.
Resultados: Nenhuma diferença em alelos de frequência de genótipos
foi encontrada entre os pacientes controles e as populações com transtornos bipolares. Nos controles, o alelo 5R no hper3 e o alelo C no
relógio foram representados como indivíduos noturnos (p<0,05). E
indivíduos homozigóticos no alelo 4R do hper3 tiveram pontuações
mais baixas (preferência noturna) do que os alelos 5R entre os grupos
controle (p<0,05) e indivíduos com transtornos bipolares (p<0,001).
Conclusões: Confirmamos uma forte relação de polimorfismo do
hper3 VNTR com preferência diurna dentro da população local mesmo desconsiderando a classificação de cronótipos. Tal correlação foi
reproduzida com mais robustez entre portadores de transtornos bipolares. Estas conclusões vêm ao encontro do que tem sido levantado
em estudos anteriores, que indicam o papel importante que o hper3
desempenha na fisiologia circadiana humana.
Descritores: Transtornos cronobiológicos; Transtorno bipolar; Polimorfismo genético; Sono/fisiologia; Fenótipo; Repetições minissatélites
INTRODUCTION
Circadian rhythm is a widespread property of living organisms. A number of circadian biological clocks and the genetic machinery involved in the generation and modulation
of these rhythms have been extensively described and are
still under active study. In mammals, the biological clock
that controls circadian rhythms is located at the suprachiasmatic nuclei of the hypothalamus (SCN). The genes that
Study carried out at Fundación Favaloro, Buenos Aires, Argentina.
1
Departamento de Ciencia y Tecnología da Universidad Nacional de Quilmes, Buenos Aires, Argentina.
2
Instituto de Neurociencias, Programa de Trastornos Bipolares da Fundación Favaloro, Buenos Aires, Argentina.
Corresponding author: Diego Andrés Golombek – Departamento de Ciencia y Tecnología, Universidad Nacional de Quilmes – Roque S. Peña, 352 – (1876) Bernal, Pcia
de Buenos Aires, Argentina – Tel.: +54 11 4365-7100, ext. 4154 – Fax: +54 11 4365-7132 – E-mail: [email protected]
Received: December 18, 2009; Accepted: February 21, 2010
Sleep Sci. 2010;3(1):�����
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Casiraghi LP, Martino D, Marengo E, Igoa A, Ais E, Strejilevich S, Golombek DA
govern circadian functioning within SCN neurons are well
conserved among mammals, and include clock, bmal1, the
per and cry families and a large array of complementary
genes (1,2). It has been demonstrated that mutations affecting
these so-called “clock genes” originate circadian alterations
in several animal models.
The sleep-activity cycle is the most evident circadian
rhythm in humans, and a classification of phenotypes,
known as chronotypes, according to diurnal preferences of
sleep and activity timing has been proposed. Several polymorphisms in human clock genes have been shown to influence chronotype (3-6), and some of them have also been
linked to the appearance of sleep disorders like Familial
Advanced Sleep Phase Syndrome (FASPS) (7,8) or Delayed
Sleep Phase Syndrome (DSPS) (5,6,9), and more recently it
has been shown that clock genes can also affect sleep architecture (10).
The link between Bipolar Disorders (BD) and the circadian system has been studied for the last decades; indeed,
several circadian-related issues have been reported, mainly sleep disorders, both before and after the onset of the
illness (11-15). Another strong evidence of this link is that
lithium, the main drug used in the treatment of BDs, has
been shown to affect circadian rhythms in rodents and flies
by inhibiting GSK3b, a kinase involved in the secondary
loop of the molecular circadian clock (16,17). Moreover, certain clock gene polymorphisms have been recently linked
to diverse BDs features, as age of onset and certain treatment responses (18-23).
Since it has been suggested that photoperiod related to
latitude may have an effect on circadian adaptation (6), we
considered it important to try to replicate previous findings
related to the effect of clock genes polymorphisms on chronotypes on a local population of healthy individuals. At the
same time, we also extended this research onto a BDs patient
population in order to assess whether this reported genetic
correlation could persist even under this circadian-disturbing psychiatric condition.
We chose two chronotype-related polymorphisms, a variable number of tandem repeats (VNTR) in exon 18 of hper3
and a single nucleotide polymorphism (SNP) in the 3´UTR
region of clock, which had been both previously reported to
affect diurnal preference (3,5,6). We analyzed their influence on
the diurnal preference of an Argentinean group of healthy
volunteers and a population of BD patients, comparing the
frequencies of alleles and genotypes present among groups.
METHODS
Volunteers and patients
Subjects with BD (n=37; age 46.09±14 years; 30% males)
were consecutively selected from the outpatients popula-
tion of the Bipolar Disorder Program of the Favaloro Foundation (Buenos Aires, Argentina) with the following inclusion criteria: age between 18 and 75 years old; diagnosis
of BDI or BDII according to Diagnostic and Statistical
Manual of Mental Disorders – Fourth edition (DSM-IV)
using Structured Clinical Interview for DSM-IV (SCID)
(24)
; euthymic (defined by Hamilton Depression Rating
Scale ≤8 and Young Mania Rating Scale ≤6) for at least 8
weeks. Exclusion criteria were: other diagnosis in axis I,
antecedent history of substance abuse, history of mental
retardation or neurological disease. A total of 53% of patients included in the study suffered from BDI, while the
remaining 47% suffered from BDII. All patients signed a
special informed consent providing information regarding
study objectives, methodologies and secrecy of all personal
data. All procedures regarding patients were approved by
a local Ethics Committee before starting the study. Patients were on pharmacological treatment at the time of
the study: 94.7% of patients were taking mood stabilizers,
18.4% antidepressants, 42.1% antipsychotics and 13.1%
benzodiazepines.
A Control Group (n=39; age 28.5±9 years; 25% males)
was recruited among students at the National University of
Quilmes, near Buenos Aires, Argentina (latitude -58°23’).
All of them signed an informed consent and were interviewed to control for psychiatric or sleep disorders before
being analyzed. The Horne & Ostberg Morningness-Eveningness Questionnaire (HO) (25) was applied to all selected
subjects and patients. The HO questionnaire gives a score of
16 to 86 points for each tested subject: low scores indicate
evening preference and high scores indicate morning preference.
For the chronotype analysis among the control population, we selected the 9 lower scores in the HO test to conform the Evening-type Group, the 9 higher scores for the
Morning-type Group, and the middle 10 scores for the Intermediate Group. Individuals with scores falling between
these groups were discarded from chronotype analysis to
avoid bias from border behavior, but they were later included in the morningness-eveningness distribution according
to genotype analysis.
Genotyping
Fingertip, blood or mouth epithelium tissue samples were
collected from healthy volunteers and BD patients and
placed on Whatman FTA Cards (Whatman, UK), which
were later processed for DNA extraction. Polymorphisms
studied were a VNTR (4-repeat or 5-repeat possible alleles;
4R or 5R, respectively) in exon 18 of hper3, previously reported by Ebisawa et al. (9), and a T/C SNP in the 3´UTR
region of clock reported by Katzenberg et al. (3), and PCR
Sleep Sci. 2010;3(1):�����
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23
24
Human period-3 gene involvement in diurnal preference
primers and conditions were replicated. Determination of
hper3 VNTR alleles was performed by settling the molecular weight of the PCR product on 2% agarose gels. The
clock SNP genotypes were obtained by restriction fragment
length polymorphism technique (RFLP) using Bsp1286i restriction enzyme (Promega, WI), which cleaves exclusively
the C allele, leaving the T allele intact.
Allele frequencies among chronotypes
We found that both hper3 5R allele and clock C allele were
significantly sub-represented in evening-types as compared
to intermediates (two-tailed Fisher’s exact test p<0.05;
Odds Ratio: 5.00; 95% confidence interval 1.095-22.828).
No differences were detected between Morning and Intermediate groups (Figure 1).
Statistical analysis
Differences in allele frequencies and genotype between
groups were analyzed by two-tailed Fisher’s Exact test
(Graph Pad Software). Differences in HO scores between
genotype groups in control volunteers and BD patients were
analyzed by two-tailed Student’s t test or Two-Way ANOVA
using Graph Pad Prism software. Results are expressed as
Mean ± SEM.
RESULTS
Allele and genotype frequencies in healthy controls
and bipolar disorders patients
No significant difference in age or gender distribution was
detected between the control group and the population of
patients. Table 1 shows the allele and genotype frequencies
found in controls and in BD patients for both hper3 and
clock polymorphisms. No significant differences were detected between groups for alleles or genotypes distributions,
even when a slightly reversed trend in genotype frequencies,
as determined by presence or absence of the less frequent allele for each gene (i.e. the 5R allele for hper3 and the C allele
for clock), were appreciated.
Table 1: Allele and genotype frequencies in both Control and Patient
Group
Allele
Group
p value
n
4R
5R
0.63
0.37
Control 39
(49/78) (29/78)
0.72
0.28
BD
34*
(49/68) (19/68)
T
C
0.77
0.23
Control 39
(60/78) (18/78)
0.69
0.31
BD
36*
(50/72) (22/72)
0.29
0.35
Genotype
hper3
4/4
4/5
5/5
0.41
0.44
0.15
(16/39) (17/39) (6/39)
0.59
0.26
0.25
(20/34) (9/34) (5/34)
Clock
T/T
T/C
C/C
0.59
0.36
0.05
(23/39) (14/39) (2/39)
0.44
0.50
0.06
(16/36) (18/36) (2/36)
p value
Figure 1: Comparison of allele frequencies of (A) hper3 4R/5R VNTR
polymorphism and (B) clock T/C SNP among chronotype groups, as described in the Methods section. A clear significant sub-representation of
the hper3 5-repeats and the clock C alleles in evening-types, as compared
to intermediates, can be appreciated.
0.16
0.25
Values in brackets represent absolute values (allele/genotype appearances
over total present). Two-tailed Fisher’s exact test p values are presented
for both allele and genotype (as 5R or C allele carriers and non-carriers)
frequencies.
*Not all BDs patients could be genotyped for both polymorphisms
since we were unable to get a new biological sample after a failure in the
genotyping process.
Sleep Sci. 2010;3(1):�����
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Two-tailed Fisher’s exact test, *p<0.05; Odds Ratio: 5.00; 95%
confidence interval 1.095-22.828; see text for details;
Numbers inside bars indicate specific allele appearances over total alleles
present.
Morning-types, n=9, Intermediates, n=10 and Evening-types, n=9.
Morningness-eveningness scores according to
genotype
We classified both control and BD populations into genotypes for each gene according to the presence or absence of
the less frequent allele of each polymorphism and analyzed
the mean HO score for each group. We found a significant
effect of genotype of hper3 VNTR on mean scores. 5R allele
carriers (n=23) had significantly higher scores, i.e. morning type scores, than non-carriers (n=16; 50.23±1.69 versus
42.93±2.78; p<0.05) (Figure 2). No differences were found
between clock C allele carriers and non-carriers (not shown).
It should be noted that in this analysis no subjects are discarded as in the chronotype groups study.
Casiraghi LP, Martino D, Marengo E, Igoa A, Ais E, Strejilevich S, Golombek DA
Figure 2: Effect of hper3 genotype on Horne-Ostberg questionnaire score
in control and BD groups. Mean HO score for both populations classified in genotypes according to the presence (5+ genotype) or absence
(5- genotype) of the 5-repeat allele of hper3. Significantly higher scores
(i.e. morning preference) are found in 5+ individuals, as compared to 5subjects. A very significant (*p<0.0001 for effect of genotype) effect of
5R allele presence on scores is detected, but no effect of health status and
no interaction, as reported by a two-way ANOVA.
Among BD patients, we found an even stronger correlation of hper3 genotype on mean HO scores. The 5R allele carriers (n=14) had a mean score of 52.93±2.46, while
non-carriers (n=20) had a 37.00±2.86 mean score (p<0.001)
(Figure 2). Again, no differences were detected regarding
the presence of the clock C allele (not shown).
A two-way ANOVA revealed a significant (p<0.0001)
effect of hper3 genotype on HO score, but no effect of bipolar disorders condition.
DISCUSSION
Our finding about the influence of both hper3 VNTR and
clock SNP polymorphisms on diurnal preference among
healthy individuals confirms previously published results
(3,5,6)
. The importance of the first study of this kind on Argentinean populations lies on the hypothesis first proposed
by Pereira et al. (6), based on their work on the length polymorphism hper3 and DSPS, which states that differences in
latitude (i.e., in seasonal photoperiods) may have different
effects on circadian functioning in relation to this gene variants. There have been some confronting results with regard
to the influence of clock 3111 T/C polymorphism on diurnal
preference (26,27), but, as previously noted, differences in photoperiod may account for apparently opposing results.
Regarding our hper3 results, it is important to highlight
the fact that, even without taking into account chronotype
classification, we have shown that the length polymorphism
studied has an effect per se on diurnal preference determined
by HO questionnaire, suggesting that hper3 variants effect
may exist not only on extreme phenotypes. These findings
add to several recent studies that attribute an important role
for hper3 in human physiology in relation to sleep and circa-
dian functioning (10,28-30), which may also include a relationship with cancer processes (31,32).
The effect on diurnal preference of hper3 VNTR polymorphism was replicated on a population of bipolar disorder patients, with more robust score differences between 5R
allele carriers and non-carriers. It is very interesting that
BD patients, who are known to suffer from several types of
circadian disturbances (mainly sleep problems), and who
are under pharmacological treatments that affect circadian
rhythms, show a similar diurnal preference to that found in
healthy individuals. Previous works by other groups have
looked for an effect of hper3 variability on BD features and
predisposition (19,22), and recently some groups have studied chronotypes among BD patients and reported that they
display an evening preference as compared to controls (33,34).
Our present results should be taken into account in such
analysis, as the eveningness reported may hold relation with
hper3 variants.
A probable cause of the strong correlation among BD
patients could be the above mentioned circadian disturbances. It can be hypothesized that, although all patients
are treated in order to minimize these disturbances, these
internal disturbances are driving individual subjects more
strongly to evening or morning-like behavior. We are currently evaluating if these polymorphisms affect the need of
bipolar disorder patients for pharmacological treatment for
sleep disorders.
Altogether, the present paper confirms previous findings
related to the effect of clock and hper3 polymorphisms on
chronotypes, and shows for the first time that the correlation between hper3 VNTR variants and diurnal preference is
maintained on BD patients.
ACKNKOWLEGDMENTS
This paper was Supported by ����������������������������
Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Agencia Nacional de Promoción Científica y Tecnológica (ANPCyT) and
Universidad Nacional de Quilmes (UNQ).
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Sleep
Science
ORIGINAL ARTICLE
Anticipatory behavioral rhythm to scheduled
glucose availability in rats
Ritmo comportamental antecipatório à disponibilidade
programada de glicose em ratos
Breno Tercio Santos Carneiro1, Fabiano Santos Fortes1, John Fontenele Araujo1
ABSTRACT
Background and objective: Food-anticipatory behavioral rhythms
are observed in several species, including mammals. It is reported that
acute ingestion of glucose phase-shifts the food-entrainable oscillator
(FEO) in rats. Here, we intended to extend the knowledge about the
role of glucose on anticipatory behavioral rhythms by submitting rats
to a long period of scheduled glucose availability. Methods: Adult
Wistar rats were under 12:12 hours light-dark cycle for three days
with ad libitum feeding (Baseline) prior to being submitted to ten days
of scheduled glucose restriction (GR) (three hours duration) starting
at Zeitgeber time (ZT) 06 and two days of meal (glucose) omission.
Results: The animals anticipated glucose availability by increasing
motor activity in the two hours preceding ZT 06. The anticipatory
behavior observed during the glucose restriction persisted on the second day of glucose omission (i.e. when glucose was not offered). That
is, this anticipatory behavior was self-sustained, evidencing the true
entrainment of the rhythm. Conclusion: Glucose ingestion was a sufficient temporal cue for inducing endogenously-generated circadian
anticipatory behavior in the rat.
Keywords: Glucose/administration & dosage; Circadian rhythm;
Feeding behavior; Rats; Models, neurological; Signal transduction
RESUMO
Introdução e objetivo: Ritmos comportamentais antecipatórios
ao alimento são observados em várias espécies, incluindo mamíferos.
Foi reportado que a ingestão aguda de glicose muda a fase do oscilador sincronizado por ciclo de alimento em ratos. Aqui, pretendeu-se
estender o conhecimento sobre o papel da glicose nos ritmos comportamentais antecipatórios submetendo ratos a um longo período
de disponibilidade programada de glicose. Métodos: Ratos Wistar
adultos ficaram sob ciclo claro-escuro de 12:12 horas por três dias com
alimentação à vontade (linha de base) antes de serem submetidos a
dez dias de restrição de glicose (RG) (três horas de duração), começando na hora do Zeitgeber (HZ) 06 e dois dias de omissão de comida
(glicose). Resultados: Os animais anteciparam a disponibilidade de
glicose aumentando a atividade motora nas duas horas precedentes à
HZ 06. O comportamento antecipatório observado durante a restrição
de glicose persistiu no segundo dia de omissão de glicose (i.e., quando
a glicose não foi oferecida). Ou seja, este comportamento antecipatório era autossustentado, evidenciando a sincronização real do ritmo.
Conclusão: A ingestão de glicose foi uma pista temporal suficiente
para induzir um comportamento circadiano antecipatório gerado endogenamente no rato.
Descritores: Glicose/administração & dosagem; Ritmo circadiano;
Comportamento alimentar; Ratos; Modelos neurológicos; Transdução
de sinal
INTRODUCTION
For the organisms living on or near earth’s surface, the main
Zeitgerber is the cyclic alternation between light and dark periods. In mammals, the pacemaker located in the suprachiasmatic nucleus (SCN) is entrained by the light-dark cycle.
The output from the SCN to other regions in the brain leads
to the expression of behavioral and physiological parameters
adjusted to long day (LD) (1). It has been reported, however,
that under conditions of scheduled restricted feeding, clock
gene oscillations in different brain regions and in peripheral
tissues become entrained to feeding time (2-6).
During periods of scheduled restricted feeding, rats
and other species show increased activity (e.g. locomotion,
wheel-running activity, food-bin activity) in the two to three
hours preceding food availability (7,8). Under natural conditions, periods of food scarcity may occur. In this situation, it
must be important for species to be behaviorally and physiologically prepared when food sources become available in a
determined time of day.
The anticipation to feeding time was named food-anticipatory activity (FAA) in early years, and its oscillatory and
circadian properties were well demonstrated (7,8). The loca-
Study carried out at Laboratório de Neurobiologia e Ritmicidade Biológica, Departamento de Fisiologia, Universidade Federal do Rio Grande do Norte – UFRN, Natal
(RN), Brazil.
1
Laboratório de Neurobiologia e Ritmicidade Biológica, Departamento de Fisiologia, Universidade Federal do Rio Grande do Norte – UFRN, Natal (RN), Brazil.
Corresponding author: Breno Tercio Santos Carneiro – Centro de Biociências, Departamento de Fisiologia, Universidade Federal do Rio Grande do Norte – Campus
Universitário, Lagoa Nova – Caixa Postal 1506 – CEP 59078-970 – Natal (RN), Brazil – Phone: (84) 3215-3409 (Ext 218); Fax: (84) 3211-9206 – E-mail: brenotercio@
yahoo.com.br, [email protected].
Received: January 7, 2010; Accepted: February 24, 2010
Sleep Sci. 2010;3(1):�����
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Anticipatory behavioral rhythm
tion of the oscillator responsible for this circadian behavior
has been investigated during the last 30 years. In late 1970’s,
Stephan et al. (9) first showed that the pacemaker entrained
by light located in the SCN was not necessary for the expression of food-anticipatory activity. Since then, many investigators have tried to locate the so-called food-entrainable
oscillator (FEO) in central or peripheral tissues (3,7,8,10).
Nonetheless, the search is still intense and discussions on
the organization of the FEO have increased in the last years
(10-12)
. Besides the importance of the location of the FEO,
another important question to be answered is: what are the
mechanisms by which the anticipatory behavioral rhythm
is entrained? In other words, what are the signals associated
with feeding that transmit information to the brain about
scheduled food availability?
In face to the relevance of different humoral signals acting in many brain sites to regulate food intake, their daily
fluctuation and, more importantly, their fluctuation in response to feeding schedules, we recently proposed that foodanticipatory behaviors would be generated by the coordinated activity of different brain areas which are directly or
indirectly entrained by an array of humoral signals which
have their concentration cycling in response to food ingestion. These include both orexinergic and anorexinergic signals in the blood (11).
It has been reported that a minimum caloric content,
but not gastric stuffing, is a necessary cue for inducing
food-anticipatory behavior (13,14). Stephan and Davidson (15)
also showed that, despite high caloric content of mineral
and vegetable oil, they are not capable of phase-shifting
the food-entrainable oscillator as glucose is. These authors
initially entrained rats to scheduled regular chow and then
phase-delayed the feeding time in eight hours. On the first
two days of delay, rats received mineral or vegetable oil, saccharin or glucose+saccharin. The animals receiving glucose
showed larger phase-delays and reentrained faster to mealtime, which suggests a role for this nutrient in the oscillatory mechanism of the FEO. Based on our proposal, in
this study we intended to test one of the signals that might
be involved in timing the FEO in the brain (i.e., glucose)
by submitting rats to scheduled glucose availability in the
middle of light phase for a long period (ten days).
METHODS
The research was approved by the Ethics Committee for Use
of Animals of Universidade Federal do Rio Grande do Norte
(protocol nº 026/2009). Ten 5-month-old Wistar rats (five
males and five females), at the beginning of the experiment,
were used. The animals were single-housed in polypropylene cages (32x40x17 cm) inside ventilated wooden boxes
with controlled temperature (23±1°C) and light-dark cycle
Sleep Sci. 2010;3(1):�����
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(12:12 hours). Recordings of the motor activity started at
ZT 11.5 (by convention, ZT 12 is defined as the time of
lights-off) on day 0 (Figure 1).
Under a 12:12 hours light-dark cycle (lights-on at 6h),
the animals were submitted to three days of Baseline with
ad libitum tap water and rodent chow (Labina, Purina®). The
chow was withheld at ZT 11.5 on day 3. From day 4 to
13, without access to regular chow, each rat had 20 mL of
glucose solution (50%) (Isofarma®, Industrial Farmacêutica
Ltda.) in two plastic flasks of 10 mL available from ZT 06
to ZT 09 (Glucose restriction). Then, the glucose solution
was not offered for two days (14 and 15) (Omission). To allow the animals to learn how to drink in the glucose plastic
flasks, 10 mL was offered at ZT 11.5 and remained available
for 24 hours on days 0, 1 and 2.
The motor activity rhythm was recorded by infrared motion sensors positioned 15 cm above the cage lids connected to a personal computer where data were collected each
5 minutes using Aschoff software. Motor activity data were
transformed in percentage of total daily activity. Anticipation was defined as the amount of activity during the two
hours preceding the glucose availability (i.e. between ZT 04
and ZT 06). Actograms were drawn on El Temps software (A.
Díez-Noguera, Universitat de Barcelona, 1999). For statistical analyses, repeated measures ANOVA followed by Tukey
HSD test, t test for dependent samples and Pearson’s correlation were performed on Statistica 7 software. Analyses were
considered statistically significant when p<0.05. Data are
expressed as means±standard error of mean (SEM).
Baseline
Days
28
Glucose
restriction
Omission
Zeitgeber time
Figure 1: A double-plotted actogram of the motor activity rhythm from
a representative rat. Zeitgeber time (ZT) is indicated at the bottom. Bar
indicating the light-dark cycle is showed on the top. Days 1 to 3 were
Baseline. Glucose restriction (vertical grey bar, ZT 06-09) started on day
4 and finished on day 13. On days 14 and 15, the glucose solution was not
offered (omission). Anticipatory behavior is seen during glucose restriction and on days of omission.
Carneiro BTS, Fortes FS, Araujo JF
RESULTS
The animals learned how to drink the glucose solution in
their flasks. Glucose ingestion was 2.85±0.67 mL on day
4 and 7.7±0.73 on day 13 (p<0.001, t test for dependent
samples). An anticipatory behavioral rhythm is evidenced by
**p<0.01, post-hoc Tukey HSD test after ANOVA for repeated measures.
Figure 2: (A) Averaged motor activity between ZT 04-06 (anticipation)
over three days of baseline and glucose restriction (GR) and on omission
2nd day. High level of motor activity is evident in the last three days
of GR and this anticipatory behavior persists on the second day of meal
omission. (B) Correlation between glucose ingested and the anticipation
on the next day. R=0.445, p<0.001, Pearson’s correlation.
visual inspection of the actogram (in this representation, the
days are xy graphs of 48 hours plotted sequentially from the
top to the bottom) on Figure 1.
Figure 2A shows averaged anticipation over the baseline
days and the last three days of glucose restriction and on the
second day of omission. Gathering data from all days of glucose restriction, we found a statistically significant correlation between glucose ingestion and the level of anticipation
on the next day (R=0.445, p<0.001, Pearson’s correlation)
(Figure 2B).
DISCUSSION
We found that scheduled glucose ingestion for ten days is a
cue sufficient to induce anticipatory behavior in rats. Also,
the volume ingested was correlated with the anticipatory
behavior observed, corroborating the hypothesis that the
caloric content is necessary for the expression of food-anticipatory behaviors (13).
The most common measure for observing food anticipation is the locomotor behavior in a running wheel. In rats with
scheduled food availability in the middle of the light phase, for
example, a pronounced increase in wheel-running activity is observed at the hours preceding feeding time, almost no running
after, and again high activity levels in the dark phase, mainly
in the first half. The pattern observed in general motor activity is a bit different from that in a running wheel. As the motion sensor detects all kinds of movement (locomotion, feeding,
grooming), the records are more spread. Most of the animals
in this study showed a pattern similar to Figure 1, with high
levels of activity after the glucose availability. Some showed a
pattern more similar to that observed in running wheels, with
a lower level of activity when compared to other animals in the
hours succeeding glucose availability. Regarding the anticipatory behavior, we observed that it is not different between rats
anticipating glucose and rats anticipating chow at the same circadian phase (unpublished data).
Mistlberger et al. (16) reported that feeding paradigms of
protein and fat or protein and carbohydrate, each one available for one hour daily in the light phase and separated by
seven hours, induces anticipatory food-bin activity in rats.
They also showed that these macronutrients were ineffective
to induce anticipation if the animals were not under caloric restriction. Later, Stephan and Davidson (15) showed that
glucose ingestion, but not other caloric substances (mineral
or vegetable oil), rapidly phase-shifts the food-entrainable
oscillator in rats. This suggests that glucose concentration
could directly alter the activity of the food-entrainable oscillator. Alternatively, as a consequence of glucose ingestion,
the concentration of other humoral signals (e.g. hormones)
would change and this could serve as a phase-shifting cue for
the FEO, or the two processes could occur simultaneously.
The observation by Stephan and Davidson that fat ingestion
is not effective in phase-shifting the FEO might seem contradictory to the Mistlberger et al. report that fat induces
anticipatory behavior. However, we should consider methodological differences in the two studies. Rats in Mistlberger
et al. study were maintained on the feeding paradigm for 14
days. In Stephan and Davidson study, however, the animals
were fed for only two days with fat and phase-shifts were
measured. In the second study, the rats were maintained on
the feeding paradigm for 14 days. It is possible that fat ingestion triggers the signals necessary to entrain the FEO in a less
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30
Anticipatory behavioral rhythm
effective manner, thus being required a longer period to observe anticipation, and consequently phase-shifts would not
be evident in only two days of ingestion. On the other hand,
glucose ingestion could be more effective in altering the internal signals necessary for entrainment (the rise in plasma
glucose itself, for example). It is not completely described,
actually, which signals associated with food ingestion are
necessary or indispensable for entrainment of anticipatory
circadian behaviors.
In a recent report, Waddington Lamont et al. (2) showed
that rats receiving sucrose (fructose+glucose) in the middle
of the light phase do not express anticipatory behavior.
Again, methodological differences must be accounted. Here,
we used concentrated glucose at 50% while they used sucrose at 32%; in the present study, the behavioral measure
was the general motor activity while Waddington Lamont
et al. used wheel-running activity; and the most important
difference we consider is that in the study by Waddington
Lamont et al., the animals had chow ad libitum plus sucrose
at midday while our rats had glucose as their only food
source. We discussed that a caloric restriction is required
for the expression of food-anticipatory behaviors (16), thus
the absence of anticipatory behavior in Waddington Lamont
et al. study could be due to the absence of caloric restriction, since their rats had chow ad libitum. The same research
group has, actually, reported later that a highly palatable
food does not induce anticipation unless the animal is in a
negative metabolic state (17).
Blood glucose is one of the humoral parameters that
change in response to food ingestion. It rises specifically after
a meal. In this paper, we could not attribute the entrainment
of anticipatory behavior to glucose only, since during the
glucose restriction period other signals might contribute in
some way for its expression. Some of these signals have been
shown to fluctuate in response to scheduled feeding (e.g.,
ghrelin, leptin, glucagon, insulin) (18-20) and it is likely that
they would also respond to long-term scheduled glucose ingestion. Also, it has been shown that some of these signals affect variables that entrain to mealtime and/or modify the expression of food-anticipatory behaviors (4,21,22). Our hypothesis
is that the change in the concentration of a range of humoral
signals (related to the regulation of food intake) after a meal
is responsible for timing the food-entrainable oscillator in the
brain, which is responsible for the expression of anticipatory
behavioral rhythms (11).
Regarding glucose itself, it has been shown that its extracellular concentration alter the firing rate of neurons in
important hypothalamic regions involved in the regulation
of food intake, promotion of arousal and which entrain to
feeding schedules, such as the arcuate nucleus (ARC), the
ventromedial nucleus (VMN) and the lateral area (23). This
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points to the relevance that the fluctuation in glucose concentration probably has in timing different brain regions. A
recent work has suggested a role for the VMN in the initiation of the food-anticipatory behavior (24). Previous reports
have also pointed the importance of the LH on the expression of food anticipation (25). We must mention, however,
that there are controversies regarding the importance of the
integrity of these and many other areas for the expression
of food-anticipatory behavioral rhythms (11). The results obtained in most lesion studies accomplished until now should
indicate that the FEO, instead of constituted by one single
area in the brain, is rather composed of a network of different areas (11) or an emergent oscillator born due to the feeding/fasting cycle present during feeding schedules (12).
Reports discussed above point that highly palatable food
associated with food deprivation induces anticipatory behaviors in rats. In this study, we eliminated any other nutrient
from feeding and showed that glucose is able to induce similar food-anticipatory rhythms. We could say that a rise in
plasma glucose constitutes at least one of the signals responsible for entrainment of anticipatory behavioral rhythms.
Perhaps, the change in the concentration of many humoral
signals due to food intake is the entraining cue to the foodentrainable oscillator in the brain (11).
We were able to show that glucose ingestion is a temporal cue sufficient to induce anticipatory behavioral rhythm
in rats without access to any other food source and that anticipation varies as a function of volume ingested and, consequently, the caloric content.
ACKNOWLEDGMENTS
Grant sponsors: Conselho Nacional de Desenvolvimento
Científico e Tecnológico (CNPq) and Fundação de Amparo
à Pesquisa do Estado do Rio Grande do Norte (FAPERN).
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Sleep
Science
ORIGINAL ARTICLE
Effects of chronobiology on prostate cancer cells growth in vivo
Efeitos da cronobiologia no crescimento de células cancerígenas da próstata in vivo
Abraham Haim1, Adina Yukler1, Orna Harel1, Hagit Schwimmer1, Fuad Fares1
ABSTRACT
Background and objective: The increased risk of developing prostate cancer (PC) observed in recent decades in industrialized countries has
showed to be related at least partially to the elevated exposure to artificial
light at night and to long photoperiod during all the year. However, the
precise effects of light and photoperiod manipulations on prostate cancer cell proliferation in vivo have not been reported to the same extent.
Methods: We exposed male C57BL/6 mice to short or long photoperiods
(short day, SD, and long day, LD). After inoculation of TRAMP-C2 cells,
half of the SD mice were also exposed to light interference at night while
half of the LD mice were treated with melatonin. Results: Under LD-acclimation, tumours were significantly larger compared to SD conditions.
Melatonin treatment to LD mice reduced tumour size significantly, while
light interferences to SD mice tended to increase it. Conclusions: we
conclude that exposure to LD and light interference may promote cancer
growth via changes in melatonin production and secretion. Our results
strongly support a novel link between temporal variables and cancer incidence. Accordingly, we anticipate our findings to increase the awareness
of scientists as well as health officials and policy makers to the adverse
effects of illumination misuse during the night.
Keywords: Light; Circadian rhythm; Climate; Sunlight; Prostatic
neoplasms; Environment; Melatonin; Photoperiod; Chronobiology
disorders
RESUMO
Introdução e objetivos: O aumento de exposição à luz artificial
durante a noite e, portanto, aumento do foto-período anual, tem sido
apontado como fator de risco, mesmo que parcial, para o crescimento
da ocorrência de risco de câncer da próstata (CP) observado nas últimas
décadas em países industrializados. Entretanto, apesar das evidências,
os efeitos da luz e da manipulação do foto-período sobre a proliferação
in vivo de células cancerígenas da próstata não têm sido descritos com
exatidão nos estudos que abordam o tema. Métodos: Camundongos
machos C57BL/6 foram expostos a períodos luminosos curtos (PLC)
e longos (PLL) de luz. Após a inoculação destes animais com células
TRAMP-C2, metade dos camundongos PLC foi exposta à interferência luminosa durante a noite, enquanto a outra metade, dos camundongos PLL, recebeu tratamento com melatonina. Resultados: Ficou
evidente que, durante a aclimação, os tumores dos animais PLL eram
significativamente maiores do que os de animais PLC. O tratamento
com melatonina em animais PLL reduziu o tamanho dos tumores de
forma significativa, enquanto a interferência luminosa em animais
PLC levou a uma tendência ao aumento de tamanho. Conclusões: É
possível que PLL e interferência luminosa possam promover o crescimento de células cancerígenas da próstata através de alterações na síntese e secreção de melatonina. Nossos resultados evidenciam uma associação desconhecida entre variáveis temporais e a incidência de câncer.
Com isto, esperamos que esta descoberta sensibilize pesquisadores,
profissionais e legisladores que atuam na Saúde Pública com relação os
efeitos negativos que a luminosidade noturna tem sobre o organismo.
Descritores: Luz; Ritmo circadiano; Clima; Luz solar; Neoplasias
da próstata; Meio ambiente; Melatonina; Fotoperíodo; Transtornos
cronobiológicos
INTRODUCTION
Seasonality is a basic temporal environmental variable to
which organisms are adapted. Such adaptation includes:
anatomical, physiological, biochemical, immune function
and behavioural pattern features. Outside of the tropical
photoperiod changes are the initial and the most important environmental cue for seasonal acclimatization of such
features. In mammals, photoperiod signals are transferred
to tissues and cells by the neurohormone melatonin (MLT)
produced and secreted from the pineal gland during the
dark phase of the 24-hour cycle. An increase in plasma
MLT levels for an extended duration is a signal for winter,
while a decrease in levels and secretion duration signals the
incoming summer. Like other animals, humans are seasonal
in birth rates, mortality, suicide rates, and many more (1,2).
The invention of the incandescent light bulb by Thomas Edison some 130 years ago led to a dramatic change in
human life style, which spread throughout the entire world
during the 20th century. Light at night (LAN) was of great
importance in changing human social and behavioural habits
towards an expansion of their waking hours, thus resulting in
an extended exposure to illumination during the hours when
human ancestors have typically been in the dark over millions of years of primate evolution. LAN has also impacted on
daily rhythms, as light is the main zeitgeber of the internal
Study carried out at University of Haifa, Mount Carmel, Haifa, 31905, Israel.
1
Department of Evolutionary and Environmental Biology, Department of Biology; The Israeli Centre for Interdisciplinary Research in Chronobiology, University of Haifa,
Mount Carmel, Haifa, 31905, Israel.
Corresponding author: Abraham Haim – Department of Evolutionary and Environmental Biology – University of Haifa – Mt. Carmel – Haifa, Israel 31905 – E-mail:
[email protected]
Received: January 8, 2010; Accepted: April 12, 2010
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Haim A, Yukler A, Harel O, Schwimmer H, Fares F
biological clock. The exposure to LAN, therefore, leads to a
disruption of the daily rhythms, particularly if it occurs at the
middle of the night. Since illumination has practically abolished seasonality and disrupted the daily rhythm schedules,
it is worth asking: what are the implications, if any, of this
change in temporal environment on human health?
The prevalence of prostate cancer (PC) has increased significantly in the last decades and became the second main
cause of death among men in many industrial countries, constituting about 33% of the diagnosed cancers in males, and it
is estimated that 50% of the diagnosed patients will develop
metastases (3). In recent years, the PC risk is of great concern
not only in developed countries but also in developing countries. Environmental changes are considered as a factor possibly responsible for the increase in PC incidence. Can environmental temporal changes, emerging from illumination and
the avoidance of daily rhythm and seasonality, be responsible
at least in part for such increase in PC incidence?
A recent study carried out on a global scale (4) revealed an
increase in the risk of PC incidence with the increase in LAN,
whereas the incidence of lung or colon cancers was not affected.
These results suggest that changes in photoperiod caused by
LAN, which decreases the number of dark hours as well as pineal
MLT secretion, an anti-oncogenic agent, can also be a cause for
the increased PC incidence in the heavily illuminated countries.
We suggest the following hypothesis: “if MLT is an anti-oncogenic agent then in mice acclimated to short day (SD) conditions
the proliferation of PC cells will be slower than in long-day (LD)acclimated mice”. Moreover, MLT treatment to LD-acclimated
mice should have a similar effect as SD acclimation, while light
interference (LI)-exposed mice should present an effect similar
to LD. The objective of our study was to test this hypothesis in
mice under in vivo conditions by exposing inoculated mice with
cancer cells to four different conditions: 1) LD, 16L:8D; 2) SD,
8L:16D; 3) LD and MLT-treatment; 4) SD and LI.
METHODS
Animal maintenance
Male mice C57BL/6, five weeks of age were purchased from
Harlan Laboratories (Jerusalem, Israel). The animals were
housed in polycarbonate cages (4-6 mice/cage) and maintained
at room temperature of 25±1ºC. Mice were fed (rat pellets,
Koffolk 1949, purchased from Koffolk, Inc., Tel Aviv, Israel)
and given tap water ad libitum. All procedures were conducted
with approval of Haifa University Institutional Animal Ethics Committee and the ministry of Health, Israel. In regards
to MLT production by C57BL/6 mice, please see discussion.
Effect of photoperiod, LI and MLT on tumour development
Cool white fluorescent illumination (at an intensity of 450 lux
and dominant wave length of 469 nm) was provided during
photophase and red dim light (intensity of 25 lux and dominant
wave length of 680 nm) during scotophase. Thirty-six mice
were randomly assigned to the following photoperiod regimes
for an acclimation period of four weeks prior to inoculation:
LD (16L:8D), lights were on between 8 and 24h, (n=18). SD
(8L:16D), lights were on between 8 and 16h, (n=18). TRAMPC2 cells were suspended at a concentration of 3 107 cells/mL
medium. Aliquots of 0.1 mL (2’ 106 cells) were injected subcutaneously into the mouse hind dorsal part, using a 27-gauge
needle. Following inoculation, each group was divided into
two subgroups (n=9 each). LD mice were divided into melatonin treated (MLT mice) and control (untreated). For the first
two weeks, 0.2 mL MLT, resuspended in 7% ethanol (M5250,
Sigma-Aldrich, Rehovot, Israel), were injected intraperitoneally every day, six hours before lights went off (5 mg/kg.Wb,),
whereas during the rest of the experiment, MLT was offered in
the drinking water (10 mg/kg.Wb) beginning six hours before
lights went off until the end of the dark period. Tap water was
offered to the mice at the rest of the light period. SD mice were
divided into control and light interfered (LI mice). The latter
ones were exposed every day, seven hours after lights went off
to 30 min of light (450 lux and a dominant wave length of 469
nm). Mice body mass (Wb) was measured using semianalytical
scale (1907 MP8 Sartorius, Germany) throughout the experiment, which lasted for 59 days post-inoculation. Tumour size
was measured twice a week using a calliper and the volume was
calculated by the formula: length x width2 x 0.52 (5).
Statistics
All data are expressed as mean ± standard deviation (SD). A
two-way ANOVA analysis was used for testing all photoperiod treatments, while Student’s t-test was used for comparing results within a photoperiod (SD or LD) given group.
For this analysis, we used the SPSS 12.0 software.
RESULTS
Effect of photoperiod, LI and MLT on PC tumour growth
Tumours first appeared after about three weeks. Significant
(p<0.001) differences in tumour volumes were observed between LD and Short Day mice at and beyond 36 days postinoculation of TRAMP-C2 cells. At 59 days post-inoculation,
the average tumour volume was 5.92±2.07 cm3 in LD mice
and only 0.85±0.41 cm3 in Short Day mice, indicating a faster growth rate in the former group (Figure 1A). In LD mice
treated with MLT, the average tumour volume at day 59 was
0.62±0.14 cm3, significantly (p<0.001) smaller than in control
untreated LD mice (Figure 1B). Survivorship at day 59 postinoculation was 55.5% (five out of nine) in the MLT-treated
group as compared to only 33.3% (three out of nine) in the
untreated group. Relative to the Short Day mice, significant increases (p<0.05) in tumour volumes were detected in LI mice;
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34
Chronobiology affects prostate cancer cells growth in vivo
tumour volumes were 0.85±0.41 cm3 and 1.84±0.25 cm3, respectively (Figure 1C). There were no differences in survivorship (44%) between the two groups.
DISCUSSION
For millions of years, humans and their ancestors were limited
in their activities by the unavailability of night-time lighting.
Although fires, candles and oil lamps allowed our predecessors
to continue some functions after dark, these light sources lacked
Figure 1: Growth rate of TRAMP-C2 cells (2 x 106) s.c. inoculated into
the posterior dorsal part of male C57BL/6 mice. A: Effect of photoperior - comparison between long day exposed mice (LD, triangles) or short
day (SD, diamonds) as described in materials and methods. B: Effect of
Melatonin (MLT) administration. Animals were exposed to LD with MLT
(circles) or control (triangles) as described in materials and methods. C:
Effect of Light interference (LI). Animals were exposed to short day (SD,
circles) or SD with LI (squares) as described in materials and methods.
All mice were followed for the development of tumours for 59 days post
inoculation. Results are expressed as tumour volume (cm3) ± SD. * Different scale, compared to A and B (up to 3 cm3), was used in figure C to plot
tumour volume against days after inoculation. In B, standard deviation
values for tumor volume under LD + MLT (circles) conditions are to o
small to be seen in the figure.
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the power and the universal access to erase the difference between night and day. As a result, for >>99% of humans history,
people have generally wound down at night. This has changed
dramatically with the introduction of illumination into modern human life, which has brought much prosperity and transformed our lifestyle forever. However, similarly to many of the
technologies that we have developed to help with our daily
lives, this change also ran against the grain of millions of years
of evolution. Over the last two decades, the results of numerous studies that questioned possible impacts of LAN and LI in
animal models revealed the potential harmful interruption of
circadian rhythms, as well as the interference with seasonality,
a phenomenon named “seasons out of time” (6). Additionally,
LI was shown to be a stressor in social voles Microtus socialis (7).
Humans all over the world are exposed to a new environment
in which the temporal variables were changed, abolishing daily
rhythms and seasonality. LAN, with all its economical blessing,
is now considered as a source of pollution (8-10). One important
outcome of this awareness is reflected by the recent decision of
the International Agency for Research on Cancer (IARC), the
cancer arm of the World Health Organization, to classify overnight shift work as a probable carcinogen factor (11).
The results of the current study can be explained based
on the progress made in our understanding of the way that
photoperiod changes are transmitted to the cells, the function
of the system as a clock (daily rhythms) and as a calendar (seasonality), and the important role of pineal MLT production
and secretion. Melatonin is a hormone that controls several
important functions and shows strong anti-oxidative and anti-oncogenic effects (12). Preliminary results of in vitro experiments carried out in our laboratory showed that MLT inhibited the proliferation of C57BL/6 derived prostate cancer cells
(TRAMP-C) (Yukler, A. Unpublished data, 2008). These
results suggest that TRAMP-C cells may contain MLT receptors. The mice that we used for our experiments, C57BL/6,
were considered as natural MLT ‘knockdown’ mice (13). However, a low but significant increase in MLT synthesis could
be observed in C57BL/6 pineal gland upon norepinephrine
stimulation, and, notably, also when animals were exposed to
long nights. The authors thus concluded that the commonly
used C57BL/6 mice are not completely MLT-deficient (14).
In LD-acclimated mice, as comparing to SD-acclimated
mice, MLT levels are assumed to be lower. This might explain the difference in tumour growth rate between the two
groups. In this sense, treatment with MLT to LD mice mimics exposure to SD. In accordance, MLT-treated LD mice
demonstrated a significant decrease in the tumour growth.
Low levels of MLT were previously measured in C57BL/6
mice under LD conditions of 14L:10D (13) and this could be
similar to the LD group in our experiments, but not to the
SD group, in which MLT levels could be higher. LI given to
Haim A, Yukler A, Harel O, Schwimmer H, Fares F
SD mice should presumably reduce pineal MLT secretion,
thus exposing the mice to MLT levels similar to LD conditions, resulting in an increase of tumour size.
Results of studies on the photoreceptors in the mammalian
retina revealed the existence of non-image forming photoreceptive cells (ipRGC) which contain melanopsin, a photopigment that is stimulated by light across the visible spectrum,
but with a maximum sensitivity in the blue range. The axons
from the ipRGCs join to form the Retino Hypothalamic Tract
that enervates the Suprachiasmatic Nuclei (SCN), and from
the SCN sympathetic nerves transfer the information to the
pineal gland (15). The dark period of the 24-hour cycle is the
signal for MLT production and secretion by the pineal gland,
even for mice with natural MLT ‘knockdown’ (13,14). Although
the inhibitory effect of exogenous MLT seems to suggest it
plays a role in photoperiodic manipulation of tumour size,
the exact underlying mechanism is largely unknown. Another
possible mechanism underlying LI effect on tumour growth
might be via stress response. This hypothesis is supported by
previous data, demonstrating light interference as a stressor (7).
Elevated levels of stress hormones may also have an effect on
cancer cells proliferation. Both mechanisms can act together,
as MLT might affect (suppress) tumour growth in the LD
mice, but LI affect (accelerate) tumour size via other mechanisms, including stress response.
Environmental variables can affect organisms through
epigenetic changes of DNA methylation patterns and/or
modification of histones in the chromatin. Among others,
such changes are proposed to be a cause for silencing tumour
suppressor genes (16). Furthermore, if MLT may have the potential to modify such changes, then suppression of MLT
production by LAN or LI can lead to silencing of tumour
suppressor genes and, thus, promote cancer. If this is the
case, then LD acclimation, in modern life style, reduces MLT
production and may possibly be the reason for PC incidence
increase in men in the industrialized world, as recently
shown by some authors (14). The bright side is that since this
could be an epigenetic effect it can be reversed. Therefore,
understanding the mechanism underlying the effect of light
and photoperiod on PC cells can lead to finding novel noninvasive treatment to PC patients. In our study, environmental
conditions were imposed on existing PC tumour. Another
step in this research should be focused on the prevention of
this kind of tumour. Therefore, it will be crucial to study
the effects of LAN on PC in respect to light intensity, wave
length as well as duration of exposure and to compare them
with the epigenetic status of PC-related genes.
Acknowledgements
The authors wish to express their many thanks to Shula Nachmias from Oranim campus of the University of Haifa for her
assistance. This work was made possible by the support of the
Research authority of the University of Haifa. Also, we extent
our thanks to the reviewers of this manuscript for their useful
remarks on a former version of this manuscript.
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3. Jemal A, Murray T, Ward E, Samuels A, Tiwari RC, Ghafoor A, et
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level of transgene expression and inhibition of tumor growth and
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6. Haim A, Shanas U, Zubidad Ael S, Scantelbury M. Seasonality
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8. Pauley SM. Lighting for the human circadian clock: recent research indicates that lighting has become a public health issue.
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12.Blask DE, Brainard GC, Dauchy RT, Hanifin JP, Davidson LK,
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HW, et al. Transcription factor dynamics and neuroendocrine
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for melatonin? J Pineal Res. 2008;44(1):41-4.
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Sleep
Science
ORIGINAL ARTICLE
The presence of neuronal-specific nuclear
protein (NeuN) in the circadian timing system
of the capuchin monkey (Cebus apella)
A presença da proteína nuclear específica neuronal (NeuN) no sistema
de temporização circadiano do macaco capuchinho (Cebus apella)
Rayane Bartira Silva do Nascimento1, Janaína Siqueira Borda1, Rovena Clara Galvão Januário Engelberth1, Raysa Oliveira de
Medeiros1, Renata Frazão2, Luciana Pinato2, André Luiz Bezerra de Pontes1, Expedito Silva do Nascimento Jr2, Maria Inês
Nogueira2, Roelf Justino Cruz-Rizzolo3, Miriam Stela Maris de Oliveira Costa1, Jeferson de Souza Cavalcante1
ABSTRACT
Background and objective: The circadian timing system (CTS) is
composed of a group of specialized neuronal structures that establish a
temporal organization of physiological and behavioral processes within
precise patterns. The central components of this system are the suprachiasmatic nucleus of the hypothalamus (SCN) and the intergeniculate
leaflet of the thalamus (IGL). The objective of this study was to verify the
presence of the neuron-specific nuclear protein (NeuN) in the circadian
timing system of capuchin monkeys (Cebus apella) using immunohistochemical techniques. Methods: Capuchin monkeys (Cebus apella) were
anesthetized and transcardially perfused with 4% paraformaldehyde in
0.1 M phosphate buffer, and then their brains were removed and frozen. A
microtome was used to make 30 μm sections in the coronal plane. One of
the series was used for Nissl staining (thionin) to demarcate the cytoarchitecture, and the remainders of the sections were processed for immunohistochemical detection of NeuN (ABC protocol). Results: NeuN-positive
neurons were observed in the suprachiasmatic nucleus of the capuchin
monkey. The pregeniculate nucleus (PGN), a structure equivalent to the
ventral lateral geniculate nucleus (vLGN) and to the IGL in rodents, did
not have any NeuN-positive neurons. Conclusions: In this primate species, only the suprachiasmatic nucleus neurons of the central structures of
the circadian timing system express the NeuN protein.
Keywords: Circadian rhythm; Suprachiasmatic nucleus; Nuclear
proteins; Immunohistochemistry/methods; Geniculate bodies; Neurons; Animals; Cebus
RESUMO
Introdução e objetivo: O sistema de temporização circadiana (do inglês ������������������������������������������������������������������
circadian timing system, �����������������������������������������
CTS) é composto por um conjunto de estruturas neurais especializadas que estabelecem uma organização temporal
dos processos fisiológicos e comportamentos dentro de padrões precisos.
Seus componentes centrais são o núcleo supraquiasmático (SCN) do hipotálamo e o folheto intergeniculado (IGL) do tálamo. O objetivo deste
estudo foi verificar, através da técnica imunoistoquímica, a presença da
proteína nuclear neuronal específica (NeuN) no sistema de temporização
circadiana do macaco-prego (Cebus apella), um primata do Novo Mundo.
Métodos: Os animais foram previamente anestesiados e submetidos à
perfusão transcardíaca com solução salina heparinizada, seguida de solução
de paraformaldeído a 4% em tampão fosfato 0,1 M. Os encéfalos foram
removidos e submetidos à microtomia por congelação, obtendo-se secções
coronais de 30 μm. Secções de uma série foram submetidas ao método
de Nissl (Thionina) para demarcar a citoarquitetura e as outras secções
foram processadas por imunoistoquímica (protocolo ABC) a fim de revelar a presença de NeuN. Resultados: Neurônios NeuN positivos foram observados no núcleo supraquiasmático do macaco-prego. O núcleo
pré-geniculado (PGN), estrutura equivalente ao núcleo geniculado lateral
ventral (GLV) e ao IGL dos roedores, não apresenta neurônios NeuN positivos. Conclusão: Nas estruturas centrais do sistema de temporização circadiana, somente os neurônios do núcleo supraquiasmático nesta espécie
de primata expressam a proteína NeuN.
Descritores: Ritmo circadiano; Núcleo supraquiasmático; Proteínas nucleares; Imunoistoquímica/métodos; Corpos geniculados;
Neurônios; Animais; Cebus
INTRODUCTION
The generation and regulation of circadian rhythms in
mammals originate from a neural-specific system, the circadian timing system (CTS). This system utilizes a central
pacemaker, which includes inputs such as retinal afferents
that allow for the synchronization of environmental cycles
and outputs that lead to behavioral effectors (1). The supra-
Study carried out at Universidade Federal do Rio Grande do Norte – UFRN, Natal (RN), Brazil; Universidade de São Paulo – USP, São Paulo (SP), Brasil; Universidade
Estadual Paulista “Júlio de Mesquita Filho” – UNESP, Araçatuba (SP) Brazil.
1
Laboratório de Cronobiologia, Centro de Biociências, Universidade Federal do Rio Grande do Norte – UFRN, Natal (RN), Brazil.
2
Departamento de Anatomia da Universidade de São Paulo – USP, São Paulo (SP), Brazil.
3
Departamento de Ciências Básicas da Universidade Estadual Paulista “Júlio de Mesquita Filho” – UNESP, Araçatuba (SP) Brazil.
Corresponding author: Jeferson de Sousa Cavalcante – Centro de Biociências, Universidade Federal do Rio Grande do Norte – Caixa Postal 1511 – CEP 59078-970 –
Natal (RN), Brazil – Phone: +55 (84) 3215-3409 – Fax: +55 (84) 3211-9206 – E-mail: [email protected]
Received: January 8, 2010; Accepted: March 28, 2010
Sleep Sci. 2010;3(1):�����
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Nascimento RBS, Borda JS, Engelberth RCGJ, Medeiros RO, Frazão R, Pinato L, Pontes ALB, Nascimento Jr ES,
Nogueira MI, Cruz-Rizzolo RJ, Costa MSMO, Cavalcante JS
chiasmatic nucleus (SCN) and the intergeniculate leaflet
(IGL) are considered to be the central components of the
CTS (1). The SCN is a small region localized at the anterior
hypothalamus bilateral to the third ventricle and above the
optic chiasm (2). In the majority of mammalian species studied, the SCN is divided into two principal cell populations:
the ventrolateral SCN, which contains neurons that secrete
vasoactive intestinal polypeptide (VIP), and the dorsomedial SCN, which contains neurons that secrete vasopressin
(VP) (2,3). In addition to VIP and VP, the SCN also contains
a large number of other neuroactive substances that can act
as neurotransmitters or neuromodulators. The SCN contains
neuropeptide Y (NPY), serotonin (5-HT), glutamate (Glu),
bombesin (BBS), gastrin-releasing peptide (GRP), cholecystokinin (CCK), substance P (SP), angiotensin II, enkephalin (ENK), somatostatin (SS), tyrosine hydroxylase (TH),
and calcium binding proteins such as calbindin (CB) and
calretinin (CR) (2). Because the SCN has a crucial role in the
temporal organization of behavior, it is considered the central circadian pacemaker in mammals. SCN lesions result in
a loss of behavioral circadian, endocrine, and physiological
rhythms (4). Arrhythmic animals with SCN lesions recover
their circadian rhythms through fetal SCN transplant; however, these animals acquire the rhythm of the donor (5).
The intergeniculate leaflet (IGL), along with the ventral
lateral geniculate nucleus (vLGN) and the dorsolateral geniculate nucleus (dLGN), form the lateral geniculate complex of the thalamus in non-primate species (6). The IGL is a
thin layer of neurons containing NPY, interposed between
the vLGN and the dLGN, and its function is to modulate
the SCN (7). The organization of the lateral geniculate complex is different in primates in relation to rodents. Although
the primate dLGN corresponds to the rodent dLGN, the
primate pregeniculate nucleus (PGN), which is a wedgedshaped cellular grouping that dorsomedially encircles the
dLGN, is considered equivalent to the rodent vLGN (8,9).
NPY-immunoreactive neurons in the PGN have been found
in Rhesus monkeys (8), common marmosets (Callithrix jacchus)
(10)
, and Cebus monkeys (9). Thus, it can be assumed that the
PGN of primates also contains a rodent-equivalent IGL . A
new neuronal marker that has been utilized to identify nerve
cells is the neuronal-specific nuclear protein (NeuN), which
is expressed in both the nucleus and cytoplasm of the majority of vertebrate nervous system cells (11). This protein has
been utilized in studies on nervous system development (12),
in morphometric studies as a postmortem neuronal marker
of human cerebral tissue (13), in histopathology diagnosis as
a marker of neuronal differentiation in cerebral tumors, and
in studies of neurodegenerative disorders (14). Although this
protein has been identified in the majority of nerve cells,
there are some NeuN-negative neurons. In development,
Cajal-Retzius neurons of the first layer of the cerebral cortex,
the medullar inferior olivary nucleus, cerebellar Purkinje
cells, mitral cells of the olfactory bulb, retinal photoreceptors, and glial cells are all negative for NeuN (11,15).
Considering the importance of the CTS in mammals and
the quantity of neuroactive substances present in the central
components of this system, the objective of this study was
to identify NeuN-protein-immunoreactive neurons in the
central components of the CTS of the capuchin monkey (Cebus apella).
METHODS
For this study, we utilized two young adult capuchin monkeys (Cebus apella) obtained from the Júlio Mesquita Filho
Primate Center of Universidade Estadual Paulista, Araçatuba (SP), Brazil. The experimental procedures were in compliance with the guidelines for the care and use of mammals
in neuroscience and behavioral research. The capuchin monkeys were housed in individual cages under natural humidity, temperature, and lighting conditions and were fed with
a standard fruit and vegetable diet.
The animals were anesthetized with sodium thiopental (30
mg/kg, i.p.) and transcardially perfused with 800 mL of 0.9%
saline containing heparin (Hipolabor Laboratories, 5000 IU/
mL). The saline was followed by 1500 mL of 4% paraformaldehyde in 0.1 M acetate buffer (pH 6.5) and subsequently
with 1500 mL of 4% paraformaldehyde in borate buffer (pH
9.0). The brains were exposed and sliced into blocks utilizing stereotaxic equipment. The blocks were removed from
the cranium and placed in cryoprotectant solution composed
of 10% glycerol and 2% dimethylsulfoxide in 0.1 M borate
buffer (pH 9.0) at 4ºC. After three days, the blocks were transferred to a similar solution containing 20% glycerol for four
more days of cryoprotection. Next, the blocks were sliced into
30-μm coronal sections with a cryomicrotome and collected
in an anti-freezing solution. One of the series was used in
Nissl staining for the cytoarchitecture study, which utilized
thionin as a dye. Another series was pretreated for antigen
recovery utilizing 1% sodium borohydrate. After the pretreatment, the slices where treated immunohistochemically
with an anti-NeuN primary antibody (1:1000, Chemicon) in
0.4-% Triton X-100 and 2-% normal rabbit serum (1:50) at
room temperature for 24 hours. Next, slices were incubated
with secondary biotinylated antibody (1:200, Sigma) for two
hours. Next, the slices were incubated in avidin-biotin immunoperoxidase complex (Elite ABC kit, Vector Laboratories) for two hours and exposed to chromogenic 2.5% 3,3’–diaminobenzidine tetrahydrochloride (DAB) (Sigma, St Louis,
MO, USA) diluted in 0.1 M phosphate buffer (pH 7.4) for
15 minutes. Between incubations, the slices underwent a sequence of eight 5-minute washes each in 0.1 M phosphate
Sleep Sci. 2010;3(1):�����
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37
38
The presence of neuronal-specific nuclear protein (NeuN) in the circadian timing system
buffer (pH 7.4). Lastly, the slices were mounted on gelatinized
glass slides, intensified in osmium tetroxide, dehydrated in a
series of alcohols, cleared by xylene, and coverslipped for optic
microscopy analysis.
RESULTS
The SCN of Cebus apella is localized in the anterior hypothalamus dorsal to the optic chiasm and bilateral to the
third ventricle. The Nissl staining showed that the SCN
cells neighbored the wall of the third ventricle (Figure 1A).
This cellular group is triangular in the rostral level but becomes more rounded as it reaches the caudal levels. NeuN
immunoreactivity was observed in the three regions (rostral,
intermediate and caudal) of the SCN. The largest number
of NeuN-positive cells was observed in the intermediate region; however, the staining of these cells was pallid when
compared to the lateral hypothalamus, which presented robustly stained cells (Figure 1B).
oc: optic chiasm; 3v: third ventricle; dLGN: dorsal lateral geniculate
nucleus.
The scale bar is equivalent to 70 µm in A, C and D, and to 85 µm in B.
Figure 1: Photomicrographs of brain sections of Cebus apella showing the
components of the CTS in bright field. (A) SCN of Cebus apella stained
by Nissl staining. (B) Panel showing the immunoreactivity for NeuN in
the SCN of the Cebus apella monkey. (C) PGN of the Cebus apella monkey
stained by Nissl staining. (D) Panel showing the negative reaction for
immunoreactivity against NeuN in the PGN of the Cebus apella monkey.
The Nissl-stained sections demonstrated that the PGN
is localized dorsomedially to the dLGN (Figure 1C). It was
observed that the PGN cells in Cebus apella do not present
NeuN immunoreactivity (Figure 1D). However, we did
confirm that the immunohistochemical process was successSleep Sci. 2010;3(1):�����
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ful in dLGN neurons, thus representing a positive control
(Figure 1D).
DISCUSSION
The SCN is composed of oscillatory neurons that control a
series of effector systems, including those that regulate the
activity and rest cycles, body temperature, neuroendocrine
function, and psychomotor performance. Its role as the central pacemaker of the CTS is modulated by its inputs, which
are topographically organized within itself (16,17). The rodent
IGL or the PGN in primates, along with the SCN, are considered the central components of the CTS because they are
the first participants in the modulation of photic and nonphotic circadian responses (1).
The present study demonstrated that the SCN of Cebus Apella is elongated in the rostro-caudal axis. Neurons
strongly stained with thionin were localized in the anterior
hypothalamus above the optic chiasm and bilateral to the
third ventricle. Furthermore, the PGN had less intense thionin staining and was localized dorsomedially to the dLGN.
This result is in accordance with our previous studies in
Cebus monkeys (9,18). The SCN and PGN cells presented a
high level of homogeneity in the Nissl staining method.
Neuron-specific nuclear protein is considered to be a nuclear
regulatory molecule specific to the nervous system, and it
has been increasingly used in central nervous system studies
(11-13)
. The present study demonstrated that SCN neurons in
Cebus apella were immunoreactive for NeuN in the whole
rostro-caudal extension. The greatest number of NeuN-positive cells was found in the intermediate level of the SCN.
The more robustly stained cells were observed in the lateral
portion, the most pallid were present in the medial portion
of the SCN, but this result could be explained by immunoreactivity variability. A study with adult rats demonstrated
that NeuN expression in the SCN was low, particularly in
the dorsal region when compared to the neighboring hypothalamic neurons. However, detectable levels of NeuN
were observed in the ventral portion of the SCN when it
was co-localized with doublecortin, which is a microtubuleassociated protein that is also considered a marker for neurogenesis (19). The NeuN immunohistochemical labeling in
the PGN of Cebus apella was negative.
In summary, our study demonstrated that the localization
of the SCN and the PGN of Cebus apella is similar to other primate species. In addition, SCN neurons were immunoreactive
for NeuN, whereas the PGN did not exhibit labeled neurons.
Taking into consideration that some neurons of the nervous
system are NeuN negative (18,15), it still remains to be determined whether Cebus apella SCN contains specific subpopulations of neurons that are not immunoreactive for NeuN. The
data in this study are preliminary, but provide a perspective
Nascimento RBS, Borda JS, Engelberth RCGJ, Medeiros RO, Frazão R, Pinato L, Pontes ALB, Nascimento Jr ES,
Nogueira MI, Cruz-Rizzolo RJ, Costa MSMO, Cavalcante JS
for the comparative studies between primate and non-primate
species when it comes to understanding the role of NeuN as a
neuronal marker in this physiological system.
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4. Van den Pol AN. The suprachiasmatic nucleus: morphology and
cytochemical substrates for cellular interaction. In: Klein DC,
Moore CRY, Reppert SM. Suprachiasmatic nucleus: the mind’s
clock. New York: Oxford University Press; 1991. p. 17-50.
5. Ralph MR, Foster RG, Davis FC, Menaker M. Transplanted
suprachiasmatic nucleus determines circadian period. Science
1990;247(4945):975-8.
6. Jones EG. The thalamus. 2ª ed. New York: Cambridge University
Press; 2007.
7. Hickey TL, Spear PD. Retinogeniculate projections in hooded and albino rats: an autoradiographic study. Exp Brain Res
1976;24(5):523-9.
8. Moore RY. The geniculohypothalamic tract in monkey and man.
Brain Res 1989;486(1):190-4.
9. Pinato L, Frazão R, Cruz-Rizzolo RJ, Cavalcante JS, Nogueira
MI. Immunocytochemical characterization of the pregeniculate
nucleus and distribution of retinal and neuropeptide Y terminals
in the suprachiasmatic nucleus of the Cebus monkey. J Chem
Neuroanat 2009;37(4):207-13.
10.Costa MS, Moreira LF, Alones V, Lu J, Santee UR, Cavalcante JS,
et al. Characterization of the circadian system in a Brazilian species of monkey (Callithrix jacchus): immunohistochemical analysis and retinal projections. Biol Rhythm Res 1998;29(5):510-20.
11.Mullen RJ, Buck CR, Smith AM. NeuN, a neuronal specific protein in vertebrates. Development 1992;116(1):201-11.
12.Preusser M, Laggner U, Haberler C, Heinzl H, Budka H, Hainfellner JA. Comparative analysis of NeuN immunoreactivity in
primary brain tumours: conclusions for rational use in diagnostic
histopathology. Histopathology 2006;48(4):438-44.
13.Gittins R, Harrison PJ. Neuronal density, size and shape in the
human anterior cingulated cortex: a comparison of Nissl and
NeuN staining. Brain Res Bull 2004;63(2):155-60.
14.Falke E, Nissanov J, Mitchell TW, Bennett DA, Trojanowski JQ,
Arnold SE. Subicular dendritic arborisation in Alzheimer’s disease correlates with neurofibrillary tangle density. Am J Pathol
2003;163(4):1615-21.
15.Kumar SS, Buckmaster PS. Neuron-specific nuclear antigen
NeuN is not detectable in gerbil subtantia nigra pars reticulata.
Brain Res 2007;1142:54-60.
16.Moga MM, Moore RY. Organization of neural inputs to the suprachiasmatic nucleus in the rat. J Comp Neurol 1997;389(3):
508-34.
17.Abrahamson EE, Moore RY. Suprachiasmatis nucleus in the
mouse: retinal innervation, intrinsic organization and efferent
projections. Brain Res 2001;916(1-2):172-91.
18.Pinato L, Allemandi W, Abe LK, Frazão R, Cruz-Rizzolo RJ,
Cavalcante JS, et al. A comparative study of cytoarchitecture and
serotonergic afferents in the suprachiasmatic nucleus of primates
(Cebus apella and Callithrix jacchus) and rats (Wistar and Lond
Evans strain). Brain Res 2007;1149:101-10.
19.Geoghegan D, Carter DA. A novel site of adult doublecortin expression: neuropeptide neurons within the suprachiasmatic nucleus circadian clock. BMC Neurosci [Internet]. 2008 [cited 2010
Mar 30];9:2. Available from: http://www.ncbi.nlm.nih.gov/pmc/
articles/PMC2253543/?tool=pubmed
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39
Sleep
Science
ORIGINAL ARTICLE
Mathematical model of the interaction between the dorsal
and ventral regions of the suprachiasmatic nucleus of rats
Modelo matemático da interação das regiões dorsal e
ventral do núcleo supraquiasmático de ratos
Bruno da Silva Brandão Gonçalves1, Breno Tercio Santos Carneiro1,
Crhistiane Andressa da Silva1, Diego Alexandre da Cunha Fernandes1,
Fabiano Santos Fortes1, João Miguel Gonçalves Ribeiro1, Rafaela Cobuci Cerqueira1,
Sergio Arthuro Mota Rolim2, John Fontenele Araújo1
ABSTRACT
Background and objective: In mammals, the main biological clock
that is synchronized by light is located in the suprachiasmatic nucleus
of the hypothalamus, which can be divided into two distinct regions:
the ventrolateral and the dorsomedial. Both behave as separate oscillators that interact with each other to form the circadian rhythm.
Methods: Our objective was to develop a mathematical model to
understand how these regions of the suprachiasmatic nucleus coordinate the circadian rhythm of motor activity in rats. To accomplish
this, we performed simulations with light-dark cycles of 24 (T24)
and 22 hours (T22) and simulations with constant darkness (CD). In
the model, we developed equations to describe the circadian rhythm
of a clock protein. Results: For the two light-dark and constant darkness cycles, the model was able to reproduce the synchronization with
T24, the dissociation with T22, and the free-running rhythm with
constant darkness. The results show that the intensity of coupling
between the two oscillators and their periods define the output of the
rhythm. Conclusions: The proposed model is consistent with data in
the literature and suggests new experimental approaches. This model
will contribute to a better understanding of the interaction between
the two regions of the suprachiasmatic nucleus.
Keywords: Mathematical models; Circadian rhythms; Motor activity; Suprachiasmatic nucleus; CLOCK proteins; Biological clocks/
physiology; Animal; Rats
RESUMO
Introdução e objetivos: No hipotálamo se encontra o principal relógio biológico sincronizado pela luz, que pode ser dividido em duas
regiões distintas: uma chamada ventrolateral e outra dorsomedial.
Ambas se comportam como osciladores distintos que se relacionam
para formar os ritmos circadianos. Métodos: Desenvolver um modelo
matemático para entender como essas regiões do núcleo supraquias-
mático coordenam o ritmo circadiano da atividade motora em ratos.
Para isso, foram realizadas simulações com ciclo claro-escuro de 24
(T24) e de 22 horas (T22), e em condição de escuridão constante (EE).
No modelo desenvolvido, foram utilizadas equações que descrevem o
ritmo circadiano dos níveis de uma proteína-relógio fictícia. Resultados: Para os diferentes ciclos de claro-escuro e escuro constante, o modelo foi capaz de reproduzir a sincronização ao T24, a dissociação em
T22 e o curso livre em EE. Os resultados apontaram que a intensidade
do acoplamento entre os dois osciladores e seus períodos define a saída
do ritmo. Conclusões: O modelo proposto foi capaz de reproduzir
dados da literatura e sugerir novas abordagens experimentais. Essas
novas manipulações podem contribuir para uma melhor compreensão
de como ocorre a interação entre as duas regiões do núcleo supraquiasmático.
Descritores: Modelos matemáticos; Ritmo circadiano; Atividade
motora; Núcleo supraquiasmático; Proteínas CLOCK; Relógios biológicos/fisiologia; Animais; Ratos
INTRODUCTION
In mammals, the suprachiasmatic nucleus (SCN) is responsible for generating the circadian expression of several physiological and behavioral variables such as locomotor activity,
sleep-wake cycle and body temperature (1). Electrophysiological data suggest that each cell of the SCN should be considered as self-sustained (2) and might have different periods,
albeit within a circadian limit (3). Because the SCN is necessary and sufficient to generate the circadian rhythm, it is
known as the principal circadian oscillator in mammals. The
SCN can be divided into morphologically and functionally
distinct dorsal and ventral regions (4). The dorsal, or dorsomedial (dm), region, contains a large population of neurons
Study carried out at Universidade Federal do Rio Grande do Norte – UFRN, Natal (RN), Brazil.
1
Center for Research on Rhythm, Sleep, Memory and Emotion, Department of Physiology, Biosciences Center, Universidade Federal do Rio Grande do Norte – UFRN,
Natal (RN), Brazil.
2
Center for Research on Rhythm, Sleep, Memory and Emotion, Department of Physiology, Biosciences Center, Universidade Federal do Rio Grande do Norte – UFRN,
Natal (RN), Brazil; Edmond and Lily Safra International Institute of Neuroscience of Natal, Natal (RN), Brazil.
Corresponding author: Bruno da Silva Brandão Gonçalves – Departamento de Fisiologia, Centro de Biociências, Universidade Federal do Rio Grande do Norte – Campus
Universitário, Lagoa Nova – Caixa Postal 1506 – CEP 59078-970 – Natal (RN), Brazil – Phone: (84) 3215-3409 (Ext 218); Fax: (84) 3211-9206
Received: December 12, 2009; Accepted: March 10, 2010
Sleep Sci. 2010;3(1):�����
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Gonçalves BSB, Carneiro BTS, Silva CA, Fernandes DAC, Fortes FS, Ribeiro JMG, Cerqueira RC, Rolim SAM, Araújo JF
that produce vasopressin, while the ventral, or ventrolateral
(vl), region is mainly comprised of neurons that produce vasoactive intestinal peptide (4). Generally, the two regions are
coupled oscillators, but this condition may change when the
light-dark cycle is delayed or when the period of illumination is reduced (5).
When rats are kept in a 22-hour (11:11) symmetrical light-dark cycle, there is a stable oscillation that uncouples these regions (6). During in vivo dissociation, the
rhythm of locomotor activity has two distinct components; one component is synchronized with the light, and
the other free-runs with a period longer than 24 hours
(6)
. By analyzing the expression of clock genes (Per and
Bmal1) at specific phases, de la Iglesia et al. (7) showed
that the component synchronized with the 22-hour lightdark cycle is associated with activity of the vl region,
while the free-running rhythm depends on variations in
the dm.
In the present study, we developed a mathematical model of the SCN to study how vl and dm regions work together
to create the circadian rhythm of motor activity in rats. The
model’s equations describe the circadian rhythm of a clock
protein.
METHODS
Mathematical model
Figure 1A shows the two oscillators that form the SCN. Following the system proposed by Schwartz et al. (8), light acts
directly on the vl-SCN region, and its output is projected to
the oscillator that represents the dm-SCN region.
(A)
(B)
Figure 1: (A) Scheme proposed for the SCN. The light-dark cycle directly
affects the vl-SCN oscillator, and this output synchronizes the dm region.
(B) Mathematical equations that describe the dynamics of the vl-SCN and
dm-SCN oscillators.
To simulate circadian oscillation, we used the Goodwin
model (9) with three variables (Figure 1B). X is the concentration of mRNA of a particular clock gene, Y is the concentration of the protein produced by gene X, and Z represents
an inhibitor of X synthesis in the cell nucleus. This model
was used to describe the vl-SCN and dm-SCN oscillators,
with changes in some constants.
The constants ax,y,z and bx,y,z correspond to the production
and degradation of the X, Y, and Z variables. The constant
‘c’ modulates the output of the oscillator and influences both
vl-SCN and dm-SCN. For the vl-SCN oscillator, a constant
‘j’ was associated with light (j=0 for dark and j=0.4 for
light), and for the dm-SCN oscillator, ‘j’ was associated with
the vl-SCN output (j=c * vl-SCN output).
The oscillators were adjusted so that the period of the
v1-SCN oscillator were equal to 24.25 hours and the period
of the dm-SCN oscillator were equal to 24.4 hours, as proposed by Schwartz et al. (8). In order to do that, the constant
‘bx’ was set to 0.33 and 0.325 for vl-SCN and dm-SCN,
respectively. The other constants were set as follows: ax=ay
=az =0.7; by=bz=0.35.
To calculate the output of the oscillators, we considered
the value of the variable Y. When the value of Y remains below a threshold, the output is equal to 0. When the value
exceeds this threshold, the output has the same value as Y.
The threshold was defined as 2/3 of the maximum value of Y.
Data analysis was performed using the program “El
temps” (A. Díez-Noguera, Universitat de Barcelona, 1999).
With this tool, we discovered existing periods in the rhythm
that are consistent with the periodogram described by Sokolove and Bushell (10). Graphical representations of rhythm
(actograms) were constructed from the output of the dmSCN and vl-SCN oscillators separately.
RESULTS
The simulations were performed with a light-dark cycle
of 24 (T24) and 22 (T22) hours and in conditions of constant darkness (CD). In each condition, we used three values for the constant ‘c’. This enabled the analysis of weak
(c=0.0094), medium (c=0.15), and strong (c=0.4) interactions between the oscillators. For the weak interaction, the
value of ‘c’ was adjusted so that, in all conditions, the oscillators presented different periods. For the medium interaction, the constant was chosen so that the dm-SCN oscillator showed two periods in T22. For the strong interaction,
we chose a value of ‘c’ so that both regions presented the
same period in all cycles.
T24
For all types of interactions, the period of the vl-SCN region
was of 24 hours (Figure 2A, B, and C). For strong interSleep Sci. 2010;3(1):�����
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41
42
Mathematical model of the interaction between the dorsal and ventral regions
actions, the dm-SCN region had an average period of 24
hours (Figure 2A and B) and a phase difference of 7.4 and
7.9 hours in comparison to the vl-SCN region. The dm-SCN
oscillator had a period of 24.6 hours when the interaction
was weak (Figure 2).
(A)
(B)
(C)
(A)
(B)
(C)
Figure 3: The vl-SCN oscillator (gray) and dm-SCN oscillator (black)
were submitted to constant darkness. Under this condition, strong (A),
medium (B) and weak (C) interactions between the oscillators were simulated.
(A)
Figure 2: Simulation of the output of the vl-SCN (gray) and dm-SCN
(black) oscillators with a photoperiod of 24 hours. The simulations were
made with strong (A), medium (B) and weak (C) interactions between
the oscillators.
(B)
Constant darkness
Only the weak interaction between the vl-SCN and dm-SCN
oscillator caused their periods to be uncoupled, at 24.33 and
24.5 hours, respectively (Figure 3C). For the medium and
strong interactions, the oscillators have the same period,
24.33 hours (Figure 3A and B). The lag of the oscillators
was of 7.2 hours for strong interactions and 7.5 hours for
medium interactions.
T22
As with the T24 cycle, the vl-SCN oscillator had the same
period as the external stimulus, in this case, 22 hours (Figure 4A, B and C). When the interaction between regions
is strong, the dm-SCN and vl-SCN regions have the same
period and a phase difference of 7.6 hours (Figure 4A). Two
periods (22 and 24.67 hours) were found in the dm-SCN osSleep Sci. 2010;3(1):�����
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(C)
Figure 4: Vl-SCN (gray) and dm-SCN (black) oscillators during a photoperiod of 22 hours. The simulations were made with strong (A), medium
(B) and weak (C) interactions between the oscillators.
Gonçalves BSB, Carneiro BTS, Silva CA, Fernandes DAC, Fortes FS, Ribeiro JMG, Cerqueira RC, Rolim SAM, Araújo JF
cillator with a medium interaction (Figure 4B). The dm-SCN
oscillator had a period of 24.75 hours when there was a weak
interaction (Figure 4C).
We reduced the value of the constant ax to 0.5 to study
how t (the endogenous period) interferes with dissociation.
After this reduction, the vl-SCN and dm-SCN oscillators
had periods of 23.5 and 23.8 hours, respectively. We then
reduced ax to 0.5 for the T22 cycle for a medium interaction
between the regions. In this case, both oscillators showed the
same period as the external stimulus, 22 hours (Figure 5A).
When the period of simulation was changed to 20 hours, the
dm oscillator presented a free-running rhythm.
(A)
(B)
Figure 5: Variation of t in the vl (in gray) and dm (black) regions. For a
22 hours photoperiod (A), the oscillators have the same period of stimulation. By submitting the oscillator to a 20-hour photoperiod (B), the dm
region enters a free-running rhythm while the vl region adjusts to the
new stimulus.
DISCUSSION
In this work, we simulated the two main oscillators that
make up the SCN. The first oscillator received stimulus
from the light-dark cycle, and the second had as its input
the output of the first oscillator. Anatomically, the vl region,
represented by the first oscillator, receives a dense projection
from the retina (11) (Figure 1A). Thus, we assumed that the
intensity of incoming light synchronizes the vl oscillator in
all of the simulated cycles.
The connection between the two oscillators caused us to
consider data showing a large synaptic projection from the
vl-SCN region to the dm-SCN region with little evidence of
reciprocity (12). In our model, the dm-SCN oscillator receives
the output of the vl-SCN oscillator as input.
At the cellular level, circadian oscillation depends on
the sequential activation of clock genes (13). This complex
mechanism also depends on proteins that regulate transcription, binding, and entry of the core clock proteins (14). To
simplify these steps and maintain the behavior of molecular
oscillation, we used a model that simulates the production
of a hypothetical clock gene.
Experimental data shows that light leads to increased
transcription of genes in neurons of the SCN (15). We modified our equation from the original model, increasing the
effect of light for the vl-SCN oscillator. Synaptic activation,
which increases the production of neuronal genes (16,17), was
simulated by adding the output of the vl-SCN oscillator in
the production of the gene to the dm-SCN oscillator.
The behavior of the dm-SCN oscillator is related to the
coupling constant ‘c’. In other words, the intensity of the
output of the photo-responsive oscillator modulates the output of the dm-SCN oscillator. There is a total separation
between the regions only when the interaction is weak for
all levels of illumination. For constant darkness, Kunz et al.
(18)
showed that a reduction in interaction between the two
groups of oscillators led to results similar to ours (Figure
3C).
With a strong interaction, there is a synchronization of
the two oscillators for all photoperiods simulated. When
the intensity is increased, Granada and Herzel (19) showed
that the oscillators synchronized only with stimuli of different frequencies. Therefore, when we changed the photoperiod from T24 to T22, a greater interaction was required to
achieve synchronization between the oscillators.
Despite being composed of multiple oscillators, the vl
and dm regions show a unique period under stable conditions (20). Herzog et al. (21) showed that neurons in culture
have periods ranging from 21.5 to 26 hours (21). As the coupling between neurons increases, the variability decreases
(21)
. For this reason, we chose to represent each region as a
single oscillator.
According to simulations carried out by Schwartz et al.
(8)
, synchronization of oscillators depends on the relationship
between the photoperiod and t. By reducing the oscillators
to T22, there was synchronization with medium interaction.
A 20-hour simulation period was required to dissociate the
oscillators.
The model uses some simplifications, such as a single hypothetical clock gene and only two oscillators, but it is able
to show how the interaction between two anatomically and
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43
44
Mathematical model of the interaction between the dorsal and ventral regions
functionally distinct regions interact to render the output
of the SCN.
Although simplified, the model presented reproduces
experimental (1,6-8) and computational (5,18,19) results that are
similar to those described in the literature. Additionally,
this study suggests that the intensity of the interaction between the oscillators and their periods define the output of
the simulated rate. Thus, this work suggests new experimental approaches for the study of circadian rhythm in the
dorsomedial and ventrolateral regions of the SCN.
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16.Bito H, Deisseroth K, Tsien RW. Ca2-dependent regulation in
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Sleep
Science
ORIGINAL ARTICLE
Evening chronotypes experience poor sleep quality
when taking classes with early starting times
Estudantes com cronótipo vespertino apresentam pior
qualidade do sono quando as aulas iniciam mais cedo
Ádison Mitre Alves de Lima1, Gabriela Carminhola Gomes Varela1,
Hosana Aparecida da Costa Silveira1, Renata Davin Gomes Parente1, John Fontenele Araujo1
ABSTRACT
Background and objective: The sleep-wake cycles of medical
students are affected by their academic demands and class starting
times. Despite their different sleep needs and characteristics (such
as chronotype), all students must follow the same schedule. Therefore, it is necessary to know whether chronotype is related to sleep
quality and daytime sleepiness. Methods: A study was carried out
with the assessment of 234 students in their 1st through 4th years of
medical school at the Federal University of Rio Grande do Norte. The
Pittsburgh Sleep Quality Index (PSQI) was used to evaluate subjective sleep quality. Values below 5 indicated good sleep quality, while
values above 5 indicated poor sleep quality. The Epworth Sleepiness
Scale (ESS), in which values above 10 indicate extreme daytime sleepiness, was used to evaluate daytime sleepiness. A Portuguese version of
a questionnaire for the “eveningness-morningness” dimension developed by Horne and Östberg (1976) was used to classify participants
with regard to their chronotypes. Results: Chronotype had a normal distribution. Students of second through fourth years of medical
school had sleep quality values above 5, while students of the first year
had sleep quality values below 5. Daytime sleepiness was homogenous
across groups, with an average of 8.9 (SD=3.55). Chronotype and
sleep quality had a linear relationship (p<0.001); evening types had
worse sleep quality than morning types. The relationship between
chronotype and daytime sleepiness was not statistically significant.
Conclusions: The medical students evaluated in this study had low
sleep quality, particularly those of extreme evening type.
Keywords: Sleep; Students, medical; Sleep stages; Chronobiology
phenomena
RESUMO
Introdução e objetivo: O ciclo sono-vigília dos estudantes de Medicina é afetado por suas demandas acadêmicas e pelo horário de início
das aulas. Apesar de suas diferentes necessidades e características do
sono (como o cronótipo), todos os alunos devem seguir o mesmo calendário. Portanto, é necessário saber se o cronótipo está relacionado à
qualidade do sono e à sonolência diurna. Métodos: O estudo foi realizado com 234 estudantes, desde o primeiro até o quarto ano da faculdade de Medicina da Universidade Federal do Rio Grande do Norte.
O Índice de Qualidade de Sono de Pittsburgh (IQSP) foi utilizado
para avaliar a qualidade subjetiva do sono. Valores abaixo de 5 indicam boa qualidade de sono, enquanto os valores acima dos 5 indicam
qualidade ruim. A escala de Epworth (ESE), em que valores acima de
10 indicam sonolência extrema, foi utilizada para avaliar a sonolência
diurna. Uma versão em Português de um questionário para a avaliação
do cronótipo desenvolvida por Horne e Östberg (1976) foi utilizada
para classificar os participantes em seus respectivos cronótipos. Resultados: O cronótipo apresentou uma distribuição normal. Os alunos do
segundo ao quarto ano do curso de medicina apresentaram uma média
no IQSP acima de 5, enquanto os estudantes em seu primeiro ano
tiveram no IQSP uma média inferior a 5. A sonolência diurna foi homogênea entre os grupos, com uma média de 8,9 (DP=3,55). Cronótipo e qualidade de sono apresentaram uma relação linear (p<0,001);
cronótipos vespertinos apresentaram qualidade de sono pior do que os
matutinos. A relação entre cronótipo e sonolência durante o dia não
foi estatisticamente significativa. Conclusões: Os estudantes de Medicina avaliados neste estudo apresentaram baixa qualidade do sono,
particularmente aqueles classificados como vespertinos extremos.
Descritores: Sono; Estudantes de Medicina; Fases do sono; Fenômenos cronobiológicos
INTRODUCTION
Several studies have shown that sleep quality is important
for the normal functioning of individuals. Epidemiological
studies have shown that approximately 30% of adults suffer
from sleep disorders (1). Sleep disorders cause deficits of cognition, attention and memory, thus impairing performance
in daily tasks and increasing the propensity for psychiatric, cardiovascular and metabolic disorders, as well as other
health problems (2). In addition, we know that irregularities
in the sleep-wake cycle pattern affect sleep quality.
Among the general population, university students are
a group that frequently presents sleep disorder complaints.
These complaints have been reported mainly by students in
programs with high academic demands, such as medicine,
Study carried out at Universidade Federal do Rio Grande do Norte – UFNR, Natal (RN), Brazil.
1
Research Group on Circadian Rhythms, Sleep, Memory and Emotion, Universidade Federal do Rio Grande do Norte – UFNR, Natal (RN), Brazil.
Corresponding author: Renata Parente – P.O. Box 1524, Campus Universitário, Lagoa Nova, CEP 59072-970 – Natal (RN), Brazil – E-mail: [email protected]
Received: January 8, 2010; Accepted: March 30, 2010
Sleep Sci. 2010;3(1):�����
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Evening chronotypes experience and poor sleep quality
in which sleep-wake cycle regularity is compromised as a
result of academic requirements, causing a delay in sleep onset. Individual chronotypes are also relevant, as those with
evening chronotypes show difficulty in waking up early.
In addition to individual chronotype characteristics,
one should take into account the fact that people have different sleep needs. Studies have shown extremes of people
who need a maximum of 6 hours 30 minutes of sleep (3) or
a minimum of 8 hours 30 minutes of sleep (3), called short
and long sleepers, respectively.
This study aimed to evaluate the sleep quality of medical
students relative to these students’ chronotypes. Our hypothesis is that the evening chronotype has an increased tendency
to suffer from worse sleep quality and greater daytime sleepiness. Furthermore, we evaluated if sleep quality and daytime
sleepiness differ according to the semester of attendance.
METHODS
This was an observational, cross-sectional, individualized study.
We evaluated 234 medical students at the Federal University of Rio Grande do Norte (UFRN). The students were
attending their first four years of medical school. For the
analysis, we divided the students according to the semester
of attendance: 50 in the first, 41 in the second, 26 in the
fourth, 36 in the fifth, 29 in the sixth, 31 in the seventh and
21 in the eighth. After a brief presentation of the study, they
were invited to participate in the project voluntarily. Students who were in the third semester could not participate
in the study because they changed schedules class during
the data collection phase. As exclusion criteria, we used the
presence of a previously diagnosed sleep disorder and the use
of medications that alter sleep architecture. Incorrectly or
partially completed data were not analyzed. Thereby, we excluded the questionnaires, not the participants, therefore the
total sample is different in each questionnaire. A total of 224
students reported their gender (114 males and 110 females).
To evaluate subjective sleep quality, we used the Pittsburgh Sleep Quality Index (PSQI) (4), which consists of 19
questions, analyzed in seven components: subjective quality,
latency, duration, habitual efficiency, sleep disturbances, use
of medications for sleep and daytime dysfunction. Each component is given a score from 0 to 3, resulting in a total score
ranging from 0 to 21. Ratings between 0 and 5 indicate good
sleep quality, while those above 5 indicate poor sleep quality.
The ESS (5), used to assess daytime sleepiness, consists of
eight questions that evaluate the possibility, on a scale of 0 to 3,
that an individual will fall asleep in various everyday situations.
After applying the scale, the values given to each situation are
summed, and a total value is obtained, quantifying daytime
sleepiness. The scale has a maximum of 24 points, and a total
value greater than 10 is considered excessive sleepiness.
Sleep Sci. 2010;3(1):�����
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To classify the students’ chronotypes, we used a Portuguese version of the questionnaire developed by Horne and
Östberg (6) (1976), under which individuals are classified according to their score: extreme evening type (EE), 16 to 30;
moderate evening type (ME), 31 to 41; intermediate (I), 42
to 58; moderate morning type (MM), 59 to 69; or extreme
morning type (EM), 70 to 86.
The independent variables analyzed were ‘semester’ and
‘chronotype’; the dependent variables were ‘daytime sleepiness’ and ‘sleep quality’. For data analysis, we used the SPSS
program, version 8.0 (SPSS Inc., Chicago, IL, USA) and oneway ANOVA for non-repeated measures with Bonferroni
verification and the linear analysis between chronotype and
sleep quality. P-values<0.05 were considered significant.
The study was conducted after approval by the Ethics Committee of the Federal University of Rio Grande do
Norte (UFRN) and signature of the informed and free consent statements by the students.
RESULTS
The chronotype distribution pattern was normal both in the
total sample and in terms of gender and semester of attendance. In the classification, we found 12 (5.33%) extreme
evening types, 32 (14.22%) moderate evening types, 133
(59.11%) intermediate types, 45 (20%) morning types and
3 (1.33%) extreme morning types.
The average PSQI obtained in the sample was 5.58±2.58.
Considering the entire sample, 130 (57.2%) had good sleep
quality, and 98 (42.98%) had poor sleep quality. We observed the best scores on two components of this index:
subjective sleep quality (1.17±0.74) and sleep duration
(0.81±0.91). With regard to the semester of attendance,
only students in the first and second semesters had good
sleep quality (4.00±2.02 and 4.70±1.71, respectively). Students in their fifth semester had the worst sleep quality, with
an average of 7.25±3.04 (Figure 1, Table 1).
<0
O205
10
*153
O174
O139
O121
0
PSQI
46
-10
N-
46
1
40
2
Semester
Figure 1: PSQI by semester.
26
4
35
5
29
6
31
7
21
8
Lima ÁMA, Varela GCG, Silveira HAC, Parente RDG, Araujo JF
Table 1: PSQI and ESS distributions by semester
Semester
1
2
4
PSQI
4±2.02
4.7±1.71
5.96±2.52
ESS
7.97±3.02
9.05±3.54
9.38±3.83
PSQI: Pittsburgh Sleep Quality Index; ESS: Epworth Sleepiness Scale.
5
7.26±3.04
9.8±3.75
6
5.83±2.83
8.51±3.69
7
6.29±2.07
8.64±3.66
8
6.14±2.37
9.38±3.51
Table 2: PSQI and ESS distributions according to chronotype
Chronotype
EM
MM
I
ME
EE
PSQI
4.67±2.52
4.96±2.55
5.38±2.29
6.52±2.61
8.5±3.5
ESS
10.33±6.43
8.96±3.23
8.5±3.54
9.09±3.46
11.92±3.58
EM: extreme morning; MM: moderate morning; I: intermediate; ME: moderate evening; EE: extreme evening; PSQI: Pittsburgh Sleep Quality
Index; ESS: Epworth Sleepiness Scale.
A regression analysis between chronotype and sleep
quality showed a linear correlation (R=0.296, p<0.001).
Individuals with a tendency toward eveningness presented
poorer sleep quality (Table 2). A more detailed analysis
showed that this relationship depended on the components
C1 (subjective quality of sleep, p=0.02) and C3 (sleep duration, p<0.001).
Data analysis of the Epworth Sleepiness Scale (ESS)
showed a mean score of 8.9±3.55, suggesting that this
population experiences little daytime sleepiness. Considering the whole sample, 71 students (31.14%) had excessive
daytime sleepiness, while 157 (68.86%) did not have it.
However, when evaluating the ESS scores, we found that the
medical students classified as extreme evening and extreme
morning types showed extreme sleepiness (11.92±3.58 and
10.33±6.43, respectively) (Table 2). With regard to the semester of attendance, we found no statistically significant
differences in ESS scores between semesters.
DISCUSSION
In these studies, we found that the assessed medical students
had poor sleep quality beginning in their fourth semester
of attendance. Students in their fifth semester had worse
sleep quality and PSQI scores significantly higher than students in other semesters. In terms of daytime sleepiness, we
found no differences between students attending different
semesters. When we analyzed sleep quality in relation to
chronotype, we found an inverse linear correlation between
chronotype scores and sleep quality scores; in other words,
individuals with a tendency toward eveningness presented
poorer sleep quality.
In our study, the average ESS score (8.9±3.55) was close
to that obtained in another study with medical students,
which showed an average score of 9.38±4.06 at the beginning of the semester and 10.72±4.03 at the end of the semester (7).
Concerning the PSQI, 42.98% of the students showed
poor sleep quality. This result confirmed what was found
in two previous studies with medical students in Brazil, in
which approximately 40% of respondents experienced poor
sleep quality (8,9). In one of these studies, the statistical analysis revealed large contributions of components 1 (subjective
sleep quality) and 3 (sleep duration) of the PSQI, a result
that corroborates the data obtained in our study. However, a
study assessing North American university students showed
that over 60% had poor sleep quality (10). The higher prevalence of students with poor sleep quality in that study may
be related to different cultural habits.
In our study, the two first semesters, during which classes
started one hour later than in other semesters, were the only
ones with average PSQI values compatible with good sleep
quality (PSQI<5). In a study from the Chronobiology Laboratory of the UFRN, 42.3% of students with a class schedule that began earlier showed poor sleep quality, whereas
only 11.5% of those with later class start times showed poor
sleep quality (9). This result shows that daily class start times
are extremely important in determining the sleep quality of
students.
The correlation between chronotype and sleep quality
scores found in our study was in accordance with a study relating chronotype to sleep quality in nurses working in shifts,
highlighting chronotype as a strong predictor in the determination of sleep quality, where evening type individuals had
worse sleep quality (11).
Concern with the regularity of the sleep-wake cycle, as
well as with sleep hygiene, is not well established yet in
medical schools. However, several studies have emphasized
this problem, which seems to be a far more serious problem
than originally considered by medical schools. For example,
a survey conducted in our university found that students
with greater irregularities in their sleep-wake cycles and
shorter sleep durations had poorer academic performance
(10)
. Furthermore, experimental evidence of the importance
of biological rhythm regularity led the World Health Organization to consider shift jobs, which cause disturbances in
circadian rhythms, carcinogenic factors (12).
Sleep Sci. 2010;3(1):�����
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47
48
Evening chronotypes experience and poor sleep quality
Those who are responsible for organizing activities in
medical schools should take into account the individual characteristics (such as chronotype) and design an activity schedule that does not lead to sleep deprivation or irregularity.
REFERENCES
1. Partinen M, Hublin C. Epidemiology of sleep disorders. In:
Kryger MH, Roth T, Dement WC, editors. Principles and practice of Sleep Medicine. 4th ed. Philadelphia: Elsevier; 2005. p.
626-47.
2. Walsh JK, Dement WC, Dinges DF. Sleep medicine, Public Policy and Public Health. In: Kryger MH, Roth T, Dement WC,
editors. Principles and practice of Sleep medicine. 4th ed. Philadelphia: Elsevier; 2005. p. 648-56.
3. Almondes KM, Araujo JF. Padrão do ciclo sono-vigília e sua relação com a ansiedade em estudantes universitários. Estud Psicol
(Natal). 2003;8(1):37-43.
4. Buysse DJ, Reynolds CF 3rd, Monk TH, Berman SR, Kupfer
DJ. The Pittsburgh Sleep Quality Index: a new instrument for
psychiatric practice and research. Psychiatry Res. 1989;28(2):
193-213.
5. Johns MW. A new method for measuring daytime sleepiness: the
Epworth sleepiness scale. Sleep. 1991;14(6):540-5.
6. Horne JA, Ostberg OA. Self-assessment questionnaire to determine morningness-eveningness in human circadian rhythms. Intern J Chronobiol. 1976;4(2):97-110.
7. Rodrigues RND, Viegas CAA, Silva AA, Tavares P. Daytime
sleepiness and academic performance in medical students. Arq
Neuropsiquiatr. 2002;60(1):6-11.
8. Medeiros ALD, Mendes DBF, Lima PF, Araujo JF. The relationship between sleep-wake cycle and academic performance in medical students. Biological Rhythm Research. 2001;32(2):263-70.
9. Lima PF, Medeiros ALD, Araujo JF. Sleep-wake pattern of medical students: early versus late class starting time. Braz J Med Biol
Res. 2002;35(11):1373-7.
10.Lund HG, Reider BD, Whiting AB, Prichard JR. Sleep patterns
and predictors of disturbed sleep in a large population of college
students. J Adolesc Health 2010;46(2):124-32.
11.Chung M, Chang F, Yang CH, Kuo TBJ, Hsu N. Sleep quality and morningness-eveningness of shift nurses. J Clin Nurs.
2008;18(2):279-84.
12.Straif KBR, GrosseY, Secretan B, El Ghissassi F, Bouvard V, Altieri A, et al. Carcinogenicity of shift-work, painting, and firefighting. Lancet Oncol. 2007;8(12):1065-6.
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Sleep
Science
ORIGINAL ARTICLE
Masking effect in rats bearing partial lesions
of the suprachiasmatic nucleus
Efeito máscara em ratos apresentando lesões parciais do núcleo supraquiasmático
Manuel Ángeles-Castellanos1, Jorge M. Amaya1, Ruud M. Buijs2, Carolina Escobar1
ABSTRACT
Background and objective: To explore the role of the suprachiasmatic nucleus (SCN) for masking by light in rats bearing bilateral
complete or partial lesions of the suprachiasmatic nucleus. Methods:
Rats were submitted to electrolytic SCN lesions, a������������������
nd their spontaneous activity was monitored to verify arrhythmia in different lighting
conditions, under light/dark cycle and constant darkness. Results:
Rats���������������������������������������������������������������
bearing a partial suprachiasmatic nucleus lesion presented arrhythmic activity patterns when assessed in constant darkness; however, in a light-dark cycle, rats show a diurnal pattern. Partial lesions
were evidenced through histological analysis. Conclusions: We conclude that when behavioral indicators are used evidence suprachiasmatic nucleus lesions, animals should be tested at least in two lighting conditions; we also conclude that the suprachiasmatic nucleus is
necessary for masking by light.
Keywords: Circadian rhythm; Darkness; Light; Suprachiasmatic
nucleus/injuries; Perceptual masking
RESUMO
Introdução e objetivo: Averiguar, por meio do uso de luz, o papel
desempenhado pelo núcleo supraquiasmático de ratos quando estes
animais sofreram lesões completas ou parciais do núcleo supraquiasmático. Métodos: Os ratos sofreram lesões eletrolíticas no núcleo
supraquiasmático e sua atividade espontânea foi monitorada com
o intuito de averiguar a existência de arritmia em condições de luz
variada, mais precisamente em ciclos de luz/escuridão e escuridão
completa. Resultados: Ratos que sofreram lesão parcial do núcleo supraquiasmático exibiram padrões de arritmia quando foram testados
em escuridão completa, porém, durante os ciclos de luz/escuridão, demonstraram um padrão duplo. As lesões foram evidenciadas por meio
de análise histológica. Conclusões: Concluímos que, quando indicadores comportamentais são utilizados para tornar lesões no núcleo
supraquiasmático evidentes, os animais precisam ser testados em pelo
menos duas condições de luz e que o núcleo supraquiasmático desempenha um papel fundamental no efeito máscara provocado pela luz.
Descritores: Ritmo circadiano; Escuridão; Luz; Núcleo supraquiasmático/ lesões; Mascaramento perceptivo
INTRODUCTION
The suprachiasmatic nucleus (SCN) of the anterior hypothalamus is the site of the mammalian circadian pacemaker. In 1972,
lesions in the SCN of rats were reported to abolish behavioral
(wheel running and drinking) and endocrine (corticosterone)
circadian rhythms (1,2). The SCN endogenous rhythms of glucose
utilization, measured by deoxyglucose uptake (3) and rhythms
of electrical activity (4), were demonstrated in intact rats. Also,
transplantation of fetal hypothalamic tissue containing the SCN
restored locomotor circadian rhythmicity in arrhythmic rats
bearing bilateral lesion of the SCN. The new restored period corresponded to the phenotype of the transplanted SCN (5,6). Such
findings confirmed the SCN as the anatomical structure that is
capable of generating and transmitting circadian rhythms, thus
being considered the master biological clock.
In rats, the SCN is subdivided into two subregions: the
ventrolateral SCN, constituted mainly by Vasoactive Intestinal Peptide immunoreactive (VIP) cells and arginine vasopressin peptide immunoreactive (AVP-ir) cells, respectively
(7)
. Under constant darkness, nocturnal mammals express their
endogenous period, which drifts progressively from the external cycles. When individuals are exposed to the LD cycle, this
daily drift is corrected by environmental cues, resetting the
clock every cycle and imposing phase and period of 24 hours.
Lighting information is conveyed by the retinohypothalamic
tract (RHT) which emerges from the optic chiasm and enters
the ventral and lateral regions of the SCN (8).
Entrainment is the process that directly influences period
and phase in the SCN and is mainly determined by the LD
cycle whereas the masking effect results from environmental stimuli that require immediate response and affect the
overt expression of behavioral rhythms for a unique occasion
without modifying the SCN rhythmicity. Due to this independence of processes, it is suggested that masking relies on
Study carried out at Universidad Nacional Autónoma de México – UNAM, México DF 04510, México.
1
Departamento de Anatomía, Facultad de Medicina, México DF 04510, México.
2
Departamento de Biología Celular y Fisiología, Instituto de Investigaciones Biomédicas, México DF 04510, México.
Corresponding author: Manuel Ángeles Castellanos – Departamento de Anatomía – Facultad de Medicina da Universidad Nacional Autónoma de México – Edificio B,
4º Piso – México DF 04510 – E-mail: [email protected] – Phone: ++5255-5623-2422
This study was supported by grants PAPIIT-UNAM IN 205809. IN- 203907 and by CONACyT 82462.
Received: January 8, 2010; Accepted: March 12, 2010
Sleep Sci. 2010;3(1):�����
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50
Masking effect in rats
structures outside the SCN (9). Previous studies suggest that
a functional SCN is necessary to express masking, and that
masking to light is mediated by the same RHT projections
from the retina to the hypothalamus (10,11).
The RHT also projects to other hypothalamic areas, including the paraventricular nucleus and anterior hypothalamic area
(12-14)
. The role of these extensive extra SCN connections is not
clear, although it is known that it may be related to light effects
on homeostatic functions overriding the signals of the SCN and
possibly producing masking (9).
The present study explored the role of the SCN for masking by light in rats bearing bilateral complete or partial lesions
of the SCN. We report that the SCN is necessary for mediating
masking effects, since complete SCN lesions abolished the daily
adjustment to the LD cycle.
METHODS
Adult male Wistar rats weighing 200 to 250g at the beginning of the experiment were housed in individual transparent acrylic cages and maintained in a soundproof monitoring room with a 12:12 h LD cycle, lights on at 7h, defined
as ZT0, and lights off at 19h defined as ZT12. The room was
maintained at a constant temperature of 22±1°C and with
continuous air flow. Rats had free access to food (Rodent
Laboratory Chow 5001) and water. Experimental procedures carried out during this study were in strict accordance
with the Mexican norms for animal handling Norma Oficial
Mexicana NOM-062-ZOO-1999, which conforms to international guidelines for animal handling, and were approved
by the Ethics Committee of the Medicine Faculty UNAM.
All efforts were made to minimize the number of animals
and their suffering.
EXPERIMENTAL DESIGN
Complete SCN lesion group (SCNX) (n=5). After surgical
procedures, rats were allowed to recover for at least a month
before starting the study. All SCNX rats were monitored with
a movement detection system in LD conditions to confirm
loss of rhythmicity. Rats exhibiting arrhythmia under LD and
DD were classified as ‘complete lesion’. Partial SCN lesion
group. Rats exhibiting arrhythmia in DD but proving to be
rhythmic in LD were also included in this group (n=6).
GENERAL ACTIVITY MONITORING SYSTEM
The individual rat cages (45 x 30 x 35 cm) were placed on
plates with movement sensors in soundproof lockers with
controlled lighting conditions. The detection system was
developed in our group with the contributions from Nico
Bos in Amsterdam, the Netherlands, and the Mexican biomedical company Omnialva. Sensors as previously reported
(15)
. Behavioral events were collected with a digitized system
Sleep Sci. 2010;3(1):�����
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and automatically stored with a PC in 15-minute intervals
for further analysis with a system developed for our laboratory SPAD9 based on Matlab. For each rat, double-plotted
actograms were obtained, and analyses with the chi-square
periodogram were included.
SURGICAL PROCEDURE FOR SCN LESIONS
Rats were anesthetized with a mixture of ketamine (90 mg/
kg) and xylazine (10 mg/kg), then being placed in a stereotaxic frame for rats (David Kopf model 900). Rats received bilateral lesions directly in the SCN, using stainless steel insect
pins insulated with epoxy paint, except at the tips (0.20 mm).
The pin tips were lowered according to coordinates bregma 0,
lateral ±0.2 from the midsagital sinus, and -9.4 ventral from
the dural surface. One mA of direct current was delivered for
30 seconds with a lesion maker elaborated in our group. Rats
were left to recover for at least two weeks in their home cages.
Their spontaneous activity was monitored to verify arrhythmia in a DD cycle during ten days after rats went through a
12:12 cycle (light on at 8h) for seven days.
IMMUNOHISTOCHEMISTRY
At the last day of the entrainment protocol, rats were anesthetized with an overdose of sodium pentobarbital (Anestesal 65
mg/mL), and were transcardially perfused with 250 mL of 0.9%
saline followed by 250 mL of fixative 4% paraformaldehyde in
phosphate buffer saline (PBS, 0.1M, pH 7.2). Brains were removed, after fixed for 24 hours and cryoprotected in 30% sucrose for 3 to 4 days. Brains were frozen and cut in sections of
40 mm at -18°C. Sections were serially collected in 4 sets; one
set of sections was processed for Vasoactive Intestinal Peptide
(VIP-ir) and a second set was processed for Arginine vasopressin peptide (AVP-ir). Free floating sections were incubated in
VIP or AVP antibody raised in rabbit (1:1000) in phosphate
buffer 0.1M, ph 7.2 with 0.9% saline,1% goat serum, and
0.3% Triton X-100 (PBSGT) for 48 hours. This was followed
by incubation in secondary antibody, goat anti-rabbit (Vector
Laboratories) 1:200 in PBSGT for 2 hours at room temperature,
followed by incubation in avidin-biotin complex (0.9% avidin
and 0.9% biotin solutions; Vector Laboratories) in PBSGT for
2 hours at room temperature. Between incubations, the sections
were rinsed 3 times for 10 minutes in PBS. Tissues were reacted
with diaminobenzidine (50 mg/100 mL) and hydrogen peroxide (35 ml, 30% H2O2) to obtain a reddish brown color. Tissues
were mounted, dehydrated and coverslipped with microscopy
Entellan (Merck). The primary antibodies were kindly donated
by Dr. Ruud Buijs.
Images of selected sections were digitized at a 10X magnification using a computerized image system (Image-Pro
plus 5.1; mediaCibernetic) attached to a light microscope
(Olympus BX41).
Ángeles-Castellanos M, Amaya J M, Buijs RM, Escobar C
Figure 1: Representative double plotted actograms for general activity in a partial SCN lesion rat (A). Mean activity profiles of six rats in DD condition
(B) and after switching to LD (C). Analysis through the chi-square periodogram in DD (D) and LD condition (E). Continuous black bars represent the
DD condition; white and black horizontal bars represent the LD cycle.
RESULTS
Actograms indicated complete loss of rhythmicity in DD
conditions for all rats and recovery of rhythmicity in LD
cycle for partial lesion rats (Figure 1A). Visual inspection of
the activity profiles in DD indicated no differences between
day and night activities (Figure 1B), while in LD cycle the
rats recovered their diurnal rhythm showing significantly
lower activity in the day and high activity in the dark phase
(Figure 1C). The�����������������������������������������
chi-square
����������������������������������������
periodogram �����������������
confirmed the arrhythmic pattern in DD conditions and the diurnal activity
rhythm in LD cycle (Figure 1D and E). Sections processed
with AVP-ir and VIP-ir confirmed complete and partial lesions in the SCN (Figure 2A and B, respectively). The majority of partial injured rats showed a consistent amount of
VIP cell in the ventral area, while the AVP-ir was substantially decreased (Figure 2A and B).
DISCUSSION
In agreement with previous reports, SCN lesions abolished
circadian rhythms of general activity (16). A main finding
herein reported is that the rats tested under conditions of
constant darkness exhibited clear arrhythmic patterns.
However, when changing the conditions to a LD cycle arrhythmic animals showed a very robust diurnal pattern, the
histological analysis clearly indicated remnants of the SCN.
Data previously revealed evidenced that the masking effect
of light and dark alternation on activity requires the SCN,
and that the presence of the SCN allows the suppression of
locomotor activity by light as previously described (17).
Figure 2: Photomicrographs of a representative incomplete SCN lesion
exhibiting the immunoreactivity for AVP (A) and VIP (B). Clear remnants of SCN can be observed for both cell groups.
Sleep Sci. 2010;3(1):�����
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51
52
Masking effect in rats
Previous studies suggested that the SCN is not necessary
for the direct effects of light on locomotor rhythmicity and
that extra SCN retinal projections may provide timing signals to other hypothalamic nuclei (18).
The neural sites mediating the masking effect have not
been identified yet; however, the neuronal activation produced by light in the SCN is modulated by activity and
arousal (19,20), suggesting that the ventral SCN may be involved in the masking process.
The histological examination of partial lesions confirmed
that the SCN of rats recovering rhythmicity after a switch
to the LD cycle contained mainly VIP cells corresponding to
the retinorecipient area of the SCN.
REFERENCES
1. Moore RY, Eichler ME. Loss of circadian corticosterone rhythm
following suprachiasmatic lesions in the rat. Brain Res.
1972;42(1):201-6.
2. Stephan FK, Zucker I. Circadian rhythms in drinking and locomotor activity of rats are eliminated by hipothalamic lesions. Proc
Natl Acad Sci USA. 1972;69(6):1583-6.
3. Schwartz WJ, Gainer H. Suprachiasmatic nucleus: use of 14clabeled deoxyglucose uptake as a functional marker. Science.
977;197(4308):1089-91.
4. Inouye ST, Kawamura H. Persistence of circadian rhythmicity in
a mammalian hypothalamic “island” containing the suprachiasmatic nucleus. Proc Natl Acad Sci USA. 1979;76(11):5962-6.
5. Menaker M, Vogelbaum MA. Mutant circadian period as a marker of suprachiasmatic nucleus function. J Biol Rhythms. 1993;8
Suppl:S93-S8.
6. Ralph M. Suprachiasmatic nucleus transplant studies using the
Tau mutation in golden hamsters. In: Klein DC, Moore RY, Reppert SM, editors. Suprachiasmatic nucleus: the mind’s clock. Oxford: Oxford University Press; 1991. p. 341-8.
7. Moore RY. Entrainment pathways and the functional organization
of the circadian system. In: Buijs RM, Kalsbeek A. Romijn HJ,
Pennartz CMA, Mirmiram M, editors. Hypothalamic integration
of circadian rhythms. Amsterdam: Elsevier; 1996. p. 103-19.
(Progress in Brain Research, 111).
8. Morin LP, Goodless-Sanchez N, Smale L, Moore RY. Projections of the suprachiasmatic nuclei, subparaventricular zone
and retrochiasmatic area in the golden hamster. Neuroscience.
1994;61(2):391-410.
9. Redlin U. Neural basis and biological function of masking by
light in mammals: suppression of melatonin and locomotor activity. Chronobiol Int. 2001;18(5):737-58.
10.Mrosovsky N. Masking: history, definitions and measurement.
Chronobiol Int. 1999;16(4):415-29.
11.Mrosovsky N, Foster RG, Salmon PA. Thresholds for masking
responses to light in three strains of retinally degenerate mice. J
Comp Physiol A. 1999;184(4):423-8.
12.Youngstrom TG, Weiss ML, Nunez AA. A retinal projection to
the paraventricular nuclei of the hypothalamus in the Syrian hamster (Mesocricetus auratus). Brain Res Bull. 1987;19(6):747-50.
13.Card JP, Brecha N, Karten HJ, Moore RY. Immunocytochemical
localization of vasoactive intestinal polypeptide-containing cells
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and processes in the suprachiasmatic nucleus of the rat: light and
electron microscopic analysis. J Neurosci. 1981;1(11):1289-303.
14.Moore RY. Retinohypothalamic projection in mammals: a comparative study. Brain Res. 1973;49(2):403-9.
15.Escobar C, Martínez-Merlos MT, Angeles-Castellanos M, del Carmen Miñana M, Buijs RM. Unpredictable feeding schedules unmask a system for daily resetting of behavioural and metabolic
food entrainment. Eur J Neurosci. 2007;26(10):2804-14.
16.Angeles-Castellanos M, Salgado-Delgado R, Rodriguez K, Buijs
RM, Escobar C. The suprachiasmatic nucleus participates in food
entrainment: a lesion study. Neuroscience. 2010;165(4):1115-26.
17.Scheer FA, Ter Horst GJ, Van Der Vliet J, Buijs RM. Physiological and anatomic evidence for regulation of the heart by suprachiasmatic nucleus in rats. Am J Physiol Heart Circ Physiol.
2001;280(3):H1391-9.
18.Redlin U, Mrosovsky N. Masking by light in hamsters with SCN
lesions. J Comp Physiol A. 1999;184(4):439-48.
19.Maywood ES, Mrosovsky N, Field MD, Hastings MH. Rapid
down-regulation of mammalian period genes during behavioral resetting of the circadian clock. Proc Natl Acad Sci U S A.
1999;96(26):15211-6.
20.Mistlberger RE, Antle MC. Behavioral inhibition of light-induced circadian phase resetting is phase and serotonin dependent.
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Sleep
Science
SHORT COMMUNICATION
Effect of brief constant darkness and illumination on
mitochondrial respiratory control of the pineal gland,
Harderian gland, spleen and thymus of adult rat
Efeitos da escuridão e da luminosidade breve e constante no controle da
respiração mitocondrial das glândulas pineal, Harderiana, baço e timo
Oscar Kurt Bitzer-Quintero1, Airam Jenny Dávalos Marín2, Fermín Paul Pacheco-Moises3, Erandis Dheni Torres-Sánchez3
and Genaro Gabriel Ortíz4
ABSTRACT
Background and objectives: Constant environmental conditions
can lead to changes in the synthesis of melatonin. In vitro studies have
shown that this hormone modulates the efficiency of mitochondrial
respiration. Therefore, this work examined whether the efficiency of
mitochondrial respiration changes in rats that have been subjected
to constant illumination or darkness for a short period. Methods:
Rats were randomly distributed in three groups: Control, Constant
Illumination (72 hours) and Constant Darkness (72 hours). Upon
completion of treatment, rats were sacrificed and mitochondria from
the pineal gland, Harderian gland, thymus and spleen were isolated.
Subsequently, mitochondrial respiratory control was quantified from
the removed tissues in the three experimental groups. Results: Our
findings show that brief treatments of continued illumination or continued darkness had no significant effect on mitochondrial respiratory control in spleen, thymus or Harderian glands. In contrast, we
observed a slight increase in mitochondrial respiratory control in the
pineal gland of animals exposed to constant illumination. Conclusions: Our results suggest that brief treatment with continuous light
or darkness does not have a significant effect on the efficiency of mitochondrial activity in spleen, thymus or Harderian gland. This is probably due to the endogenous circadian rhythms that tightly regulate
mitochondrial enzymatic activity in these tissues.
Keywords: Mitochondria; Cell respiration; Pineal gland; Circadian
rhythm; Disease models, animal; Rats, Wistar
RESUMO
Introdução e objetivos: Mudanças constantes do meio ambiente
podem levar a alterações na síntese de melatonina. Estudos in vitro
têm evidenciado que o hormônio melatonina atua na modulação da
respiração mitocondrial. Este trabalho investigou se a eficiência de
tal respiração é alterada em ratos que foram submetidos à luminosidade ou à escuridão constante por um breve período. Métodos:
Os ratos foram aleatoriamente distribuídos em três grupos: Controle, Luminosidade Constante (72 horas) e Escuridão Constante (72
horas). Ao término do tratamento, os ratos foram sacrificados e as
mitocôndrias das glândulas pineais, Harderiana, timo e baço foram
isoladas. Em seguida, o controle da respiração mitocondrial dos tecidos dos quais foram removidos foi quantificado nos três grupos.
Resultados: Os resultados deste estudo mostraram que tratamentos
breves utilizando luminosidade ou escuridão contínua não tiveram
nenhum efeito significativo no controle da respiração mitocondrial
das glândulas pineais, Harderiana, timo e baço. Em contrapartida,
verificou-se um pequeno aumento de controle na respiração mitocondrial na glândula pineal de animais expostos à luminosidade
constante. Conclusões: Os resultados deste estudo indicam que tratamento breve com luminosidade ou escuridão contínua não exerce
efeito na eficiência da atividade mitocondrial das glândulas do baço,
timo ou Harderiana, e que tal fato provavelmente se deve aos ritmos
circadianos endógenos que exercem um controle rigoroso da atividade enzimática mitocondrial existente nesses tecidos.
Descritores: Mitocôndrias; Respiração celular; Glândula pineal; Ritmo circadiano; Modelos animais de doenças; Ratos Wistar
INTRODUCTION
Mammals possess a physiological system that coordinates all
metabolic functions, operates as a pacemaker, is sensitive to
light and that regulates circadian rhythms, seasonal cycles
and neuroendocrine responses in many species, including
humans. This system consists of the retina, the suprachiasmatic nucleus (SCN) and the pineal gland. The retina con-
Study carried out at Centro de Investigación Biomédica de Occidente – CIBO, IMSS, Guadalajara, Jalisco, México.
1
Laboratorio de Neuroinmunomodulación, División de Neurociencias, Centro de Investigación Biomédica de Occidente – CIBO, IMSS, Guadalajara, Jalisco, México.
2
Laboratorio de Neuroinmunomodulación, División de Neurociencias, Centro de Investigación Biomédica de Occidente – CIBO, IMSS, Guadalajara, Jalisco, México;
Departamento de Bioquímica, Instituto de Ciencias Biológicas, Facultad de Medicina, Universidad Autónoma de Guadalajara, Guadalajara, Jalisco, México.
3
Departamento de Química, Centro Universitario de Ciencias Exactas e Ingenierías – CUCEI, Universidad de Guadalajara, Guadalajara, Jalisco, México.
4
Laboratorio de Desarrollo, Envejecimiento y Enfermedades Neurodegenerativas, División de Neurociencias – CIBO, IMSS, Guadalajara, Jalisco, México.
Corresponding author: Genaro Gabriel Ortiz – Laboratorio de Desarrollo, Envejecimiento y Enfermedades Neurodegenerativas, División de Neurociencias, Centro de
Investigación Biomédica de Occidente (CIBO), IMSS – Sierra Mojada # 800 – Colonia Independencia – Cp 44340 – Guadalajara, Jalisco, México – Tel. (33) 36-68-30-00
ext. 31951 – Fax. (33) 36-18-17-56 – E-mail: [email protected]
Received: January 8, 2010; Accepted: April 4, 2010
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Effect of brief constant darkness and illumination on mitochondrial respiratory control
veys light information to the SCN through the retinohypothalamic projection, which connects to the pineal gland
through an additional pathway (1).
One of the consequences of the activation of these pathways by light is the circadian modulation of melatonin production by the pineal gland. Melatonin secretion is high at
night and low during the day and can be suppressed by a
short, continuous pulse of light (2). This has important metabolic consequences because melatonin influences the production of two hormones that play key roles in lipid and
carbohydrate metabolism: insulin and cortisol (3). Activity
of the pineal gland has also been proposed to influence the
endocrine, nervous and immune systems (4). In vitro studies have shown that melatonin increases the efficiency of
oxidative phosphorylation in the liver and brain (5). Whereas
some of the in vitro effects of melatonin on mitochondria
have been well characterized, little is known about the in
vivo consequences of light-regulated changes in melatonin
levels. However, changes in the efficiency of mitochondrial
respiration (or respiratory control) in the rat brain follow a
circadian pattern (5).
This study examines the effect of a short period (72 hours)
of continuous illumination or darkness on mitochondrial respiration in the pineal and Harderian glands, the spleen and
the thymus of rats.
METHODS
Subjects and experimental treatments
Adult male Wistar rats (280-300 g) were used for this
study. All animals were housed under controlled temperature (22±1 °C) and had free access to standard food (Purina)
and water.
All animal experiments were approved by the ethical
committee (Mexico) and were conformed to international
guidelines on the ethical use of animals, according to the
“NORMA Oficial Mexicana NOM-062-ZOO-1999”.
Rats were divided into three experimental groups,
each containing 20 animals. The first group (Control) was
housed under a normal 12-hour light/dark cycle, and the
second and third groups were housed under either constant
illumination or darkness for three days. Illumination for
the Control and the second experimental group was provided by Vita-Lite fluorescent lights. At the end of the
experimental period, rats were sacrificed by decapitation
and the spleen, thymus, pineal and Harderian glands were
removed immediately.
To prevent any circadian variability of mitochondrial
metabolic activity, treatments for all groups were initiated
at the same time (10h). All animals were sacrificed exactly
72 hours after the onset of treatment for harvesting of the
tissues of interest.
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Mitochondrial isolation
The removed glands and organs were homogenized in SHE
medium (250 mM sucrose, 25 mM Hepes (pH 7.5), 1 mM
EGTA) using a Potter-Elvehjem homogenizer (6). Mitochondrial proteins were quantified by Lowry’s method (7).
Mitochondrial respiration
Oxygen uptake of mitochondrial suspensions was determined
at 30ºC using a Clark-type oxygen electrode (Hansatech, UK)
in an air saturated media (0.5 ml) containing 125 mM KCl, 20
mM MOPS (pH 7.6), 0.1 mM EGTA, 5 mM KH2PO4, 2 mM
MgCl2 and 0.1 mg protein. The rate of mitochondrial respiration in state 3 was determined in the presence of 1 mM ADP
and 3 mM succinate. The rate of respiration in state 4 (basal
oxygen consumption) was determined in the presence of 3 mM
succinate, without the addition of exogenous ADP or after endogenous ADP had been consumed by the mitochondria. The
respiratory control index was calculated as the ratio between the
rate of oxygen consumption in state 3 and that in state 4.
Statistical analysis
Data are shown as mean±SEM, being analyzed by one-way
analysis of variance (ANOVA). The Student-Newman-Keuls
test was used to compare the experimental groups with the
Control, if appropriate. The level of statistical significance
was set at p<0.05.
RESULTS AND DISCUSSION
Figure 1 shows the respiratory control in mitochondria from
the spleen, thymus, pineal gland and Harderian gland of
control rats and experimental rats exposed to either constant
illumination or darkness for 72 hours. A significant, albeit
2.5
Control
Constant light
Constant darkness
2.0
Respiratory control
54
1.5
1.0
0.5
0.0
Thymus
Pineal
Harder
Spleen
Figure 1: Respiratory control of the indicated organs and glands of rats
subjected to brief treatment of continuous illumination or continuous
darkness. Respiratory control was measured as indicated in Methods. Asterisks indicate a significant difference from the control group (p<0.05).
The bar indicates the mean±SEM.
Bitzer-Quintero OK, Dávalos-Marín AJ, Pacheco-Moisés FP, Torres Sánchez ED, Ortíz GG
small, increase in the respiratory control of the pineal gland
in the constant illumination group compared with the control and constant darkness groups was found. This means
that under relatively brief, constant illumination, there is a
slight increase in the efficiency of mitochondrial ATP synthesis. No significant differences were found in the spleen,
thymus or Harderian gland for any of the treatments. In the
thymus, a trend towards an increase in respiratory control
under constant darkness was observed, but this tendency
was not statistically significant.
It is well known that light is a dominant signal for entrainment of the circadian system and, in particular, mitochondrial metabolism (8-11). However, our results suggest
that oxidative phosphorylation in the mitochondria remains
fully functional when rats are subjected to brief (72 hours)
constant darkness or illumination. In particular, we did not
observe any differences in mitochondrial efficiency in the
Harderian gland, thymus or spleen. The only difference
observed was an increase in the efficiency of mitochondrial
ATP synthesis in the pineal gland in the constant illumination condition.
These results were explained by the presence of endogenous circadian clocks. The free-running period of the rat
is longer than 24 hours. Under brief constant environmental conditions (either darkness or illumination), the rat’s
endogenous circadian clocks can express their endogenous
periodicity. Therefore, when rats are submitted to constant
environmental conditions for a brief period, this normal
rhythmicity is preserved.
7. Lowry OH, Rosebrough NJ, Farr AL, Randall RL. Protein
measurement with the Folin phenol reagent. J Biol Chem
1951;193(1):265-75.
8. Brooke H, Miller, McDearmon El, Panda S, Hayes KR, Zhang J,
et al. Circadian and CLOCK-controlled regulation of the mouse
transcriptome and cell proliferation. Proc Natl Acad Sci USA
2007;104(9):3342-7.
9. Akhtar RA, Reddy AB, Maywood ES, Clayton JD, King VM,
Smith AG, et al. Circadian cycling of the mouse liver transcriptome, as revealed by cDNA microarray, is driven by the suprachiasmatic nucleus. Curr Biol 2002;12(7):540-50.
10.Panda S, Antoch MP, Miller BH, Su AI, Schook AB, Straume M,
et al. Coordinated transcription of key pathways in the mouse by
the circadian clock. Cell 2002;109(3):307-20.
11.Storch KF, Lipan O, Leykin I, Viswanathan N, Davis FC, Wong
WH, et al. Extensive and divergent circadian gene expression in
liver and heart. Nature 2002;417(6884):78-83.
REFERENCES
1. Wehr TA. The durations of human melatonin secretion and sleep
respond to changes in daylength (photoperiod). J Clin Endocrinol
Metab 1991;73(6):1276-80.
2. Horowitz TS, Cade BE, Wolfe JM, Szeisler CA. Efficacy of bright
light and sleep/darkness scheduling in alleviating circadian
maladaptation to night work. Am J Physio Endocrinol Metab
2001;281(2):E384-91.
3. Koller M, Härma M, Laitinen JT, Kundi M, Piegler B, Haider M.
Different patterns of light exposure in relation to melatonin and cortisol rhythms and sleep of night workers. J Pineal Res 1994;16(3):
127-35.
4. Markus RP, Ferreira ZS, Fernandes PA, Cecon E. The immunepineal axis: a shuttle between endocrine and paracrine melatonin
sources. Neuroimmunomodulation 2007;14(3-4):126-33.
5. Martín M, Macías M, León J, Escames G, Khaldy H, Acuña-Castroviejo D. Melatonin increases the activity of the oxidative phosphorylation enzymes and the production of ATP in rat brain and
liver mitochondria. Int J Biochem Cell Biol 2002;34(4):348-57.
6. Moreno-Sánchez R, Torres-Márquez ME. Control of oxidative
phosphorylation in mitochondria, cells and tissues. Int J Biochem
1991;23(11):1163-74.
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55
Sleep
Science
CASE REPORT
Circadian variations in the capacity to adjust
behavior to environmental changes
Variações circadianas na capacidade de ajuste
comportamental em face de alterações ambientais
Aída García1, Candelaria Ramírez1, Pablo Valdéz1
ABSTRACT
Background and objective: Circadian variations have been found in
the performance of many human activities. The performance of many
tasks depends on basic cognitive processes, such as executive functions.
One of the components of these functions is the capacity to adjust behavior to environmental changes. The objective of this study was to
identify circadian rhythms in the capacity to adjust behavior to environmental changes. Methods: Three college students were recorded
in a constant routine protocol for 29 hours, starting at noon. Rectal
temperature was measured every minute, and their performance on a
tracking task was assessed every 1 hour and 40 minutes. In this task,
each participant observed a circle following a linear path with a constant speed. Each time the circle appeared, the participant had to place
a cursor inside the circle and press the left button of the mouse. After a
variable number of circles, the path and speed were modified, and the
participants’ capacity to efficiently respond to these changes was measured. Results: All participants showed a decreased capacity to adjust
their behavior to changes in the tracking task at night and early in the
morning. Conclusions: Circadian variations were observed in people’s
capacity to adjust their behavior to changes in the tracking task. This
capacity was reduced at night and early in the morning. This impairment might lead to errors and accidents in night-shift workers.
Keywords: Circadian rhythm; Environment; Human activities
RESUMO
Introdução e objetivo: Variações circadianas têm sido verificadas no
desempenho de várias atividades humanas. A execução de muitas tarefas
depende de processos cognitivos básicos, tal como a função executiva,
sendo que um destes componentes é a capacidade de ajustar o comportamento em face de alterações encontradas no ambiente. O objetivo
deste estudo foi identificar ritmos circadianos que influenciam a capacidade de se ajustar a tais alterações ambientais. Métodos: Três alunos
de graduação se submeteram a um protocolo de rotina por 29 horas ao
registro que deu início ao meio-dia. A temperatura retal foi medida a
cada minuto e o desempenho em uma tarefa de rastreamento foi avaliado a cada 1 hora e 40 minutos. A tarefa executada por cada um dos
participantes consistiu no acompanhamento de um círculo em trajetória
linear a velocidade constante. Sempre que o círculo aparecia, o partici-
pante era requisitado a colocar o cursor dentro dele e clicar o botão da
esquerda do mouse. Após um número variado de círculos, a trajetória e
a velocidade foram alteradas e a capacidade dos participantes em responder a estas variações foram medidas. Resultados: Os três participantes
apresentaram menor eficiência ao acompanhar o círculo durante a noite
e pelas primeiras horas da manhã, demonstrando menor capacidade de
ajuste comportamental nestes períodos. Conclusão: Variações circadianas foram verificadas na capacidade de ajuste comportamental na tarefa
de acompanhamento do círculo. A redução desta capacidade ocorreu durante a noite e nas primeiras horas a manhã. Tal deficiência pode levar
trabalhadores de turno noturno a sofrerem acidentes e cometerem erros.
Descritores: Ritmo circadiano; Meio ambiente; Atividades humanas
INTRODUCTION
Circadian rhythms have been found in human physiology as
well as behavior. Circadian variations in human performance
have been observed in many activities. For example, in the
execution of sensory (1) and motor tasks (2,3), reaction time (4),
a continuous performance task (5), memory tasks (6-8), reading
comprehension (9), solving arithmetic problems (10), shifting
criteria tasks (11), and in time estimation (12,13). Performance
improves during daytime, reaching the highest level between 20 and 22h, while it declines at nighttime, reaching
the lowest level between 4 and 6h (14,15).
Kleitman related physiological rhythms with rhythms
in performance; he proposed that rhythms in metabolic activity (measured by body temperature) produce rhythms in
performance (16,17). In this manner, metabolic oscillations can
affect brain activity, modulating cognitive processes and,
consequently, producing changes in performance.
Although evidence has been collected in support of
Kleitman’s hypothesis (4), there are several exceptions. Some
cognitive processes have been shown circadian variations
that are not synchronized with the circadian rhythm of body
Study carried out at Universidad Autónoma de Nuevo León, Monterrey, NL, Mexico.
1
Laboratory of Psychophysiology, School of Psychology, Universidad Autónoma de Nuevo León, Monterrey, NL, Mexico.
Corresponding author: Aída García – Mutualismo, 110, Col. Mitras Centro – Monterrey, NL México – CP 64460 – Tel.: (52) 81 8348-3866 – Fax: (52) 81 8333-7859
– E-mail: [email protected]
Received: December 21 , 2009; Accepted: March 21, 2010
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García A, Ramírez C, Valdez P
temperature (18). In the same way, other cognitive processes
do not show circadian variations (10,19,20).
Kleitman’s hypothesis assumes that oscillations in metabolism modulate performance in a general manner. However,
it is possible that some brain areas are more susceptible to
metabolic variations. This sensitivity could produce circadian
variations in particular basic cognitive processes, which, in
turn, would modulate the performance of many activities that
depend on the use of that cognitive process. There are three
basic cognitive processes that can modulate performance in
many activities: attention, working memory, and executive
functions (21,22). Therefore, it is relevant to assess potential circadian variations in these basic processes, with the purpose of
determining which ones change in the course of the day.
Executive functions refer to the capacity to program,
regulate, and verify behavior, and are crucial for problemsolving and self-control (23). Executive functions depend on
the activity of the frontal cortex, specifically the prefrontal
area (24,25). This study presents an analysis of one component
of executive functions: the capacity to adjust behavior to the
environment. This capacity is crucial to detect and correct
errors during performance in order to adjust behavior to new
environmental demands. Many activities that we carry out
in daily life, such as driving a car, practicing sports, and handling tools or machinery, require the capacity to adjust our
behavior to environmental changes in order to avoid errors
that could cause accidents.
This study sought to identify circadian variations in people’s
capacity to adjust their behavior to environmental changes. In
order to analyze potential circadian variations in this capacity, it
is important to use a tracking task, which consists of following
a stimulus that changes in position and speed (26).
In previous studies with tracking tasks, a decline in efficiency was observed at night and early in the morning, with
an increase of efficiency in the afternoon (27-30). However, it
is important to point out that these studies assessed the responses to each position of the stimulus, but not the capacity to adjust behavior to changes. In the present study, a
tracking task was designed to present a stimulus with fixed
path and velocity and, after a variable number of stimuli,
introduce unpredictable changes in the direction and speed
of the stimulus. This feature allowed us to register adjustments in people’s behavior in response to each change in the
environment. The objective of the study was to identify circadian rhythms in people’s capacity to adjust their behavior
to changes in the environment.
METHODS
Participants
Three college students volunteered to participate in this
study: a 19-year-old male and two 17-year-old females, all
right-handed, with no health or sleeping problems. The participants were not taking any medication that could affect the
central nervous system during the study. All of them attended
classes at a morning shift (7 to 13h10) and did not have programmed activities after class or on weekends. Each participant signed an informed consent letter, which was also signed
by the parents of minors. The project was approved by an
academic committee and carried out in compliance with the
principles of the declaration of Helsinki for human research.
Materials
A personal computer was used to present the stimuli and
record the responses; stimuli were displayed on a 14” (600 x
800 pixels) monitor, placed at 60 cm in front of the participants. A Steri-probe® 491B thermistor probe connected to a
Mini-Logger 2000 (Philips® Respironics) was used to record
the rectal temperature.
Tracking task
The tracking task consisted of a 50-pixel diameter circle following a linear path across the screen at a constant speed of
displacement (with a fixed inter-stimulus interval). The circle
was displayed on the screen for 180-m, while the inter-stimulus interval was set at a value randomly chosen from 200 to
730 m for each trial. Each time the circle appeared, the participants had to target a cursor inside it and press the left button
on the mouse using the index finger of the right hand. After
presenting 22 or 33 circles, the path and speed of displacement (inter-stimulus interval) were modified. Sixteen changes
in path and speed of displacement were presented. This task
lasted seven hours and two minutes. To determine the degree
of adjustment made to the changes in the task, the accuracy
(i.e., correct responses) and latency to respond to the first and
fourth stimuli after a change in the trajectory were analyzed.
In addition, the number of circles required to the adjustment
to changes in path and speed of displacement was analyzed.
Procedure
At the beginning of the study, the participants answered a
questionnaire requesting general information; a Spanish version of the morningness – eveningness scale (22,31), and a questionnaire about daily caloric intake on two different days, one
during a weekday and other on the weekend. The participants
also kept a sleep diary for two consecutive weeks. The three
participants reported that they had not consumed alcoholic
beverage or drugs, or smoked tobacco, for at least three days before the recording session. The participants were then trained
in the tracking task. After the training, the participants were
individually recorded in a constant routine protocol for 29
hours in a cubicle isolated from sunlight, external environmental noise and temperature. The data recording started at
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57
58
Circadian variations in behavioral adjustments to change
12h and finished at 17h of the following day. In this protocol,
the environmental temperature was kept constant (24±1ºC),
as well as light exposure (5 lux maximum) and caloric intake.
Feeding was provided after each task application and consisted of one portion of nutritional supplement or fruit juice and
whole-wheat flour cookies proportional to 1/14 of the 75%
of their average daily caloric intake. Additionally, body posture remained constant, as the participants remained reclined
in an armchair (45° angle) for the entire study period, only
standing up to go to the restroom when necessary. Moreover,
participants remained awake and did not have access to clock
time. Participant rectal temperature was recorded every minute using a thermistor probe inserted 10 cm in the rectum and
connected to a minilogger. The tracking task was performed
every 1 hour and 40 minutes, for a total of 18 applications. In
addition to the tracking task, other tasks – not presented in the
Results section of this study – were assessed during the study.
Recording sessions were carried out on Tuesdays for participants 1 and 2, and on Thursday for participant 3.
Data analysis
Mean values of bedtime, waking time and sleep duration
were calculated from the sleep diary. The median rectal temperature per hour was calculated for each participant. These
data were smoothed with a three-point moving average, and
a Cosinor analysis was applied to calculate the fitting percentage of the data to a 24-hour sinusoidal curve.
To obtain indices of behavioral adjustment to changes in
the tracking task, the number of circles required to adjust
(i.e., three consecutive correct responses) to the 16 changes
of each task application were averaged. The accuracy (i.e.,
percent of correct responses) and the median response latency to the first and fourth circles after these changes of
path were calculated. In addition, the moment of the day at
which the maximum and minimum values were reached on
these indicators was obtained for each participant.
RESULTS
The three participants were classified as intermediate on the
morningnesss – eveningness scale (54, 47 and 53). Before the
recording session, the participants slept an average of 7 hours
and 24 minutes, 6 hours and 55 minutes, and 8 hours and 9
minutes per night, bedtimes were 01h54, 01h46, and 01h34;
and waking times were 7h53, 8h07, and 8h39, respectively.
The day before the recording session, the participants took 3
hours, 1 hour and 30 minutes and 1 hour naps during daytime and slept for 9 hours and 10 minutes, 6 hours and 50
minutes and 8 hours during nighttime.
The participants showed circadian variations in rectal
temperature (Acrophases: 17h, 16h20, 19h50; %R: 75.94,
94.29, 92.98; p<0.001) (Figure 1A).
Sleep Sci. 2010;3(1):�����
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Figure 1: Rectal temperature and capacity to adjust behavior to environmental changes in a tracking task, for each participant during the recording session, in a constant routine protocol. (A) Changes in rectal temperature. (B) Number of stimuli required by the participants to successfully
match the cursor with the circle in the place where the circle appeared
after a trajectory change. In (C) and (D), the white circles represent the
accuracy and latency to respond to the first stimulus after a change in path
and speed, while the black circles represent the accuracy and latency to respond to the fourth circle after the change. The three participants showed
a higher capacity to adjust to environmental changes in the afternoon,
with lower capacity at night and early in the morning.
In the afternoon, the participants required, on average,
3.06, 3.56, and 4.5 circles to adjust after a change. However, during the night and early morning, the participants
required more circles (averages of 7.94, 17.81 and 17.56
circles) to adjust after a change (Figure 1B).
The accuracy with which participants responded to the
first circle after a change in trajectory was 0% for the three
participants at all times of day, with response latencies between 350 and 650 m (Figures 1C and 1D). Participants
demonstrated a good adjustment to the fourth circle in
the afternoon, showing higher accuracy (68.75, 37.5, and
43.73% correct responses) and faster response latency (56,
126 and 48 ms) at this time of day. Worse adjustment to the
fourth circle was observed at night and in the early morning,
when participants showed decreased accuracy (18.75, 6.25,
and 0% correct responses) and slow response latency (239,
331.5, and 240.5 ms) (Figures 1C and 1D).
DISCUSSION
This study presents a specific analysis of people’s capacity
to adjust behavior to environmental changes. These findings are consistent with the results of previous studies that
García A, Ramírez C, Valdez P
analyzed efficiency to respond to tracking tasks (27-30). So, capacity to adjust to changes shows circadian variations, with
higher efficiency in the afternoon and lower efficiency at
night and early in the morning.
Some individual differences were observed. Participant
1 showed higher accuracy as well as lower response latency
across the entire session when compared to participants 2
and 3. However, all of them showed circadian variations
within their own execution level.
The constant routine protocol used in this study allows
the detection of circadian rhythms by controlling factors
that may affect these rhythms (32). This protocol involves
sleep deprivation, which could potentially result in diminished performance over time. Nevertheless, the time of day
at which the participants showed the lowest capacity to adjust to changes was at night and early morning, not at the
very end of the session. These results are consistent with
previous studies showing that both sleep deprived and nonsleep deprived participants presented a decline in efficiency
in execution of a tracking task at these times of day (28,29).
Only three participants were included in this study,
thus it will be necessary to replicate these data with a larger
group. However, circadian variations were observed in each
participant, so it is reasonable to expect that similar patterns
would emerge from data recorded from other people. The
individual pattern is crucial when circadian rhythms are analyzed (33). The participants were adolescents and, although
a delay in the phase of the sleep-wake cycle were observed
for this age group (34), the period of the circadian rhythms is
similar to that of adults (35). Therefore, it is likely that the
rhythms observed in this study are also present in adults.
The capacity to adjust to changes is a component of executive functions that depends on the activity of the frontal lobe,
suggesting that this brain region is sensitive to changes in
body metabolism. In consequence, according to Kleitman’s
hypothesis (17), metabolic circadian rhythms could influence
the frontal lobe, altering executive functions and consequently affecting the performance of most human activities. In addition, circadian variations have been found in other basic
processes: such as components of attention (22) and phonological and spatial components of work memory (8). Future studies should analyze interactions between these processes, their
daily cycles, and their relationships with body metabolism.
This type of information will provide a better understanding
of the role of the biological clock in human behavior (36).
The capacity to adjust to environmental changes is crucial to verify and correct our actions. Impairment of this
function can make people more likely to make mistakes
and less able to correct them with the accuracy and speed required, leading to dangerous situations or serious accidents.
This is an important risk factor for workers during night
shifts, especially when engaging in activities that involve
handling heavy machinery, driving vehicles, or other operations that are carried out in variable conditions and require
quick and precise responses.
The decline observed in the capacity to adjust to environmental changes corresponds with a higher incidence of
errors and lower efficiency of workers during night and early
morning hours. The error rate and low efficiency of workers
at night and dawn can also be linked to a higher frequency
of accidents during night shifts. Moreover, these accidents
are often more serious than those that happen during day
shifts (37).
In conclusion, circadian variations were observed in the
capacity to adjust behavior to environmental changes in a
tracking task. A decline in this capacity was found at night
and early in the morning. This decline might be a risk factor
promoting more errors and accidents for people working at
a night shift or early in the morning.
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1988;13(6):443-4.
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35.Carskadon MA, Labyak SE, Acebo C, Seifer R. Intrinsic circadian
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36.Valdez P. Cronobiología: Respuestas psicofisiológicas al tiempo.
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61
Authors instructions
SCOPE AND POLICY
The SLEEP SCIENCE journal (ISSN 1984-0659 print version) published
every three months, is the official organization of Associação Brasileira
de Sono (ABS) and Federação Latino-Americana de Sociedades de Sono
(FLASS) for publication of scientific papers concerning sleep, chronobiology, and related topics.
After being approved by the Editorial Board, all articles will be evaluated by two or three qualified reviewers, and confidentiality will be preserved throughout the review process. Articles that fail to present merit,
have significant errors in methodology or are not in accordance with the editorial policy of the journal will be directly rejected by the Editorial Board,
with no recourse. Original manuscripts, those that have not been published
elsewhere except in abstract form, on any aspect of sleep will be considered.
The accuracy of all concepts presented in the manuscript is the exclusive responsibility of the authors. The journal reserves the right to make stylistic,
grammatical and other alterations to the manuscript. Manuscripts must
not be concurrently submitted to any other publication, print or electronic.
Articles may be written in Portuguese, Spanish or English.
Papers should state that the protocol has been approved by the Ethics Committee of the Institution where the research was carried out. All
studies involving human subjects should inform that written consent has
been obtained from all subjects (individually).
PRESENTATION AND SUBMISSION OF MANUSCRIPTS
It is requested that the authors strictly follow the editorial guidelines of the
journal, particularly those regarding the maximum number of words, tables
and figures permitted, as well as the rules for producing the bibliography.
Failure to comply with the author instructions will result in the manuscript being returned to the authors so that the pertinent corrections can be
made before it is submitted to the reviewers. Special instructions apply to
the preparation of Special Supplements and Guidelines, and authors should
consult the instructions in advance by visiting the homepage of the journal.
Abbreviations should be used sparingly and should be limited only to
those that are widely accepted. All abbreviations should be defined at first use.
The following rules were based on the standard proposed by the International Committe of Medical Journal Editors (ICMJE) and published in the article Uniform Requirements for Manuscripts Submitted to Biomedical Journals, updated in October 2009, and available from: http://www.icmje.org/
MANUSCRIPT FORMAT
This journal publishes contributions in the following categories:
Original Articles: each manuscript should clearly state its objective
or hypothesis; the design and methods used (including the study setting
and time period, patients or participants with inclusion and exclusion
criteria, or data sources and how these were selected for the study; the
essential features of any interventions; the main outcome measures; the
main results of the study, and a section placing the results in the context
of published literature.The text should be divided into separate sections
(Introduction, Material and Methods, Results, Discussion), without a separate for conclusions. The text (excluding the title page, abstracts, references, tables, figures and figure legends) should consist of 2,000 to 3,000
words; table and figures should be limited to a total of 5 and 40 references.
Authors should state in the cover letter that the manuscript is intended to be a full-length paper.
Short Communication: a short communication is a report on a single
subject which should be concise but definitive. This scope of this section is
intended to be wide and to encompass methodology and experimental data on
subjects of interest to the readers of the journal. The text should not exceed 12
pages double-spaced, typed in 23 line each, have a maximum of two figures
or tables (or one of each) and 20 references. Authors should state in the cover
letter that the manuscript is intended to be a Short-Communication.
Review Article: a review article should provide a synthetic and critical analysis of a relevant area and should not be merely a chronological
description of the literature. The text may be divided into sections with
appropriate titles and subtitles. The text should not exceed 5,000 words,
excluding references and illustrations (figures or tables). The number of
illustrations should not exceed 8 and 60 references.
The authors should state in the cover letter that the manuscript is
intended to be a Review Article.
Case Report: a case report should have at least one of the following
characteristics to be published in the journal: of special interest to the
clinical research community; a rare case that is particularly useful to demonstrate a mechanism or a difficulty in diagnosis; new diagnostic method;
new or modified treatment; a text that demonstrates relevant findings and
is well documented and without ambiguity.
Case Reports should not exceed 1,500 words, excluding title page,
abstract, references and illustrations. The number of references should not
exceed 20.
Overview: an Overview does not contain unpublished data. It presents the point of view of the author(s) in a less rigorous form than in a
regular review or mini-review and is of interest to the general reader. The
text should not exceed 5,000 words, excluding references and illustrations
(figures or tables). The number of illustrations should not exceed 8 and
60 references.
MANUSCRIPT PREPARATION
The title page should include the title in English and in Portuguese; a
running title to be used as a page heading, which should not exceed 60
letters and spaces; the full names and institutional affiliations of all authors; complete address, including telephone number, fax number and
e-mail address, of the principal author; and a declaration of any and all
sources of funding.
Abstract: The abstract should present the information in such a way
that the reader can easily understand without referring to the main text.
Abstracts should not exceed 250 words. Abstracts should be structured
as follows: Objective, Methods, Results and Conclusion. Abstracts for review articles and case reports may be unstructured.
Abstracts for Short Communications and Case Reports should not
exceed 100 words and should not be structured.
Keywords: Three to six keywords in English defining the subject of
the study should be included.
Tables and Figures: All tables and figures should be in black and white,
on separate pages, with legends and captions appearing at the foot of each.
All tables and figures should be submitted as files in their original format.
Tables should be submitted as Microsoft Word files, whereas figures should
be submitted as Microsoft Excel, .tiff or .jpg files. Photographs depicting
surgical procedures, as well as those showing the results of exams or biopsies,
in which dying and special techniques were used will be considered for publication in color, at no additional cost to the authors. Dimensions, units and
symbols should be based on the corresponding guidelines set forth by the Associação Brasileira de Normas Técnicas (ABNT, Brazilian Association for the
Establishment of Technical Norms), available from: http://www.abnt.org.br.
Sleep Sci. 2010;3(1):�����
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62
Legends: Legends should accompany the respective figures (graphs,
photographs and illustrations) and tables. Each legend should be numbered with an Arabic numeral corresponding to its citation in the text.
In addition, all abbreviations, acronyms, and symbols should be defined
below each table or figure in which they appear.
References: References should be listed in order of their appearance
in the text and should be numbered consecutively with Arabic numerals.
The presentation should follow the Vancouver Style, updated in October
of 2004, according to the examples below. The titles of the journals listed
should be abbreviated according to the style presented by the List of Journals
Indexed in the Index Medicus of the National Library of Medicine, available
at: http://www.ncbi.nlm.nih.gov/entrez/journals/loftext.noprov.html.
A total of six authors may be listed. For works with more than six
authors, list the first six, followed by ‘et al.’.
Examples:
Journal articles
1. Tufik S, Lindsey CJ, Carlini EA. Does REM sleep deprivation induce
a supersensitivity of dopaminergic receptors in the rat brain? Pharmacology. 1978;16(2):98-105.
2. Andersen ML, Poyares D, Alves RS, Skomro R, Tufik S. Sexsomnia:
abnormal sexual behavior during sleep. Brain Res Rev. 2007;56:271-82.
Abstracts
3. Moreno CRC, Carvalho FA, Matuzaki LA, Louzada FM. Effects of irregular working hours on sleep and alertness in Brazilian truck drivers
[abstract]. Sleep. 2002;25:399.
Chapter in a book
4. Andersen ML, Bittencourt LR. Fisiologia do sono. In: Tufik S, editor.
Medicina e biologia do sono. São Paulo: Manole; 2007. P. 48-58.
Official publications
5. World Health Organization. Guidelines for surveillance of drug resistance in tuberculosis. 2nd ed. Geneva: WHO; 2003. p. 1-24.
Thesis
6. Bittencourt L. Avaliação davariabilidade do Índice de apnéia e hipopnéia em pacientes portadores da síndrome da apnéia e hipopnéia do sono
obstrutiva [tese]. São Paulo: Universidade Federal de São Paulo; 1999.
Electronic publications
7. Abood S. Quality improvement initiative in nursing homes: the ANA
acts in an advisory role. Am J Nurs [Internet]. 2002 [cited 2002 Aug
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12];102(6):[about 3 p.]. Available from: http://www.nursingworld.org/
AJN/2002/june/Wawatch.htm
Homepages/URL
8. Cancer-Pain.org [Internet]. New York: Association of Cancer Online
Resources, Inc., c2000-01 [updated 2002 May 16; cited 2002 Jul 9].
Available from: http://www.cancer-pain.org/
Others situations
In other situations not mentioned in these author instructions, the recommendations given by the ICMJE should be followed, specifically those
in the article Uniform Requirements for Manuscripts Submitted to Biomedical Journals: Writing and Editing for Biomedical Publication (Updated October 200 9), available from: http://www.icmje.org/. Additional
examples for special situations involving references can be obtained at:
www.nlm.nih.gov/bsd/uniform_requirements.html
SUBMISSION OF MANUSCRIPT
The manuscript must be accompanied by a letter signed by all authors,
with permission for publication and a statement that is unprecedented
and has not been submitted for publication in another journal or book.
That letter must include: a) conflicts of interest; b) certificate of approval by the ethics committee of the institution where the research was
carried out when the investigation involves experiments on humans or
animals; c) documentation of the possible sources of funding work; d) a
statement that participants provided signed consent forms, in the case
of medical research on humans; e) letter of transfer of copyright to the
Journal Sleep Science.
Important note: the journal Sleep Science in support of policies for the
registration of clinical trials of the World Health Organization (WHO)
and the ICMJE, recognizing the importance of such initiatives for recording and promoting international information on clinical studies, open access, will only accept for publication from August 2009 articles of clinical
research that have received an identification number to one of the Clinical
Trial Registry validated by the criteria established by WHO and ICMJE,
available from: http://clinicaltrials.gov or the Pubmed website.
All manuscripts submissions for the Sleep Science must be submitted
via e-mail, to [email protected]
Associação Brasileira de Sono – Sleep Science
Rua Marselhesa, 500 – 13º andar – Vl. Clementino
São Paulo, SP – Brazil
CEP 04020-060
Fax no.: +55 11 5908 7111