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Title: Effects of experimental sleep deprivation on anxiety-like

behavior in animal research: systematic review and
meta-analysis
Author: Gabriel Natan Pires Andreia Gomes Bezerra Sergio
Tufik Monica Levy Andersen
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DOI:
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S0149-7634(16)30145-2
http://dx.doi.org/doi:10.1016/j.neubiorev.2016.06.028
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13-3-2016
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Please cite this article as: Natan Pires, Gabriel, Gomes Bezerra, Andreia, Tufik, Sergio,
Levy Andersen, Monica, Effects of experimental sleep deprivation on anxiety-like
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Biobehavioral Reviews http://dx.doi.org/10.1016/j.neubiorev.2016.06.028
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Effects of experimental sleep deprivation on anxietylike behavior in animal research: systematic review and
meta-analysis

Gabriel Natan Pires*, Andria Gomes Bezerra, Sergio Tufik, Monica Levy
Andersen

Departamento de Psicobiologia, Universidade Federal de So Paulo So

Paulo, Brazil

Corresponding author:
Gabriel Natan Pires
Departamento de Psicobiologia - Universidade Federal de So Paulo
Rua Napoleo de Barros, 925
Vila Clementino - SP- 04024-002
So Paulo
Brazil
Phone # (55-11) 2149-0155
Fax # (55-11) 5572-5092
E-mail address: gnspires@gmail.com

HIGHLIGHTS - Effects of experimental sleep deprivation on anxiety-like

behavior in animal research: systematic review and meta-analysis

Sleep deprivation leads to increase in anxiety levels in humans.

Animal research has failed to reproduce this effect in a consistent

manner.

The reasons for the inconsistencies between human and animal studies
are discussed.

Elevate plus-maze is not sensible to evaluate anxiety due to sleep

deprivation.

New tools are needed to evaluate sleep deprivation-induced

anxiogenesis in rodents.

ABSTRACT
Increased acute anxiety is a commonly reported behavioral consequence of
sleep deprivation in humans. However, rodent studies conducted so far
produced inconsistent results, failing to reproduce the same sleep deprivation
induced-anxiety observed in clinical experiments. While some presented
anxiogenesis as result of sleep deprivation, others reported anxiolysis. In face
of such inconsistencies, this article explores the effects of experimental sleep
deprivation on anxiety-like behavior in animal research through a systematic
review and a series of meta-analyses. A total of 50 of articles met our inclusion
criteria, 30 on mice, 19 on rats and one on Zebrafish. Our review shows that

sleep deprivation induces a decrease in anxiety-like behavior in preclinical

models, which is opposite to results observed in human settings. These results
were corroborated in stratified analyses according to species, sleep deprivation
method and anxiety measurement technique. In conclusion, the use of animal
models for the evaluation of the relationship between sleep deprivation lacks
translational applicability and new experimental tools are needed to properly
evaluate sleep deprivation-induced anxiogenesis in rodents.

Keywords: Animal behavior; Animal models; Anxiety; Elevated-plus-maze;

Meta-analysis; Sleep; Sleep deprivation; Systematic Review.

1. INTRODUCTION
Increased acute anxiety has been reported as one of the most common
behavioral consequences of sleep deprivation in humans. This effect was first
reported in the classical article by William Dement (1960), who reported anxiety,
together with lack of attention and irritability, as a major neurobehavioral
consequence of REM (rapid eye movement) sleep deprivation. Since then,
several studies have addressed the relationship between sleep deprivation and
anxiety, and the results suggest sleep deprivation is an anxiogenic condition
(Baum et al., 2014; Goldstein et al., 2013; Matzner et al., 2013; Minkel et al.,
2012; Motomura et al., 2013; Schuh-Hofer et al., 2013). Such effects have been
mainly observed as a consequence of total sleep deprivation (Babson et al.,
2010; Minkel et al., 2012; Sagaspe et al., 2006; Selvi et al., 2007; Vardar et al.,

2007), but also due to REM sleep deprivation (Greenberg et al., 1972; Nielsen
et al., 2010) and to sleep restriction (Talbot et al., 2010; Wu et al., 2008).
Reflecting the relevance of the relationship between sleep deprivation
and anxiety in humans, several studies on the same topic have been conducted
in basic preclinical studies, mainly employing rodent models. Considering that
the mechanisms underlying sleep deprivation-induced anxiety are poorly
understood, this is a natural choice, especially when considering the possible
value of animal models for research on anxiety. However, rodent studies
conducted so far have produced inconsistent results, failing to reproduce the
same sleep deprivation-induced anxiety observed in humans (Pires et al.,
2012b; Silva et al., 2004b). This is specially observed in studies that employed
the elevated plus-maze (the gold-standard technique for assessing anxiety-like
behavior in rodents) in sleep-deprived animals. While some studies showed
anxiogenesis as a result of sleep-deprivation (Kumar and Kalonia, 2007; Kumar
and Singh, 2008; Nair et al., 2011; Silva et al., 2004b) others reported anxiolytic
behavior due to REM sleep deprivation (Andersen et al., 2005; Pokk and Vli,
2002; Suchecki et al., 2002; Zielinski et al., 2013), oppositely to what has been
observed in humans.
These inconsistencies, either among rodent studies or between results in
animal models and humans, put in doubt the translational applicability of the
elevated plus-maze and other anxiety assessment tools in rodents, when
associated to sleep deprivation paradigms. Such methodological problems have
already been discussed in the literature, both in their theoretical and practical
aspects (Pires et al., 2012b, 2013, 2015b; Silva et al., 2004b). However, the
results are still not clear and a more detailed and consistent analysis is required

to evaluate the true value of basic rodent research in the relationship between
sleep deprivation and anxiety.
Animal meta-analysis has arisen in the last few years as a very useful
and applicable way to summarize data from basic studies. More than only a way
to analyze the global effect of a given protocol or treatment, they also involve
practical and ethical aspects. Such analyses are able to evaluate if a specific
field of study in basic research has enough translational potential to be used as
a model of some human condition or disease (Hooijmans and Ritskes-Hoitinga,
2013; Sena et al., 2014; van Luijk et al., 2014). Thus, the present study uses
meta-analyses to evaluate the results of studies that assessed the effects of
experimental sleep deprivation on anxiety-like behavior in animal research. This
was done with two main aims: 1. To assess the global effects of sleep
deprivation on anxiety-like behavior in animal studies and; 2. To evaluate under
which conditions experimental sleep deprivation in animal models lead to a
consistent increase in anxiety-like behavior levels, thus replicating humans
studies and presenting translational applicability.

2. METHODS
2.1 Systematic review
Studies reporting the effects of experimental sleep deprivation on
anxiety-like behavior in laboratory animals were identified by searching two
different electronic databases (Pubmed and Scopus), at two different times, first
in May 2014 with an update in July 2014. The primary search strategy
(Leenaars et al., 2012) used was [("sleep deprivation" OR "sleep curtailment"
OR "sleep restriction" OR "sleep loss") AND (anxi* OR "elevated plus-maze")],

with the use of additional search filters for document type. Special attention was
given to elevated plus-maze by inserting it in the search strategy, since this is
the gold-standard method for evaluation of anxiety-like behavior in rodents.
Articles were selected in a two steps process: In the first step, abstracts were
screened according to the selection criteria disclosed below and full text
manuscripts were consulted only if needed. The second step conditionally
considered full manuscripts and included data extraction. Additionally, relevant
original studies cited by the articles selected in the primary search, as well as
studies suggested by colleagues and experts in the field were also eligible to be
included.

2.2 Inclusion and exclusion criteria

We included original studies that assessed the effects of experimental
sleep deprivation on anxiety behavior in laboratory animals. For this purpose,
sleep deprivation was defined as any kind of lack of sleep, which includes total
sleep deprivation, partial sleep deprivation, sleep restriction and sleep
fragmentation. Only studies in which sleep deprivation was induced by
experimental methods were included, which means that natural observation of
lack of sleep or any other non-experimental sleep deprivation study were not
considered. To be included, studies should address sleep deprivation as cause
(independent variable) and anxiety-like behavior as outcome (dependent
variable); any other kind of study design was excluded. Studies that did not
address only sleep deprivation, but had at least one clear and unequivocal
control group and one experimental group, both free of confounding factors
were also included. Articles that presented an incompatible study design, with a

significant lack of data (even after contacting authors), and those for which we
could not find the full text were excluded.

2.3 Data extraction

As a primary option, data were extracted directly from the full text
articles. Whenever an article failed to disclose some specific information or did
not disclose data in enough detail (such as employing figures instead of tables
or textual description), authors were contacted and asked to provide the results
of their experiments or raw data. After two unsuccessful contact attempts, if
possible, data were estimated by graph and figures using a digital ruler. If none
of these options were possible, the article was excluded.
Each selected article was subdivided into experiments, which are defined
as any case when there is one control group compared to a concomitant
experimental group. Each experiment might be composed by multiple
comparisons, depending on the number of variables assessed in each case.
For each experiment, data were extracted regarding sample description, sleep
deprivation method and anxiety-like behavior. In respect of sample description,
we extracted data regarding group, species, strain, gender, age and sample
size. There were no restrictions about species (provided that they were nonhuman), strain, gender, age or sample size. In relation to sample sizes,
whenever the articles disclosed a sample range instead of the exact value of
animals per group, we considered the lower possible value in the provided
range, avoiding any overestimation of effect. Also, when two different
experimental groups shared the same control group, the sample size of the
control group was adjusted by dividing it by the number of comparisons.

Regarding sleep deprivation methods, the procedure was categorized

according to the nature of sleep manipulation, employed method and duration of
the sleep manipulation. There were no restrictions about the type of method
employed for sleep deprivation, provided that it was experimentally induced.
Concerning the nature of sleep manipulation, it was classified as sleep
deprivation when the employed procedure led to a complete suppression of the
sleep; as partial sleep deprivation, when only a specific sleep stage was
suppressed (being the REM stage the most common); as sleep restriction
whenever the protocol led to a reduction in total sleep time; or as sleep
fragmentation when the method led to intermittent awakenings throughout the
experimental period. Finally, regarding anxiety-like behavior measures, we
extracted any variable of any test that directly or indirectly assessed anxiety.

2.4 Risk of Bias and Publication bias

Possible biases affecting the selected articles were evaluated using the
SYRCLEs Risk of Bias Tool, a screening instrument specifically developed to
risk of bias assessment in animal studies (Hooijmans et al., 2014). Few
adaptation were made to this tool, in order to contextualize it to sleep
deprivation studies: the items allocation concealment (selection bias), random
housing (performance bias) and blinding (performance bias) were excluded,
because they cannot be afforded in sleep deprivation protocols. Experimental
sleep deprivation implies in having experimental group animals undergoing
specific housing characteristics or sleep deprivation apparatuses, while control
group remains in their regular (or slightly modified) home cages. Thus, group
allocation and animal groups are immediately recognized by their housing and

environmental characteristics. In total, six items were evaluated, two related to

selection bias, two related to detection bias, one about attrition bias, one about
reporting bias a final one related to other source of bias.
Regarding the publication bias, all the selected articles were evaluated
using a visual analysis of a funnel plot (Sterne and Egger, 2001) for assessing
possible publication bias. The funnel plot was constructed upon the relationship
between the studies precision (standard error) and effect sizes. In the absence
of publication bias, the less precise studies would be expected to be spread
widely at the top of the graph, while more precise studies would be
concentrated at the bottom, near to the estimated effect size value.

2.5 Statistical analysis

Considering the purposes of the present study, different meta-analyses
were performed, which were grouped in four clusters. Initially, in order to
evaluate the overall and unspecific effects of experimental sleep deprivation on
anxiety, we performed a global analysis of all selected experiments (cluster
one). We then carried out a stratified intermediate analysis, in order to evaluate
the isolated effects of some variables, such as species and kind of sleep
deprivation (cluster two). Subsequently, stratified analyses were conducted by
grouping studies by the instruments employed to measure anxiety-like behavior
(cluster three). Finally, in order to evaluate which specific experimental methods
and conditions can lead to observation of an anxiogenic condition due to sleep
deprivation, we performed a stratified analysis aiming for the minimal possible
variability among the descriptive data (cluster four). This was intended to
analyze in which specific cases lack of sleep is able to produce an anxiogenic

condition, as observed in human beings. Thus, in cluster four articles were

grouped in order to have only one category of each descriptive variable per
analysis (species, gender, age, sleep manipulation, sleep deprivation method
and anxiety test). A minimal amount of two articles per analysis was used.
For each comparison, we calculated the standardized mean difference
based on Hedges G method as a measure of effect size. This method
standardizes the magnitude of the effect of the experimental group in
comparison to the control group and allows comparisons among effects
measured on different scales (Hedges and Olkin, 1985; Vesterinen et al., 2014).
Data were handled in order to make negative effect sizes denote anxiolysis and
positive effects sizes denote anxiogenesis. We then used a DerSimonian and
Laird random effects weighted mean difference meta-analysis to calculate an
overall effect size for each analysis. Whenever we needed to use and compare
different variables to compose an analysis, the choice was made according to
the ranking disclosed in Table 1.
Heterogeneity of effect sizes within each comparison was tested using
the Cochrans Q test and I2 statistics (Higgins et al., 2003). Data are presented
as effect size confidence intervals at 95%. Results were considered as
significant when the confidence interval range was all lower or higher than zero
and associated with a Cochrans Q p value lower than 0.05. All calculations
where performed as previously suggested (Vesterinen et al., 2014), using
RevMan 5.

3. RESULTS
3.1 Selected studies and sample description
10

Our search strategy resulted in 756 articles, 407 of which were retrieved
from Pubmed/Medline and 349 exclusively found on Scopus. After selection
according to our inclusion and exclusion criteria, a total of 713 articles were
excluded and 56 remained eligible to be included in our meta-analysis. Finally,
seven more studies (including one conference abstract) that were not found by
our primary search strategy but were in accordance with the aims of this metaanalysis and with our inclusion and exclusion criteria were also included.
Overall, 50 articles composed our sample (Alvarenga et al., 2008; Andersen et
al., 2005; Araujo et al., 2006; Berro et al., 2014; Cohen et al., 2012; Cortese et
al., 2010; de Oliveira et al., 2004; Dubiela et al., 2011; Fernandes-Santos et al.,
2012; Garg and Kumar, 2008; Hajali et al., 2012; Kalonia and Kumar, 2007;
Kumar and Garg, 2008; Kumar and Kalonia, 2007; Kumar and Singh, 2007,
2008, 2009; Nair et al., 2011; Novati et al., 2011; Patti et al., 2010; Perry et al.,
2008; Pires et al., 2013; Pokk et al., 1996; Pokk et al., 2000; Pokk and Vli,
2001a, b, 2002; Pokk and Zharkovsky, 1995, 1997, 1998a, b; Polesel et al.,
2014; Porsolt et al., 1978; Rothman et al., 2013; Silva et al., 2004a; Silva et al.,
2004b; Singh and Kumar, 2008; Singh et al., 2013; Suchecki et al., 2002; Ser
et al., 2011; Takatsu-Coleman et al., 2013; Tartar et al., 2009; Torabi-Namia et
al., 2012; Vassiljev et al., 2000; Vollert et al., 2011a; Wang et al., 2014; Xu et
al., 2010; Yehuda et al., 2007; Zager et al., 2009; Zielinski et al., 2013),
containing 76 experiments. Among these, 30 articles reported experiments in
mice, 19 in rats and one in Zebrafish. Due to the uniqueness of this single study
in zebrafish (Singh et al., 2013), we decided not to include it in the calculations.
The selection process is shown in Figure 1, the full list of articles composing our
sample is available on Table 2 and a description of these articles and

11

experiments, as well as list of variables and categories available used to stratify

our analyses, is available in Table 3.

3.2 Publication Bias

During data selection, we noticed identical or very similar mean and
variance values in a series of articles by a research group, which is indicative of
duplicate publication. These articles, as well as any other selected article
authored by this group, are disclosed in Table 2. Whenever possible, the results
of each meta-analysis and specifically the overall effect sizes are presented
both including and excluding these articles, in a sensitivity analysis.
Among the seven items evaluated using the SYRCLEs Risk of Bias Tool,
the item with the highest bias risk rate was other source of bias, as a
consequence of the suspicion of duplicate publication mentioned above.
Following, incomplete outcome data (attrition bias) and selective reporting
outcome (reporting bias) also presented some degree of risk of bias. The
remaining items did not show any evident risk of bias. The results of the risk of
bias assessment can be checked on Table 4.
The funnel plot was also performed including and excluding the articles
suspected of duplicate publication (Figure 2, A and B). Visual analysis of the
funnel plot indicates some degree of publication bias in both cases, but in
opposite directions. In the first case, including all articles, a lack of studies
indicating anxiolysis can be noted. Conversely, when the aforementioned
articles are excluded, the funnel plot indicates a lack of articles indicating
anxiogenesis. Due to the uncertainties about these articles, the publication bias

12

analyses are unreliable and their impact on the results should be interpreted
carefully, taking into consideration the sensitivity analyses.

3.3 Analyses
A total of 29 meta-analyses were performed, from the first overall
analysis until the most specific ones, in which only one category per descriptive
variable was considered. For the overall meta-analysis, no effect was observed,
but a significant anxiolytic status was detected as a consequence of sleep
deprivation in the sensitivity analysis [analysis #1: -0.35 (-0.77; 0.07); I2=0.90;
p<0.01; analysis #1*: -0.78 (-1.18; -0.38); I2=0.90; p<0.01; Figure 3]. Potentially
duplicated articles accounted for 6% of total results in analysis #1. The same
anxiolytic effect was observed in five of nine second cluster analyses (Figure 4),
including overall sleep deprivation in male rats [analysis #2: -0.75 (-1.36; -0.14);
I2=0.87; p<0.01], in mice [analysis #5*: -0.95 (-1.64; -0.33); I2=0.922; p<0.01]
and in rodents in general [analysis #8: -0.87 (-1.43; -0.31); I2=0.91; p<0.01;
analysis #9: -0.85 (-1.60; -0.10); I2=0.84; p<0.01]. For the third cluster analyses,
in which data was stratified by the employed anxiety measurement technique
(Figure 5), anxiolysis as consequence of sleep deprivation was observed in the
elevated plus-maze [analysis #10*; -0,56 (-1.08; -0.05); I2=0.88; p<0.01] and in
the staircase test [analysis #15; -6.60 (-9.47; -3.73); I2=0.96; p<0.01], while the
mirror chamber test showed anxiogenesis [analysis #14; 7.41 (3.80; 11.01);
I2=0.58; p<0.01. Open field test, plus-maze discriminative avoidance task, and
zero maze showed no significant results. Other anxiety measurement tests
were employed in less than two articles and, thus, were not included in this
cluster. For the fourth cluster, 14 different analyses were performed, with high

13

variability among results. Among these, 11 showed no significant results (either

due to excessive heterogeneity or to the confidence interval range passing
though zero), while three indicated anxiolysis and none indicated anxiogenesis
(Figure 6). All the performed analysis and its results are listed and described in
detail in Table 5.

4. DISCUSSION
From the perspective of translational medicine, meta-analyses of animal
data have become increasingly relevant over the last few years. This tool has
emerged as a consequence of the need to rigorously summarize the findings of
animal studies before beginning any new clinical trial. This was the premise
taken by Sandercock and Roberts (2002), in a letter that nowadays is
considered a landmark in the importance of preclinical meta-analyses as they
are currently conceived. Since then, these analyses have evolved, mostly by
the parallel efforts of two research groups, SYRCLE and CAMARADES, who
have developed tools and techniques and advocated the importance and
relevance of meta-analyses in animal studies. It is important to note that despite
sharing a common methodological and statistical background, clinical and
preclinical meta-analyses are very different in their purposes and design
(Vesterinen et al., 2014). While clinical meta-analyses are usually based on
large trials and intended to guide decisions regarding a given treatment or
technique, preclinical meta-analyses are exploratory and hypothesis generating
tools, in which understanding the sources of bias and heterogeneity found in a
sample of similar articles is as important as verifying the efficacy of a given
treatment. Therefore, animal meta-analyses are important tools for ethical

14

animal research, since they allow the extent of the translational applicability that
certain animal models may have to be evaluated, providing direction to future
research and preventing unnecessary animal use, if it is shown that the animal
models lack translational relevance.
This seems to be the case in the studies on the relationship between
sleep deprivation and anxiety in animals. Previous studies have already
reported that animal research on the effects of sleep deprivation on anxiety-like
behavior have failed to present the same sleep deprivation-induced anxiety
observed in humans, and may not therefore have a translational value (Pires et
al., 2012b, 2013; Silva et al., 2004b). However, so far no systematic review has
addressed and criticized the conflicting results raised by such studies, and new
studies on this topic remain to be conducted and published regardless of the
lack of translational evidence. Until now we have not been able to state which
are the overall effects of sleep deprivation on anxiety-like behavior and in which
cases sleep deprivation in animal models is able to produce an unambiguous
increase in anxiety, thus being able to be used as a reliable preclinical model
the sleep deprivation-induced anxiety. Based on this, the present work intended
to evaluate the effects of experimental sleep deprivation on anxiety-like
behavior found in preclinical models, as well as to evaluate if, and in which
cases, we have animal models that successfully mimic the human increase in
anxiety levels due to lack of sleep.
In this sense, our results served as a confirmation of the previous
suspicion of a lack of translational applicability. Both in our overall analysis
(analysis #1*) as well as in most of the subsequent analysis of clusters two to
four, it was observed that sleep deprivation led to a decrease in anxiety levels,

15

an opposite effect to that which would be expected from a clinical perspective.

Of the remaining analysis, the great majority show no effect of sleep deprivation
(either due to increased heterogeneity or due to confidence interval passing
through zero). But among the significant results, 11 out of 12 analyses
presented anxiolysis as a result of sleep deprivation. Such results were
observed most in cluster two, with some in clusters three and four,
encompassing rats and mice, for different types of sleep deprivation and for a
good number of anxiety measurement techniques and devices. Thus, as a
primary conclusion, the present article demonstrates that animal research about
the effects of sleep deprivation on anxiety does not represent what is observed
in humans.
Interesting results were obtained from meta-analyses of cluster three, in
which articles were grouped by the method for anxiety measurement. Only one
single analysis presented results denoting anxiogenesis as a consequence of
sleep deprivation (analysis #14). Experiments using the mirror chamber test
presented a consistent increase in anxiety-like behavior, which corroborates the
anxiogenesis observed in clinical research. However, the data should be treated
cautiously in this case, as the results were exclusively based on those articles
for which there is a suspicion of redundant publication. If this suspicion is not
confirmed, then the mirror chamber test should be used as the only efficient
protocol to assess anxiety in sleep deprived rodents that replicates human data.
With the exceptions of the mirror chamber test, a decrease or equivalence in
anxiety was observed in all cases, with significant anxiolysis due to sleep
deprivation in the elevated plus-maze and in the staircase test.

16

The analyses of the elevated plus-maze (analyses #10 and #10*) were
naturally the biggest in cluster three, as this protocol is the gold-standard
method for evaluation of anxiety behavior in rodents (Carobrez and Bertoglio,
2005), and was composed by 39 experiments, comprising a total of 767 animals
(31 experiments and 670 animals in adjusted analysis). Among these 39
experiments, 21 presented negative effects sizes (a marker of decrease in
anxiety), while the remaining 18 presented positive effect sizes (denoting
anxiogenesis). However, one may notice that some of the experiments with
positive effects sizes came from those potentially misreported articles, which
explains the appearance of significant anxiolysis in the adjusted analysis
(analysis #10*). Another issue that deserves attention is that some of the
experiments that presented the biggest positive effect sizes seem to rely upon
uncommon behaviors of control groups, instead of a real effect of sleep
deprivation. This is the case for Nair et al., experiments 1 and 2 (2011); and
Silva et al., experiments 1 and 4 (2004b), which report time in open arms (a
measure the denotes anxiolysis) in control animals ranging from 40% to 60%.
Thus, this seems to be more a case in which the control groups were displaying
abnormal behavior rather than a real effect observed in experimental groups.
Such an observation, which theoretically reduces the power of experiments that
presented positive effects sizes, reinforces the idea that in general, sleep
deprivation leads to an anxiolytic condition when assessed in the elevated plusmaze. For the staircase test, this was the biggest effect size in cluster three,
with any included experiment showing positive effect sizes, but it should be
noted that one of the experiments included presented extremely discrepant
results, although the reasons for this are not clear (Vassiljev et al., experiment

17

1). Plus-maze discriminative avoidance task and zero maze showed nonsignificant results due to increased heterogeneity (Cochrans Q p value lower
than 0.05) and the open field test showed no effect of sleep deprivation due to
effect size, passing through zero. Some other anxiety measurement tests and
protocols were used among the selected articles, however, they were not
included in the cluster three meta-analyses as they were used and reported in
only one article each. This was the case for the hole board test (Pokk and
Zharkovsky, 1998b), the grooming analysis algorithm (Pires et al., 2013), the
open field with starter box (Tartar et al., 2009), the light-dark explorations test
(Vollert et al., 2011b), the elevated T maze (Ser et al., 2011) and the light-dark
box in zebrafish (Singh et al., 2013). Individually, the majority of these tests also
demonstrated decreased anxiety in sleep deprived animals, with the exception
of the grooming analysis algorithm and the light-dark box in zebrafish, in which
results demonstrated an anxiogenic status due to sleep deprivation.
Finally, the results raised from cluster four are of restricted applicability.
These analyses were designed to represent the most specific possible
experimental cases, providing the exact conditions in which animals models
would be used to replicate human sleep deprivation-induced anxiogenesis. For
that, only a single category of each variable was allowed to compose each
analysis. However, as a result of such specificity, very few articles and
experiments were included in each case and, thus, these may be biased by the
reduced sample size. Even so, it may be noted that significant data were
observed for some uses of the elevated plus-maze and the staircase test
(analyses #20 and #27), corroborating results from cluster three. Also, the only
analysis exclusively performed with female animals showed a significant

18

anxiolysis, which is interesting if compared with previous analyses involving

only males (such as analyses #8 and #9).
Altogether, results range from no effect of sleep deprivation on anxietylike behavior to anxiolysis, but in no case (except for the potentially biased
analysis #14), did basic rodent research replicate human data. Some
explanations can be proposed for the discrepancies between human and rodent
responses to sleep deprivation in what regards anxiety. First, it should be taken
into account that an increase in state anxiety is not the only behavioral effect of
sleep deprivation. Several clinical reports have shown increased lack of
attention (Killgore, 2010), impulsivity (Anderson and Platten, 2011), aggression
(Kamphuis et al., 2012), and the induction of a transient mania-like behavior
(Colombo et al., 1999; Wright, 1993). Indeed, it is probably this hypomanic
episode that is responsible for the acute improvement in depressive symptoms
observed due to sleep deprivation in humans (Hemmeter et al., 2010; Luca et
al., 2013). In rodents, this mania-related behavior is very well established and
easily observed. Actually, sleep deprivation has been considered as a reliable
animal model of mania (Gessa et al., 1995; Young et al., 2011). Thus, it is more
reliable to think that sleep deprivation causes a complex behavioral phenotype
rather than only isolated behavioral parameters, which is composed by both
anxiety and mania or impulsivity. It is exactly this behavioral phenotype that
should be taken into consideration whenever a sleep-deprived animal is tested
in the elevated plus-maze or in any other of these anxiety measurement tools.
The elevated plus-maze is a cross-shaped platform, elevated from the
floor (50cm for rats, 40cm for mice), with two opposing open arms and two
opposing arms closed laterally. This test deals with the antagonism of two

19

natural rodent behaviors: the tendency to explore the environment, denoting

anxiolysis, and the tendency to remain in a safe environment, represented by
the closed arms of the equipment, a circumstance that denotes anxiogenesis.
Thus, it is expected that an anxious animal will remain in the enclosed arms for
most of the time, presenting low exploratory and locomotor behaviors (Carobrez
and Bertoglio, 2005; Pires et al., 2012b; Walf and Frye, 2007). However, what
would be expected from an animal with increased impulsivity or in mania is
exactly the opposite: it would have a higher rate of time in the open arms,
presenting increased locomotor and exploratory activity. Thus, the states of
anxiety and mania or impulsivity are opposite and mutually exclusive. It is based
on this opposition that the elevated plus-maze has been a reliable, consistent
and internally valid method for the great majority of possible experimental
conditions involving the quantification of anxiety-like behavior in rodents.
Nevertheless, is also based on these features that this model fails to detect any
anxiety caused by sleep loss. In other words, the elevated plus-maze may not
be able to detect sleep deprivation-induced anxiety-like behavior. As most of the
other available methods for testing anxiety in rodents (such as the open field
test, the staircase test and the plus-maze discriminative avoidance task) draw
upon the same paradigm observed in the elevated plus-maze, the arguments
above remains valid for them all.
Obviously, the present work does not intend to harm the applicability of
the elevated plus-maze or any other method for the assessment of anxiety. Our
intentions are to argue that for this very specific condition, in which the
suppression of sleep leads to a behavioral state mutually composed by
increased anxiety and impulsivity, these methods may lack sensitivity and

20

applicability. Nonetheless, they remain valid for every other possible use.
Conversely, methods in which anxiety and impulsivity could be assessed
independently or where a single variable could represent both behavioral
conditions may be more viable to be used in cases in which sleep deprivation is
involved. This seems to be the case in self-grooming (or self-cleaning) analysis,
as this behavior increases both in cases of high anxiety and high impulsivity.
Indeed, some articles have already evaluated grooming following sleep
deprivation (Andersen et al., 2005; Nunes et al., 2012; Pires et al., 2012a; Pires
et al., 2013, 2015a). In respect of the methods currently available to evaluate
grooming behavior, one could think of three possible alternatives: the classical
evaluation by latency, frequency and duration of this behavior (Andersen et al.,
2005); the use of self-cleaning indexes (Nunes et al., 2012) and the use of the
grooming analysis algorithm (Kalueff and Tuohimaa, 2004, 2005a, b). Among
these options, the use of classical behavioral parameters has been
discouraged, since the quantity of this behavior is insufficient to distinguish
between states of high- and low-anxiety. Indeed, the relationship between selfgrooming and anxiety in rodents appear as a U-shaped curve, which means
that this behavior can be observed at similar levels in both high- and low-anxiety
rodents (Kalueff and Tuohimaa, 2005b). The second option, the use of a selfcleaning index, was proposed as a method to correlate grooming and anxietylike behavior following sleep deprivation (Nunes et al., 2012). In this method,
the animals are tinted with a yellow dye, and the color of their fur is evaluated.
The more grooming the animal performs, the less yellow its fur appears by the
end of an observational period. Despite being easy to perform, this method also
draws upon the amount of grooming, which might reduce its reliability to

21

measure anxiety, as described above. Thus, the best alternative seems to be

the grooming analysis algorithm. This method, developed and described by
Kalueff and Tuohimaa (2004, 2005a, b), discriminates levels of anxiety in rats
based on grooming characteristics and microstructure. According to this model,
while a normal non-anxious animal performs grooming in an ordered and wellstructured cephalocaudal pattern; anxious rodents perform self-grooming in a
more fragmented and chaotic pattern. Thus, this model distinguishes between
high and low anxiety animals, not by the amount of self-grooming displayed, but
rather by its sequential patterns and microstructure. In order to do so, this
method categorizes grooming in seven stages: 0) no grooming; 1) forepaw
licking; 2) nose, snout and face grooming; 3) head washing (semicircular
grooming behind the ears and over the top of the head); 4) body
grooming/scratching (including body scratching with hind paws); 5) leg licking
and; 6) tail/genital grooming. The transitions from one grooming stage to
another are counted and classified as correct (transitions between two
subsequent grooming stages in a cephalocaudal pattern) or incorrect (between
two non-adjacent grooming stages). The main indicative of anxiety according to
this method would be the number of non-cephalocaudal transitions (or incorrect
transitions), which fortunately is the same that would be expected for an
impulsive animal. Only one article using this protocol has been selected for the
present meta-analysis, presenting increased anxiety as a result of both REM
and total sleep deprivation, in accordance to what is observed in humans (Pires
et al., 2013). More studies using this technique to evaluate the reproducibility of
these results and in particular pharmacological testing to evaluate its
consistency with expected human behavioral responses, are required to

22

establish whether this is a reliable method. However, both due to the theoretical
background involved in this case and to the previous achievements, this may be
a good alternative for further studies. Other methods that were not found in the
experiments in this analysis, and even new methods yet to be developed should
also be tested in order to try to simulate the human condition of increased
anxiety due to sleep loss. It is worth saying that regardless of the method used
for assessing anxiety-like behavior, the real background etiology does not
change, since the causal factor for it is the lack of sleep, not the method used to
measure its consequences. Thus, our suggestion for new methods are just
intended to find a more sensible technique, presenting data acquired from a
different perspective. If any new technique proves to be able to provide reliable
data corroborating human anxiogenesis due to sleep deprivation, then it should
be stimulated as a most appropriate tool from a translational perspective.
However, animal studies on the effects of sleep deprivation on anxiety should
be discouraged if the results remain inconclusive or opposite to data from
human research, even after the exhaustive testing of new tools. In the face of
sustained failure to replicate clinical findings and the lack of translational
coherence, it should be assumed that rodents do not develop anxiety as a result
of lack of sleep.
Another important point related to the effect of sleep deprivation on
anxiety in rodents is the potential impact of stress on this relationship. Stress
can be defined as the physiological and behavioral response displayed by an
organism in face of a source of stress, being this response depended on
several variables, including the nature and the strength of the stressor. In what
regards behavioral research, stress always become an important point, as it

23

modulates the behavior in different ways. In fact, a whole behavioral phenotype

may emerge on stressful situations, which are impacted by the rise of
adrenergic hormone levels, activation of the HPA axis, increased activity on the
amygdala and hippocampus, among other (for review, check Everly and Lating,
2013). Sleep deprivation is by itself an important source of stress. In fact, it is
practically impossible to set sleep deprivation apart from stress and assess the
effects of sleep deprivation independently. For this reason, we must consider
the results of this study as a consequence of the sleep deprivation-stress dyad.
Moreover, the size or strength of the stress might vary according to some
characteristics of sleep deprivation, such as its duration and type. A 48h period
of sleep deprivation, for example, will logically be a stronger stressor compared
to a 24h period. In addition, total sleep deprivation seems to trigger a stronger
stress response compared to sleep restriction. The method of sleep deprivation
may also influence the stress response with some being more stressful than
others; which means that additionally to the stress directly caused by the sleep
loss, the methods used to deprive sleep may lead to additional stress
responses. Despite there not being a comprehensive study evaluating stress
across the different available sleep deprivation methods, a few intended to
evaluate the effect of these some methods on stress-related variables
(Machado et al., 2006; Medeiros et al., 1998; Suchecki et al., 1998). It might be
possible to sort these methods according to their stress potential, if the
additional stress sources that each method involves are considered, in addition
to sleep deprivation per se. From this perspective, the gentle handling method
would probably be the least stressful method, as the only source of stress,
beyond lack of sleep, would be due to handling the animal. On the other hand,

24

platform methods would be the most stressful as they encompass other

important stress sources, such as locomotion restriction, wetness and humidity,
and social isolation (in the case of single platform method).
Our data does not seem to exhibit any clear correlation between stress
and anxiety, with the only possible relationship being perhaps seen in cluster
four, as analyses employing the stressful platform methods raised significant
results (analyses #20, #27 and #29). It should be mentioned that this
observation may be biased, as we have not performed any stress-related
search or analysis. Even so, the overall results acquired in our meta-analyses
are puzzling, if considered in light of the expected relationship between stress
and anxiety. In general, stress seems to increase anxiety-like behavior, when
induced by methods such as chronic restraint stress (Crema et al., 2010),
chronic unpredictable stress methods (Crema et al., 2010; Pgo et al., 2008),
early life stress (Ishikawa et al., 2015), social defeat (Reader et al., 2015) and
social isolation (Amiri et al., 2015; Cruces et al., 2014), among others. This
stress-induced anxiety is exactly the opposite of the anxiolysis observed in
sleep deprivation. This incongruence reinforces our belief that the results
observed in these meta-analyses are due to the concomitance of anxiety and
impulsivity after sleep deprivation, as well as to the imprecision and lack of
sensitivity of the elevated plus maze and related methods. If we had a reliable
assessment tool to overcome these methodological issues, then it is likely that
an increase in anxiety would have been observed after sleep deprivation, in line
with the results in experiments with other stress sources. Until such a method
exists, the exact effect of stress on the association between sleep deprivation

25

and anxiety will remain unclear. Stress certainly must play an important role in
this relationship and should be focus of further research.
Regardless of the reason, we should be aware of the risks involved in the
discrepancies between rodent and human research. First and more obviously,
whenever a preclinical animal study makes use of a non-applicable method to
assess a human condition, it lacks coherence and meaning. It is paramount for
any kind of research to choose the best methods available, in order to acquire
the best possible results. In the present case, it may be attenuated by the fact
that the best methods are indeed being used; however, what is happening is
that these methods are not useful or lack sensitivity in this very peculiar and
specific experimental condition. Since these problems were raised both by the
present and by previous studies (Pires et al., 2012b, 2013), we hope that future
studies intending to assess anxiety in sleep deprived animals keep this in mind.
A second risk regards those experiments in which an unconfirmed or
misinterpreted increase in anxiety is considered in addition to other variables.
Studies that evaluate additional interventions to the relation between sleep
deprivation and anxiety become uncertain, since when the correlation between
these variable is not well established, the evaluation of additional variables may
be imprecise. This might be the case in studies assessing the effect of drugs on
the behavior of sleep-deprived animals. Since there is no consensus about the
direct relationship between the primary variables, any conclusion regarding the
pharmacological effect becomes inaccurate. A final concern is related to ethics
in animal experimentation. Results acquired so far about anxiety-like behavior
due to sleep deprivation seem to be consistent, though opposite to what would
be expected. Regardless of this, new experiments are generated and others are

26

replicated with the intention to observe increased anxiety in sleep-deprived

animals. One of the main purposes of preclinical meta-analysis is to avoid
unethical or unnecessary animal use (Vesterinen et al., 2014). Thus, authors
involved in new experiments dealing with the relation between sleep deprivation
and anxiety should be aware of the results herein reported, in order to reduce
animal use for unjustified purposes.
As this was a very comprehensive investigation, some caveats should be
highlighted, in order to allow a better appraisal of the presented data. First of all,
the reader should bear in mind that this was an animal meta-analysis and that
differences in design and purposes are expected when compared to the
classical human meta-analyses (Vesterinen et al., 2014). One of these
differences concerns the quality and specificity of the articles included. While a
clinical meta-analysis would include only those articles that met a pre-specified
threshold of internal validity, preclinical meta-analyses are intended to include
as much data as possible. Thus, one can observe that some of the selected
articles were not even intending to evaluate the effects of sleep deprivation on
anxiety, but they did it, either directly or indirectly, due to their study designs,
presenting at least one unequivocal control and one experimental sleep
deprived group. Even so, despite the intention to be as inclusive as possible,
one may notice that only 50 out of an initial sample of 756 articles were
considered eligible, resulting in an inclusion rate of 6.61%. This striking
reduction in the number of articles, from those resulting from the initial data
search until the final sample, is natural in meta-analysis (Leenaars et al., 2012).
This can be explained by the aims of each step of article selection in a metaanalysis, which is set up to be sensitive to avoid false negatives in the initial

27

data search; but prioritizes specificity and avoiding false positives in the full text
appraisal and data extraction stages. Moreover, three important features of our
protocol contributed to the high number of excluded records. First, we chose not
to use a search filter for terms only related to animal research, but rather to
exclude human research articles manually. Second, theoretical articles
accounted for a significant percentage of our initial sample and were promptly
excluded as they are not eligible for our analysis. Third, we had a strict interest
on studies having experimental sleep deprivation as the intervention and
anxiety-like behavior as the outcome. Studies beyond this restriction accounted
for the largest parcel of exclusions, and were considered as being incompatible
designs (Figure 1), which encompasses non-experimental sleep deprivation
(as defined in our inclusion criteria), inverse relationships (studies evaluating
the effect of anxiety on sleep patterns) or cases in which both anxiety and sleep
deprivation were outcomes of different and unrelated interventions. Thus,
despite the final numbers seeming to be a greatly reduced portion from the
initial sample, this should be understood as part of a process designed to
assure that only truly eligible data are included in the analysis. A final potential
limitation comes from the fact that some experimental protocols were used and
reported by only one research group. In these cases, more than reviewing the
effects of lack of sleep on anxiety, we were reviewing the consistency of some
research groups works. Such was the case for the staircase test, used only by
Dr. Pokks group (Pokk and Vli, 2001a, b; Vassiljev et al., 2000), plus-maze
discriminative avoidance task, used only by Dr. Frussa-Filhos group (Alvarenga
et al., 2008; Fernandes-Santos et al., 2012; Patti et al., 2010; Silva et al.,
2004a; Takatsu-Coleman et al., 2013), and for the mirror chamber test and the

28

zero maze, exclusively used by Dr. Kumars group (Garg and Kumar, 2008;
Kumar and Garg, 2008; Kumar and Singh, 2007, 2008, 2009; Singh and Kumar,
2008).
In conclusion, the present meta-analyses shows that in general,
experimental sleep deprivation in rodents leads to a decrease in anxiety levels,
the opposite of the increase in state anxiety observed in humans. Such a
discrepancy impairs the translational applicability of animal models in the
relationship between anxiety and sleep deprivation. The most probable
explanation for such a discrepancy lies in the nature of the methods that have
been used to assess anxiety-like behavior in rodents. Together with anxiety,
sleep deprivation in rodents elicits a mania-like behavior or increased
impulsivity, which means that traditional methods, such as the elevated plusmaze and other anxiety measurement tests, are unable to properly quantify
anxiety-like behavior faced with this multifaceted behavioral phenotype. It is
necessary to find a method that allows underlying anxiety to be measured
without interference from other behaviors, with the best current option seeming
to be the grooming analysis algorithm. Another possible explanation would be
that rodents do not develop any kind of anxiety due to sleep deprivation. Until
we have conclusive evidence regarding the real translational applicability of the
use of animal models for the investigation of anxiety-like behavior in sleep
deprived animals, studies conducted and published should be analyzed
carefully and treated with caution.

29

5. ACKNOWLEDGEMENTS
This article has not been submitted elsewhere for publication, in whole or
in part, and all of the listed authors have approved the final manuscript. GNP is
a current employee of Springer Nature; this position has no relation with the
present article, nor with any of this authors academic activities. Other authors
have no conflicts of interest to disclose. Funding for this study was provided by
Associao Fundo de Incentivo Pesquisa (AFIP), National Council of
Technological and Scientific Development (CNPq), and So Paulo Research
Foundation (FAPESP).

30

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36

7. FIGURE CAPTIONS

Figure 1: Flowchart of articles selection

37

Figure 2: Publication bias analysis. Funnel plots including all selected

experiments (A) and excluding potential redundant publication (B). For better
visualization, an outlier point is presented separately, which corresponds to
Vassiljev et al., 2011.

38

Figure 3: Forest plot with all included experiments (analysis #1). Horizontal
lines represent the effect size the confidence interval (95%). The diamond by
the bottom of the plot represents the effect summary (analysis #1). The
adjusted summary is also presented (analysis #1*).

39

Figure 4: Results for analyses on cluster two. The horizontal gray-shaded band
represents the overall estimated effect size (analysis #1*). The hatched bar
represents an adjusted analysis, excluding potential duplicated publication.
Data are presented as effect size 95% confidence interval. SD: general sleep
deprivation; REMSD: REM sleep deprivation; TSD: Total sleep deprivation. *:
confidence interval range not crossing zero line and p<0.05.

40

Figure 5: Results for analyses on cluster three. The horizontal gray-shaded

band represents the overall estimated effect size (analysis #1*). The hatched
bar represents an adjusted analysis, excluding potential duplicated publication.
Vertical axis was fragmented for better visualization. Data are represented as
the effect size 95% confidence interval. EPM: Elevated plus-maze; OFT:
Open field test; PMDAT: Plus-maze discriminative avoidance task; ZM: Zero
maze; MCT: Mirror chamber test; StT: Staircase test. *: confidence interval
range not crossing zero line and p<0.05.

41

Figure 6: Results for analyses on cluster four. Each bar represents one
different comparison, corresponding to Table 5. The horizontal shaded-gray
band represents the overall estimated effect size (analysis #1*). Data are
represented as the effect size 95% confidence interval. Vertical axis was
fragmented for better visualization.

42

8. TABLES

Table 1. List of behavioral tests and variables ranked by relevance

1. Elevated plus maze
3. Plus-maze discriminative avoidance task
Open arm time (%)
Time in open arms (%)
Closed arm time (%)
Total entries (n)
Entries in open arms (%)
Open arm entries (n)
4. Zero maze
Closed arm entries (n)
Time in open quadrants (s)
Anxiety index
Entries in open quadrants (n)
Time/open arm entry (s)
Time in close quadrants (s)
Immobility (%)
Entries in close quadrants (n)
Segments crossed - open (n)
Average time/open arm entry (s)
Segments crossed - closed (n)
Segments crossed - total (n)
5. Mirror chamber
Center time (%)
Time in the mirror chamber (s)
Total entries (n)
Distance walked (ft.)
Stretching (n)
Rearing (n)
Grooming (n)
Fecal bolus (n)

Entries in the mirror chamber (n)

Latency to enter the mirror chamber (s)
Average time/entry
6. Staircase test
Steps climbed (n)
Latency (s)
Rearings (n)

8. Grooming analysis algorithm

Incorrect transitions (n)
Correct transitions (n)
Total transitions (n)
Transitions ratio
Interruptions (n)
Grooming latency (s)
Grooming frequency (s)
Grooming duration (s)
9. Open field with starter box
Time in open field (%)
Entries in open field (n)
Latency to enter open field
(s)
10. Light-dark explorations
Time in light (s)
11. Elevated T maze
Baseline avoidance (s)
Avoidance I (s)
Avoidance II (s)
Escape (s)

2. Open field test

Central locomotion (n)
Time in central zone (s)
7. Hole board test
Walking in central zone (cm)
Head dippings (n)
Quadrants crossed (n)
Rearing (n)
12. Light-dark box
Total walked distance (cm)
Latency to enter (s)
Locomotion ratio
Time spent (s)
Peripheral locomotion (n)
Average entry duration (s)
Latency to first movement (s)
Number of entries (n)
Rearing (n)
Distance traveled (m)
Grooming (n)
Immobility (s)
Fecal bolus (n)
Tests ranked by number of articles in included in the meta-analyses. Variables ranked by subjective importance. This
ranking was consulted whenever it was necessary to choose a variable to be included in a given analysis

43

Table 2. List and description of selected articles

Article

Age
Sleep
Procedure Duration
(weeks) manipulation
Wistar
Male
adult
REM sleep
Multiple
96h
deprivation
platform
Wistar
Male
adult
REM sleep
Multiple
96h
deprivation
platform
Wistar
Male
Adult
REM sleep
Multiple
96h
deprivation
platform
Swiss
Female
12
REM sleep
Multiple
48h
deprivation
platform
Swiss
Male
12
REM sleep
Multiple
48h
deprivation
platform
Swiss
Male
12
Total sleep
Gentle
6h
deprivation
handling
Sprague- Male
adult
Total sleep
Gentle
6h
Dawley
deprivation
handling
Sprague- Male
10
Total sleep
Gentle
6h
Dawley
deprivation
handling
Sprague- Male
10
Total sleep
Gentle
12h
Dawley
deprivation
handling
Wistar Female
12
REM sleep
Multiple
72h
deprivation
platform
Wistar
Male
12
REM sleep
Multiple
96h
deprivation
platform
Swiss
Male
12
Total sleep
Gentle
6h
deprivation
handling
Strain

Gender

Anxiety tests
and hormones
PMDAT

Exp

Species

Alvarenga et
al., 2008
Alvarenga et
al., 2008
Andersen et
al., 2005
Araujo et al.,
2006
Araujo et al.,
2006
Berro et al.,
2014
Cohen et al.,
2012
Cortese et al.,
2010
Cortese et al.,
2010
de Oliveira et
al., 2004
Dubiela et al.,
2011
FernandesSantos et al.,
2012
FernandesSantos et al.,
2012
Garg and
Kumar, 2008*

Rats

Rats

Rats

Mice

Mice

Mice

Rats

Rats

Rats

Rats

Rats

Mice

Mice

Swiss

Female

12

Total sleep
deprivation

Gentle
handling

6h

PMDAT

Mice

Albino Laca

Both

adult

Sleep
restriction

Grid over
water

72h

Hajali et al.,
2012
Hajali et al.,
2012
Hajali et al.,
2012
Kalonia and
Kumar, 2007*
Kumar and
Garg, 2008*

Rats

Wistar

Male

16

Rats

Wistar

Female

16

72h

OFT

Rats

Wistar

Female

16

72h

OFT

Mice

Male

adult

48h

EPM

Mice

Albino Laca
Albino Laca

Both

adult

Multiple
platform
Multiple
platform
Multiple
platform
Grid over
water
Grid over
water

72h

REM sleep
deprivation
REM sleep
deprivation
REM sleep
deprivation
Sleep
restriction
Sleep
restriction

EPM, zero
maze, mirror
chamber
OFT

72h

Kumar and
Kalonia, 2007*
Kumar and
Singh, 2007*

Mice

Male

adult

Mice

Male

adult

Sleep
restriction
Sleep
restriction

Grid over
water
Grid over
water

48h

Albino Laca
Albino Laca

EPM, zero
maze, mirror
chamber
EPM

Kumar and
Singh, 2008*

Mice

Albino Laca

Male

adult

Sleep
restriction

Grid over
water

72h

Kumar and
Singh, 2009*

Mice

Albino Laca

Both

adult

Sleep
restriction

Grid over
water

72h

Nair et al.,
2011
Nair et al.,
2011
Novati et al.,

Mice

C57BL/6J

Male

adult

Mice

C57BL/6J

Male

adult

Rats

Wistar

Male

Sleep
fragmentation
Sleep
fragmentation
Sleep

Sweeper
bar
Sweeper
bar
Rotating

14 days 12h/day
14 days 12h/day
7 days -

72h

PMDAT
OFT, EPM
OFT
OFT
OFT
EPM
EPM
EPM
OFT, EPM
OFT, EPM
PMDAT

EPM, zero
maze, mirror
chamber
EPM, zero
maze, mirror
chamber
EPM, zero
maze, mirror
chamber
EPM
EPM
OFT, EPM,

44

2011
Novati et al.,
2011
Patti et al.,
2010
Patti et al.,
2010
Perry et al.,
2008
Pires et al.,
2013
Pires et al.,
2013
Pokk and Vli,
2001a
Pokk and Vli,
2001b
Pokk and Vli.,
2002
Pokk and
Zharkovsky,
1995
Pokk and
Zharkovsky,
1997
Pokk and
Zharkovsky,
1997
Pokk and
Zharkovsky,
1998
Pokk and
Zharkovsky,
1998b
Pokk et al.,
1996
Pokk et al.,
2000
Polesel et al.,
2014
Polesel et al.,
2014
Porsolt et al.,
1978
Rothman et
al., 2013
Silva et al.,
2004
Silva et al.,
2004
Silva et al.,
2004
Silva et al.,
2004
Silva et al.,
2004a
Silva et al.,
2004
Silva et al.,
2004b

restriction

disc

20h/day

ACTH

Sleep
restriction
REM sleep
deprivation
Total sleep
deprivation
REM sleep
deprivation
REM sleep
deprivation
Total sleep
deprivation
REM sleep
deprivation
REM sleep
deprivation
REM sleep
deprivation
REM sleep
deprivation

Rotating
disc
Multiple
platform
Gentle
handling
Single
platform
Multiple
platform
Gentle
handling
Single
platform
Single
platform
Single
platform
Single
platform

31 days 20h/day
72h

OFT, EPM,
ACTH
PMDAT

6h

PMDAT

72h

EPM

96h

GAA

6h

GAA

24h

Staircase test

24h

Staircase test

24h

EPM

24h

EPM

20

REM sleep
deprivation

Single
platform

24h

EPM

Male

20

REM sleep
deprivation

Single
platform

24h

EPM

Albino BALB/C

Male

adult

REM sleep
deprivation

Single
platform

24h

EPM

Mice

Albino NMRI

Male

adult

REM sleep
deprivation

Single
platform

24h

EPM, hole
board test

Mice

Male

adult

Male

adult

24h

Staircase test

Mice

Male

12

24h

OFT

Mice

Swiss

Male

12

48h

OFT

Rats

Male

adult

24h

OFT

Mice

SpragueDawley
3xTgAD

Male

56

Mice

Male

12

Mice

Male

12

72h

OFT

Mice

Male

12

24h

EPM

Mice

Male

12

72h

EPM

Mice

Male

12

72h

EPM

Mice

Male

12

72h

EPM

Mice

Swiss
EPM-M1
Swiss
EPM-M1
Swiss
EPM-M1
Swiss
EPM-M1
Swiss
EPM-M1
Swiss
EPM-M1
Swiss

42 days 6h/day
72h

Open field

Male

12

Single
platform
Single
platform
Multiple
platform
Multiple
platform
Single
platform
Multiple
platform
Multiple
platform
Multiple
platform
Multiple
platform
Single
platform
Multiple
platform
Multiple
platform
Multiple
platform

EPM

Mice

REM sleep
deprivation
REM sleep
deprivation
REM sleep
deprivation
REM sleep
deprivation
REM sleep
deprivation
Sleep
restriction
REM sleep
deprivation
REM sleep
deprivation
REM sleep
deprivation
REM sleep
deprivation
REM sleep
deprivation
REM sleep
deprivation
REM sleep
deprivation

24h

Albino NMRI
Albino NMRI
Swiss

72h

PMDAT

Rats

Wistar

Male

Mice

Swiss

Male

12

Mice

Swiss

Male

12

Rats

Male

16

Rats

Wistar
Hannover
Wistar

Male

12

Rats

Wistar

Male

12

Mice

Male

adult

Mice

Male

adult

Mice

Albino BALB/C
Albino BALB/C
Albino

Male

adult

Mice

Albino NMRI

Male

adult

Mice

Albino BALB/C

Male

Mice

Albino BALB/C

Mice

EPM

45

Singh and
Kumar, 2008

Mice

Albino

Male

adult

Sleep
restriction

Grid over
water

72h

Singh et al.,
2013
Suchecki et
al., 2002
Suchecki et
al., 2002
Ser et al.,
2011
TakatsuColeman et al.,
2014
TakatsuColeman et al.,
2014
TakatsuColeman et al.,
2014
Tartar et al.,
2009
Tartar et al.,
2009
Torabi-Namia
et al., 2012
Torabi-Namia
et al., 2012
Vassiljev et al.,
2011
Vassiljev et al.,
2011
Vassiljev et al.,
2011
Vollert et al.,
2011
Wang et al.,
2014
Wang et al.,
2014
Wang et al.,
2014
Xu et al., 2010

Zebrafish

AB

Both

adult

Rats

Wistar

Male

16

96h

EPM, ACTH

Rats

Wistar

Male

16

96h

EPM, ACTH

Rats

Wistar

Male

20

Mice

Swiss

Male

12

Constant
light
Single
platform
Multiple
platform
Multiple
platform
Multiple
platform

24h

Total sleep
deprivation
REM sleep
deprivation
REM sleep
deprivation
Sleep
restriction
REM sleep
deprivation

EPM, zero
maze, mirror
chamber
Light/dark box

21 days 18h/day
12h

Elevated T
maze
PMDAT

Mice

Swiss

Male

12

REM sleep
deprivation

Multiple
platform

24h

PMDAT

Mice

Swiss

Male

12

REM sleep
deprivation

Multiple
platform

12h

PMDAT

Rats

FischerNorway
FischerNorway
Wistar

Male

adult

Sleep
Treadmill
24h
OFT with
fragmentation
starter box
2
Rats
Male
adult
Total sleep
Treadmill
24h
OFT with
deprivation
starter box
1
Rats
Male
adult
Total sleep
Rotating
48h
EPM
deprivation
disc
2
Rats
Wistar
Male
adult
Sleep
Rotating 7 days EPM
restriction
disc
12h/day
1
Mice
Albino Male
adult
REM sleep
Single
24h
Staircase test
NMRI
deprivation
platform
2
Mice
Albino Male
adult
REM sleep
Single
24h
Staircase test
NMRI
deprivation
platform
3
Mice
Albino Male
adult
REM sleep
Single
24h
Staircase test
NMRI
deprivation
platform
1
Rats
Wistar
Male
11
REM sleep
Multiple
24h
OFT, lightdeprivation
platform
dark test
1
Rats
Sprague- Male
8
REM sleep
Multiple
18h
OFT
Dawley
deprivation
platform
2
Rats
Sprague- Male
8
Sleep
Multiple
7 days OFT
Dawley
restriction
platform
18h/day
3
Rats
Sprague- Male
8
Sleep
Multiple 14 days OFT
Dawley
restriction
platform
18h/day
1
Rats
Sprague- Male
8
Total sleep
Treadmill
72h
OFT, EPM
Dawley
deprivation
Yehuda et al.,
1
Rats
Sprague- Male
adult
REM sleep
Single
72h
EPM
2007
Dawley
deprivation
platform
Zager et al.,
1
Mice
C57BL/6J Male
9
REM sleep
Multiple
72h
OFT, EPM
2009
deprivation
platform
Zielinski et al.,
1
Mice
C57BL/6J Male
13
Sleep
Rotating 7 days EPM
2013
restriction
disc
12h/day
Exp: Experiment; EPM: Elevated plus maze; OFT: Open field test; PMDAT: Plus-maze discriminative avoidance task;
GAA: Grooming analysis algorithm; *: articles suspected of duplicate publication.

46

Table 3. Sample description

Articles

Exps

Species
Mouse
Rat
Zebrafish

30
19
1

45
30
1

Gender
Male
Female
Both/undisclosed

44
4
4

67
5
4

1
49
1

1
74
1

Age
Juvenile (6 weeks)
Adult
Old (>30 weeks)
Sleep manipulation
Total sleep deprivation
REM sleep deprivation
Sleep restriction
Sleep fragmentation

10
29
14
2

12
44
17
3

Articles

Exps

Protocol duration
6h
12-18h
24-48h
72-96h
7 days; 6-12h/day
7 days - 18-20h/day
14 days - 6-12h/day
14 days - 18-20h/day
21 days
35 days
42 days

6
3
21
20
1
2
1
1
1
2
1

7
4
27
28
2
2
2
1
1
1
1

Anxiety test
EPM
OFT
PMDAT
Zero maze
Mirror chamber
Staircase test
Hole board test
GAA
OFT with starter box
Light-dark explorations
Ligh-dark box
Elevated T maze

29
14
5
5
5
4
1
1
1
1
1
1

38
25
10
5
5
6
1
2
2
1
1
1

Procedure
Multiple platform
18
31
Single platform
15
18
Grid over the water
8
8
Gentle Handling
6
8
Rotating disc
3
5
Treadmill
2
3
Sweeper bar
1
2
Constant light
1
1
Some sums may not results in exact values, since one single article may have been used in
more than one procedure for sleep deprivation or one experiment may have been employed in
more than one anxiety test. Exps: number of experiments. EPM: Elevated plus maze; OFT:
Open field test; PMDAT: Plus-maze discriminative avoidance task; GAA: Grooming analysis
algorithm

47

Table 4: Risk of bias assessment

Appropriate

Unclear

Inappropriate

Selection bias Sequence generation

20%

80%

0%

Selection bias Baseline characteristics

44%

50%

6%

Detection bias Random outcome measurement

4%

96%

0%

Detection bias Blinding

20%

80%

0%

Attrition bias Incomplete outcome data

86%

0%

14%

Reporting bias selective outcome reporting

88%

0%

12%

Other source of bias

82%

0%

18%

Adapted from the SYRCLE's risk of bias tool (Hooijmans et al., 2014)

48

Stratifications
Overall analysis - no filters
Adjustment on analysis #1

Cluster 2

2
3
4
5
5*
6
7
8
9

Rats, Male, Adult, 6-96h

Rats, Male, Adult, REMSD, 6-96h
Rats, Male, Adult, TSD, 6-96h
Mice, Male, Adult, 6-96h
Adjustment on analysis #5
Mice, Male, Adult, REMSD, 6-96h
Mice, Male, Adult, TSD, 6-96h
Male, Adult, REMSD, 6-96h
Male, Adult, TSD, 6-96h

21
13
7
36
31
28
3
41
10

10
10*
11
12
13
14
15

EPM
Adjustment on analysis #10
OFT
PMDAT
Zero maze
Mirror chamber test
Staircase test

39
31
22
10
6
6
6

C1

#
1
1*

Cluster 3

Table 5. Description and results for all meta-analyses

Exp n-ctrl
75
742
67
697

CI-Lower CI-Upper I2
p
-0.77
0.07
0.90 <0,01
-1.18
-0.38
0.90 <0,01

n-exp
783
734

ES
-0.35
-0.78

218
141
94
365
343
308
27
452
113

239
166
70
385
354
320
34
483
112

-0.75
0.69
-0.69
-0.34
-0.95
-0.97
-1.30
-0.87
-0.85

-1.36
-1.65
-1.55
-1.02
-1.64
-1.68
-3.29
-1.43
-1.60

-0.14
0.26
0.16
0.35
-0.33
-0.27
0.68
-0.31
-0.10

0.87
0.91
0.83
0.92
0.92
0.92
0.89
0.91
0.84

<0,01
<0,01
<0,01
<0,01
<0,01
<0,01
<0,01
<0,01
<0,01

377
329
196
102
36
36
106

390
341
198
116
36
36
106

0.27
-0.56
-0.42
-0.39
7.22
7.41
-6.60

-0.31
-1.08
-1.04
-0.79
5.47
3.80
-9.47

0.85
-0.05
0.19
0.01
8.96
11.01
-3.73

0.90
0.88
0.85
0.49
0.20
0.58
0.96

<0,01
<0,01
<0,01
0.04
0.28
<0,01
<0,01

Cluster 4

16 Mice, Male, Adult, TSD, GH, 6h, EPM

2
16
23
-0.19
-0.84
0.46
0.00 0.95
17 Mice, Male, Adult, REMSD, MP, 48-72h, EPM
4
41
39
0.75
-0.46
1.96
0.83 <0,01
18 Mice, Male, Adult, REMSD, MP, 48-72h, OFT
4
31
31
-0.24
-3.13
2.64
0.93 <0,01
19 Mice, Male, Adult, REMSD, MP, 48-72h, PMDAT
2
14
20
0.32
-1.20
1.83
0.77 0.04

20 Mice, Male, Adult, REMSD, SP, 24-48h, StT

6
106
106
-6.60
-9.47
-3.73
0.96 <0,01
21 Mice, Male, Adult, REMSD, SP, 24-48h, EPM
7
59
59
-1.50
-2.08
-0.93
0.40 0.12

22 Mice, Male, Adult, SR, GW, 48-72h, EPM

5
30
31
7.50
5.79
9.20
0.00 0.87
23 Mice, Male, Adult, SR, GW, 48-72h, ZM
3
18
18
9.70
4.86
14.53
0.63 0.07

24 Mice, Male, Adult, SR, GW, 48-72h, MCT

3
18
18
9.29
6.57
12.02
0.00 0.69
25 Rat, Male, Adult, TSD, GH, 6h, EPM
2
31
24
-1.02
-1.60
-0.44
0.00 0.84
26 Rat, Male, Adult, REMSD, MP, 72-96h, OFT
3
31
30
-0.42
-1.78
0.93
0.83 <0,01

27 Rat, Male, Adult, REMSD, MP, 72-96h, EPM

3
53
52
-1.49
-2.79
-0.19
0.85 <0,01
28 Rat, Male, Adult, REMSD, SP, 72-96h, EPM
3
51
51
0.96
-3.57
1.66
0.94 <0,01

29 Rat, Female, Adult, REMSD, MP, 72-96h, OFT

3
26
26
-1.64
-2.30
-0.99
0.00 0.91
Exp: experiments; n-ctrl: sample size in control groups; n-exp: sample size in experimental group; ES: Effect size; CI-Lower:
Lower bound of confidence interval at 95%; CI-Upper: Upper bound of confidence interval at 95%; REMSD: REM sleep
deprivation; TSD: Total sleep deprivation; SR: Sleep restriction; PMDAT: Plus maze discriminative avoidance task; EPM:
Elevated-plus maze; OF: Open Field test; PMDAT: Plus-maze discriminative avoidance task; MCT: Mirror chamber test; ZM:
Zero maze; StT: Staircase test. GH: Gentle handling; MP: Multiple platform; SP: Single platform; GW: Grid over water. p
values are referent to the Cochran's Q test. *: Adjusted analysis with exclusion of the articles suspected of duplicate
publication. : Analyses in which all articles were suspected of duplicate publication. : results in which confidence interval
range is all valued higher or lower than zero, with p<0.05.

49