Self-grooming, experimental anxiety and

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Self-grooming, experimental anxiety and paradoxical sleep deprivation in rats
ORIGINAL ARTICLE
Self-grooming, experimental anxiety and
paradoxical sleep deprivation in rats
Limpeza corporal, ansiedade experimental e privação de sono paradoxal em ratos
Helga Caputo Nunes1, Fernanda Augustini Pezzato1, Katsumasa Hoshino1
ABSTRACT
Objective: Paradoxical sleep deprivation increases grooming behaviors
in rats. Discordant data on the change promoted by such deprivation
on the experimentally defined anxiety levels have made difficult to assess if the deprivation induced grooming may be used as an experimental model of repetitive behaviors, observed in different psychiatric disorders, due increased anxiety levels. In such context, the present study
aims to report data gathering grooming and anxiety in rats. Methods:
The degrees of tidiness displayed by female (n=26) and male (n=27)
rats were evaluated after a period of maintenance in home-cages provided with wood shavings that have the property to dye animal’s fur. In
the second study, the tidiness degrees were evaluated after 96 h of sleep
deprivation in anxious male rats (n=6) and compared to non-anxious
controls (n=6). Results: Females, that are believed to be more anxious
than males, displayed higher tidiness (0% colored females x 96% colored males) in the first study. All anxious male rats (100%) that displayed
wild running in response to intense acoustic stimulation showed better
tidiness than their controls at the end of 96 h sleep deprivation. Conclusions: Our data suggest that self-grooming in rats is tightly related
to anxiety and allows the interpretation that its increased occurrence in
sleep deprivation results from its anxiolytic or tranquilizing property.
Keywords: animal, anxiety, compulsive behavior, grooming, models,
rats, sleep deprivation.
RESUMO
Objetivo: A privação de sono paradoxal aumenta a emissão dos comportamentos de cuidados corporais (grooming) em ratos. A discordância quanto à ocorrência de aumento da ansiedade experimentalmente
definida nesta privação tem dificultado o estabelecimento de se o fenômeno pode constituir, ou não, um modelo experimental dos comportamentos repetitivos que ocorrem em diferentes transtornos psiquiátricos, devido à elevação dos níveis de ansiedade. Neste contexto,
o presente trabalho objetivou coletar dados indicativos da existência
de relação entre grooming e a ansiedade de ratos. Métodos: Os graus
de limpeza corporal de ratos machos (n=27) e fêmeas (n=26) foram
comparados após serem mantidos por um tempo em gaiolas-viveiro
com forração de maravalha, que possuía a propriedade de tingir o pelo
dos animais. Em um segundo estudo, o grau de limpeza corporal de
ratos machos com níveis altos de ansiedade experimentalmente definida (n=6) foi comparado ao dos ratos controles (n=6), não ansiosos,
após 96 h de privação de sono paradoxal. Resultados: As fêmeas, que
são consideradas mais ansiosas comparativamente aos machos, mostraram não ter tingimento dos pelos (0% x 96% de machos tingidos) no
primeiro estudo. Os machos mais ansiosos, selecionados por manifestarem a corrida incontrolável induzida por som intenso e considerada
fuga de pânico, mostraram (100%) permanecer mais limpos no final da
privação de sono. Conclusões: Os dados sugerem que o grooming dos
ratos pode ser emitido como componente do repertório de comportamentos ansiosos e sugerem que esta ocorrência se deve à propriedade
ansiolítica ou tranquilizadora que o cuidar do corpo tem.
Descritores: ansiedade, asseio corporal, comportamento compulsivo,
modelos animais, privação do sono, ratos.
INTRODUCTION
Frequent manifestation of a behavioral pattern is the most outstanding feature of some psychiatric disorders. Trichotillomania
(the compulsive urge to pull out one’s own hair), washing the
hands very often and bathing excessively are well-known examples. Nevertheless, available data on their causes, mechanisms
and treatments are scarce. For this reason, the advancement
of knowledge about these and other psychiatric disorders still
depends on further research carried out through experimental
models employing laboratory animals(1,2).
Washing hands and bathing are self-grooming behaviors
that have high adaptation value. Their excessive and unjustified manifestation, however, characterizes an anxiety disorder
known as obsessive-compulsive disorder. Trichotillomania, despite being classified as a manifestation of habit and impulsive
disorders, is also preceded by increasing and uncontrollable
anxiety, which is relieved by pulling out the hair (ICD-10). The
involvement of anxiety in both of these types of disorders suggests a common link between them, and this fact seems to justify classifying trichotillomania as a distorted manifestation of
grooming, as proposed in the literature(3).
The association of anxiety with grooming behavior disorders is supported by laboratory models employing animals,
where it must be emphasized that anxiety is ethologically or experimentally defined. This allows experimental paradigms for
the study of repetitive psychopathological behavioral patterns
to be established. The available data show that rats manifest
grooming ritualistically with distinguishable patterns and sequences and quantifiable intensities in several appetitive or aversive situations(4). Each grooming modality (licking, scratching,
etc.) seems to have a unique central mechanism(5), and their manifestation in situations of anxiety or stress is well established(6).
Universidade Estadual Paulista “Julio de Mesquita Filho” – UNESP – Bauru (SP), Brasil.
Corresponding author: Katsumasa Hoshino. Biological Sciences Department. Sciences Faculty – UNESP - Av. Luiz Edmundo Carrijo Coube, 14-01 - Bauru (SP), Brazil - CEP: 17033360 - E-mail: [email protected]
Received: April 13, 2011; Accepted: August 30, 2011.
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This relationship allows grooming time to be experimentally
employed to evaluate levels of anxiety(7). From this perspective,
increases in grooming as observed in rats subjected to paradoxical sleep deprivation(8,9) suggest that this is a fitting paradigm for
the study of repetitive behavior disorders. First, the grooming
behaviors of different species seem to have similar evolutionary
origins due to their high adaptation value. This suggests that
grooming behaviors comply with the criterion of homology
that is mandatory in experimental models. Second, paradoxical
sleep deprivation is recognized as stressful(10), and it involves an
increase in the level of anxiety(11) with the demonstration of behaviors suggestive of anxiety(12,13). This relationship among anxiety, sleep deprivation and grooming can manifest in different
ways. Deprivation increases the production of wild running in
audiogenic seizures(14), which indicate an abnormal increase of
anxiety because they can be viewed as episodes of panicking
escape(15). Concordantly, escape behavior that can be evoked by
high-intensity sound stimulation is expressed by the most anxious animals(16).
Interestingly, escape-inducing sound stimuli also evoke
grooming(17). The anxiogenic property of sleep deprivation is
also evident in the observation of the management of the aggression it induces. Fights induced by sleep deprivation can be
diminished with the use of fluoxetine(18) (an antidepressant drug
used in the treatment of panic syndrome(19)) and increased with
the administration of hypertonic sodium lactate(20) (this procedure is used to induce attacking to diagnose panic disorder(21)).
The fact that panic disorder is one of the anxiety disorders
supports the hypothesis that sleep deprivation is anxiogenic.
Concordantly, sleep deprivation exacerbates panic attacks in the
population affected by this anxiety disorder(22). Finally, the proposal to use sleep deprivation and its effects as an experimental
model for mania(23) validates the use of grooming in rats as a
paradigm for studying the excessive manifestation of behavioral
patterns in some psychopathological disorders.
The literature is not limited to data agreeing with the
relationships among sleep deprivation, anxiety and grooming in
rats mentioned above. The same studies indicating an increase in
the manifestation of grooming in paradoxical sleep deprivation
also report that other anxiety-indicating behaviors, evaluated
with the elevated-plus maze, do not occur(8,9). Moreover, and in
contrast to what could be expected, the increase of the time
that sleep-deprived rats spend in the open arms or the center of
the elevated plus-maze leads to the conclusion that such deprivation is anxiolytic(24). Further, sleep deprivation decreases the
manifestation of cleansing behavior, which is increased by intracerebral administration of enkephalinase inhibitors or opioid
abstinence(25). This contradiction must be resolved before sleep
deprivation–affected grooming can be used as an experimental
model; it should be noted that this model has the advantages
of being simple, quick and less expensive than models employing genetic manipulation and revealing the molecular mechanisms involved in the production of compulsive behaviors(26).
The need to quantify grooming in order to support other data
in knockout models also makes it important to confirm that
grooming is a component of anxious behaviors.
20
The existence of contradictory data necessitates the
evaluation of the validity of grooming as a paradigm for the
investigation of pathologically repetitive behaviors. In this context, the difference in the levels of anxiety between male and female rats provides an opportunity to confirm the relationships
between variables relevant to this matter. Like women(27), female
rats also manifest more anxious behaviors than males(28). Therefore, it is likely that female rats will keep themselves cleaner than
males under natural conditions due to their higher frequency
of grooming behaviors arising from a higher level of anxiety.
This hypothesis is backed by the higher rate of wild running in
females in response to high-intensity sound stimuli(29), which are
stressful, anxiogenic and grooming-inducing(17,30).
The hypothesis that greater body cleansing in females
is determined by greater anxiety might be contradicted by the
hypothesis that the degree of body cleansing results from a reduction in the chances to soil oneself due to a smaller rate of locomotor activity because of anxiety. The validity of any of these
hypotheses can be tested through the use of male rats subjected
to paradoxical sleep deprivation. Male rats manifesting wild running when subjected to high-intensity sound stimulation exhibit
higher indexes of anxiety in the elevated plus-maze test(16). Because paradoxical sleep deprivation through the single-platform
method is anxiogenic and restricts locomotion(31), the manifestation of grooming assessed by the degree of body cleansing is a
function of the individual degree of anxiety.
Due to the relevance of this subject and its potential to
improve our understanding of sleep deprivation and anxiety
disorders, this study assessed the indices of body cleansing in
male and female rats under natural living conditions and between anxious and non-anxious males subjected to paradoxical
sleep deprivation.
MATERIAL AND METHODS
Ethical requirements
This study complied with the recommendations for the use of
animals in experimentation, and it began only after obtaining
the approval of the Research Ethics Committee of the institution with which the authors are affiliated.
Experimental subjects
Sixty-five Wistar rats were used; the animals were supplied by
the Central Vivarium of Unesp, Botucatu. In the first study,
which addressed intersexual differences in cleansing, 27 males
and 26 females were used. The second study compared the degree of cleansing in 6 males that exhibited wild running induced
by high-intensity sound stimulus with 6 males that did not exhibit this manifestation after 96 hours of sleep deprivation. Sensitive animals were selected from a sample of 45 male rats.
Evaluation of body cleansing in study 1
The animals of both sexes were received at 45 days of age and
were housed in groups of 4 or 5 individuals of the same sex
in conventional propyl propylene cages (32x40x18 cm) covered
by metallic-grid lids. Commercial feed and potable water were
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Self-grooming, experimental anxiety and paradoxical sleep deprivation in rats
given ad libitum. Cages were lined with pine (Pinus elliottii) trunk
clippings that were changed every 2 days. This lining caused a
yellowish hue of varying intensity in the hair of animals, which
agrees with previous observations. Animals were kept in rooms
with automatically controlled temperature (23–25°C) and light
(7:00–19:00 h) until reaching the age of 100 days. Two evaluators with experience in handling animals classified each as having white, slightly yellow, average yellow or very yellow body
hair. For this purpose, a series of digital photographs of the
different degrees of hair color was taken against a black background. Disagreement between evaluators was resolved by a
third evaluator. The number of animals exhibiting each color
intensity was computed according to sex at the end of the study.
RESULTS
The results of the first study are described in Table 1. All females maintained their white hair color, while males exhibited
different intensities of yellow.
Evaluation of sensitivity to high-intensity sound stimulus in study 2
Animals were individually placed in a transparent acrylic cage
(30.5x30.0x18.5 cm) that was placed inside a sound isolation box
(48.0x48.0x28.0 cm) with a glass window and 11-watt incandescent lamp inside to facilitate observation. Animals were allowed
to explore the cage for 15 seconds before a 103-dB, 200-Hz
sound stimulus was applied for 60 seconds. Wild running was
operationally defined as explosive high-speed running inside the
cage that could progress into a gallop and jumps, making the
animal crash against the walls. Manifestation of this behavioral
pattern was the criterion that defined an animal as susceptible to
wild running, as previously reported(15,16,29).
The second study showed that the cleanest rats were the
ones that manifested wild running, while the rats resistant to
sound stimulation were the dirtiest. As seen in Table 2, the animals that manifested wild running induced by sound stimulation
received all of the lowest (cleanest) scores on the cleanliness
scale, while the rats that were resistant to this effect all had the
highest (dirtiest) scores.
Paradoxical sleep deprivation
The single platform technique was used to induce paradoxical
sleep deprivation because it is the classical and commonly employed method. Briefly, it consists of keeping the rat on a circular 6-cm-diameter platform 1 cm above the surface of 5-cmdeep water in a plastic vessel (32x40x27 cm). The occurrence
of muscular relaxation, which characterizes paradoxical sleep,
made the animal’s snout touch the water or eventually fall down,
which awakened them and suppressed this stage of sleep.
Index of grooming in sleep deprivation
Grooming in sleep-deprived animals was evaluated by the same
evaluators as in the first study. In this second stage, animals were
classified on a progressive scale from the cleanest to the dirtiest.
Disagreements in rating were decided by the third evaluator. At
the end, the susceptibility of each animal to intense sound stimulation was disclosed, and the distribution of animals on each
cleansing scale was evaluated using the Mann-Whitney test(32).
Data analysis
The difference in the frequencies of animals with different hair
colors stratified by sex was calculated with the chi-squared test.
The Mann-Whitney test was used to evaluate the dependence of
grooming on the susceptibility to escape induced by high-intensity sound stimulation. The level of significance was established
at 0.05 in both studies.
Table 1. Frequencies of animals with different degrees of yellow hair
color due to housing in cages lined with pine (Pinus elliottii) clippings for
45 days. Males and females were significantly different (chi-squared test 7
df, p<0.05).
Sex
Very
Average
Slight
White
Total
Males
19
7
1
0
27
Females
0
0
0
26
26
Total
19
7
1
26
53
Table 2. Sorting of rats according to the degree of body cleansing
(1=cleaner, 12=less clean). The evaluation was carried out after 96 hours
of paradoxical sleep deprivation in rats resistant (R) and susceptible (S)
to the induction of wild running by high-intensity sound stimulation. A
significant difference in scores was observed (Mann-Whitney, p<0.05).
Cleanliness score
1
2
3
4
5
6
7
8
9
10
Wild running
S
S
S
S
S
S
R R R R
11
12
R
R
DISCUSSION
Obtained data
The data obtained in this study indicate that females exhibited
a higher degree of body cleansing and that the males with the
highest levels of anxiety manifested this trait to a lesser degree.
Support of methodological validity
In this study, grooming was evaluated globally only once, which
differs from the classic quantification method of repeated
counting in standardized observation time windows. Therefore,
it is legitimate to ask whether the degree of body cleanliness of
the animals could be attributed to a higher frequency of grooming activities. Originally, this question involved the assumption
that there are different types of grooming, namely efficacious
and inefficacious, that promote body cleansing. The literature
does not reflect this assumption, which seems rather logical
given that inefficacious activities must necessarily result from
topographic differences in their execution; in this case, they
would no longer be grooming behaviors. The subjective nature
of our evaluation of body cleansing might be considered inappropriate. However, our results should still be valid because the
procedures complied with established principles of psychometric quantification(33) and the evaluation exhibited a high degree
of interobserver agreement.
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Another question relevant to our results concerns the
possibility of females having hair chemically and structurally
different from males, which confers less adhesiveness to dirt.
The literature suggests microstructural differences in hair as a
function of aging(34) and sex(35), thereby giving support to this
possibility. Our preliminary studies showed that keeping rats
for 24 hours in cages lined with red, iron oxide–rich soil stains
the hair of males and females strongly and equally. Moreover,
the possibility of an intersexual difference in cleansing resulting
from hormonal differences can be ruled out because these differences are restricted to the genital area(36), whereas this study
focused on the full surface of the body.
Function of grooming
Grooming has the adaptive function of caring for the surface
of the body in order to diminish the risk of infection and infestation. The greater degree of cleansing in females may be
attributed to the need to guarantee hygiene in the process of
procreation. Despite the likelihood of this tendency, the observation of higher degrees of cleansing also in males susceptible
to wild running indicates that at least a part of grooming is due
to higher levels of anxiety. This causal relationship suggests the
possibility that grooming has other adaptive functions besides
body cleansing. Indeed, grooming is manifested in both aversive and appetitive situations, such as new environments, social
encounters, stressors and exploratory and feeding behaviors(17).
Previous observations in such situations indicate that rodents
groom when their level of alertness is decreased(4), which suggests that grooming reduces anxiety, that is, it has anxiolytic effects, as shown in primates(37). This function gives coherence
to and supports the data obtained in this study because the increase of cleansing can be attributed to an increase of the activities aimed at reducing high levels of anxiety. This interpretation
is supported by the blockade promoted by anxiolytic agents on
the grooming induced by brain intraventricular administration
of adrenocorticotropic hormone or bombesin(38). The notion of
the evolutionary incorporation of new adaptive functions into
grooming is viable because grooming has a well-known thermolytic function, that is, it decreases body temperature through
the evaporation of saliva, which is mirrored by the activity of
grooming all over the body.
Anxiolytic mechanism of grooming
The idea that the higher degree of body cleansing observed in
this study was due to grooming’s of anxiolytic function leads
one to question how this behavior promotes the reduction of
anxiety. The answer to this question might be found in the high
dependence of rats on the detection of sensory information,
mainly through touch and olfaction, for their activities that are
predominantly nocturnal(39). To soil oneself or to have odors impregnating the body reduces or distorts the acuity of the sensory channels, reducing their ability to detect and analyze information. Risky situations are anxiogenic, and activities that unblock
and improve the acuity of the sensorial channels decrease anxiety. This process shows that the anxiolytic function of groom-
22
ing observed in stressful or aversive situations derives from its
original cleansing function. This interpretation supports the assertion that grooming activities are stereotyped and always end
in body cleansing, even when their manifestation might have
a different function at that moment, such as the regulation of
temperature.
The observation that grooming manifests in moments
of decreased alertness in stressful situations(4) seems logical. The
rise in the level of alertness signals the need to manifest defensive behaviors that have higher priority in the face of danger or
threats. An example is the need to become immobile to avoid
detection by a predator, which has the first priority, while the activity of grooming must be inhibited until the danger goes away.
This episodic trait leads one to believe that the higher degree of
cleansing of females under normal living conditions might be
attributed to their higher sensitivity to stimuli, which increases
the frequency of experienced anxiogenic episodes (state anxiety) or to their high basal anxiety rate (trait anxiety). How much
the grooming manifested by females depends on the processes
of learning through reinforcement generated by the relief of
anxiety is a matter that still requires clarification.
The observation that male rats manifesting wild running
kept themselves cleaner when deprived of paradoxical sleep
contradicts the hypothesis that body hygiene is determined by
the reduction of the possibility of soiling through the inhibition of locomotor activity caused by higher levels of anxiety.
Therefore, the association of panicked running with cleanliness
confirms the notion that anxiety is directly related to the level
of cleanliness. However, rats are awoken by wetness at the onset of paradoxical sleep in the platform method. Wetting rats
by sprinkling water on them is stressful and induces grooming(6,17). This means that paradoxical sleep deprivation through
the use of the platform method imposes an additional chronic
anxiogenic factor, and this perhaps is the main reason for the
increased grooming observed in this situation, a connection that
has been previously hypothesized(8). This interpretation makes
it necessary to re-evaluate contradictory data describing sleep
deprivation as anxiolytic(24) or having no influence on anxietyindicating behaviors in rats(8,9). In this regard, it is important to
consider that the anxiolytic property manifested in the elevated
plus-maze depended on the use of the multiple-platform method to induce sleep deprivation in rats(40).
Finally, it should be noted that the observations of a
higher degree of cleansing associated with the highest levels of
anxiety in female rats and also in the most anxious male rats
when subjected to sleep deprivation, when analyzed together
with other data reported in the literature, indicate that the aim
of the present study, namely, to increase the available data on
the validity of the use of this behavioral manifestation as an
experimental model for psychopathological repetitive behaviors,
was fulfilled.
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