Sleep deprivation impairs emotional memory retrieval in mice: Influence of sex

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Sleep deprivation impairs emotional memory retrieval in mice: In

ﬂ

uence of sex

Luciano Fernandes-Santos

a

_{, Camilla L. Patti}

a,b

_{, Karina A. Zanin}

a

_{, Helaine A. Fernandes}

a

_{, Sergio Tu}

_ﬁ

_k

a

_,

Monica L. Andersen

a

_{, Roberto Frussa-Filho}

a,b,

_⁎

a_{Departamento de Psicobiologia, Universidade Federal de São Paulo, Napoleão de Barros, 925, São Paulo, SP, Brazil} b_{Departamento de Farmacologia, Universidade Federal de São Paulo, R. Botucatu, 862, São Paulo, SP, Brazil}

a b s t r a c t

a r t i c l e

i n f o

Article history:

Received 12 March 2012

Received in revised form 28 March 2012 Accepted 29 March 2012

Available online 13 April 2012

Keywords:

Conditioning fear context Gender

Memory retrieval Passive avoidance task

Plus-maze discriminative avoidance task Sleep deprivation

The deleterious effects of paradoxical sleep deprivation on memory processes are well documented. Howev-er, non-selective sleep deprivation occurs more commonly in modern society and thus represents a better translational model. We have recently reported that acute total sleep deprivation (TSD) for 6 h immediately before testing impaired performance of male mice in the plus-maze discriminative avoidance task (PM-DAT) and in the passive avoidance task (PAT). In order to extend theseﬁndings to females, we examined the effect of (pre-test) TSD on the retrieval of different memory tasks in both male and female mice. Animals were test-ed using 3 distinct memory models: 1) conditioning fear context (CFC), 2) PAT and 3) PM-DAT. In all experi-ments, animals were totally sleep-deprived by the gentle interference method for 6 h immediately before being tested. In the CFC task and the PAT, TSD induced memory impairment regardless of sex. In PM-DAT, the memory impairing effects of TSD were greater in females. Collectively, our results conﬁrm the impairing effect of TSD on emotional memory retrieval and demonstrate that it can be higher in female mice depending on the memory task evaluated.

1. Introduction

Several studies have demonstrated the close relationship between sleep and memory in both humans and in animals (Fu et al., 2007; Lim and Dinges, 2010; Palchykova et al., 2006; Patti et al., 2010; Sterpenich et al., 2007; Stickgold, 2005; Stickgold and Walker, 2007). Memory is typically deﬁned as the ability to retain and manipulate previously acquired information by means of neuronal plasticity (de Oliveira Álvares et al., 2010; Thompson et al., 2002). It has been well-established that sleep plays a critical role in learning and memory formation. For instance, many studies have reported that paradoxical sleep deprivation in animals leads to memory deﬁ-cits in several behavioral models such as avoidance (Bueno et al., 1994; Harris et al., 1982; Skinner et al., 1976), the Morris water maze (Youngblood et al., 1999, 1997), the radial maze (Smith et al., 1998) and the plus-maze discriminative avoidance tasks (PM-DAT) (Alvarenga et al., 2008; Patti et al., 2006, 2010; Silva et al., 2004a, 2004b).

Despite the well-documented consequences of paradoxical sleep deprivation in animal models, the effects of total sleep deprivation

(TSD) on learning and memory as well as anxiety and locomotor ac-tivity in animal models have been overlooked. In this sense, TSD is a non-selective sleep deprivation and occurs more commonly in mo-dern society, thus representing a better translational model. Speciﬁ -cally, according to Fenzl et al. (2007), one has to differentiate between TSD, when the animal is prevented to from falling asleep at all, and selective sleep deprivation, where only particular sleep stages are eliminated. The later achievement is frequently used to de-prive paradoxical sleep in rodents, usually by placing the animals in small platforms surrounded by water (Alvarenga et al., 2008; Patti et al., 2010; Silva et al., 2004a, 2004b). On the other hand, TSD (para-doxical and slow-wave sleep) is less complex, simply achieved by re-moving or introducing objects within the cages or by gently handling animals, keeping them awake.

Importantly, quite recently we have demonstrated that memory deﬁcits induced by 72 h of acute paradoxical sleep deprivation, but not those induced by 6 h of TSD, are related to state-dependency phe-nomenon (Patti et al., 2010). In this study, we have also reported that TSD-induced memory deﬁcits had a greater magnitude than those in-duced by the paradoxical sleep deprivation in the PM-DAT model. In addition, mice subjected to TSD immediately before training and/or testing in the PM-DAT presented memory impairments without alterations in anxiety or locomotor activity. However, this study only examined male mice (Patti et al., 2010).

Sex differences in behavior have been extensively described in the literature. With respect to learning and memory, studies usually show better spatial learning in males (Astur et al., 2004; Blokland et al.,

–

Abbreviations:TSD, total sleep deprivation for 6 h; PM-DAT, plus-maze discrimina-tive avoidance task; PAT, passive avoidance task; CFC, context fear conditioning; M, male mice; F, female mice; CTRL, control (home cage) condition.

⁎Corresponding author at: Departamento de Farmacologia, UNIFESP, Rua Botucatu, 862, Ed. Leal Prado, 1° andar, 04023062 São Paulo, SP, Brazil. Tel.: +55 11 5549 4122; fax: +55 11 5549 4122x222.

E-mail address:frussa.farm@epm.br(R. Frussa-Filho).

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2006; Jonasson, 2005; Rilea et al., 2004) and stronger emotional memory in females (Canli et al., 2002; Hamann, 2005). Beyond these behavioral differences between the sexes, sex may also play a remarkable role in sleep. Indeed, many studies indicate that sleep is differentially regulated between sexes both in humans (Paul et al., 2008) and in animals (Fang and Fishbein, 1996; Yamaoka, 1980), sug-gesting that distinct effects may modulate their sleep patterns and thus, different behaviors could be expected (Antunes et al., 2006). In-deed, sex inﬂuences sleep–wake amount, the daily timing of the sleep–wake cycle, and the ability to restore sleep after extended wakefulness. Several lines of evidence suggest that in mammals, re-productive hormones are responsible for the effects of sex on sleep and may have organizational and activational inﬂuences on sleep regulatory mechanisms (Paul et al., 2008)

The goal of the present study was to evaluate the sex differences in mice subjected to TSD using two classic memory models (the passive avoidance task—PAT, and context fear conditioning—CFC) as well as the PM-DAT, which is used to evaluate learning, memory, anxiety, and motor activity, and their respective interactions.

2. Material and methods

2.1. Subjects

Seventy-four three-month-old male and seventy-ﬁve 3-month-old female Swiss mice (raised at CEDEME, Universidade Federal de São Paulo) were used in the experiments. Animals weighing 30–35 g were housed under controlled temperature (22–23 °C) and lighting (12 h light, 12 h dark; lights on at 6:45 a.m.) conditions. Food and water were available ad libitum throughout the entire study.

All experimental procedures were approved by the Ethics Com-mittee under protocol (#1203/2009). The mice were allowed at least 2 weeks of adaptation to the housing facilities before the start of the experiments.

2.2. TSD

Mice were subjected to TSD through the gentle handling method, which consists of keeping the animal awake by tapping on or moving the cage and, if necessary, by gently touching them with a soft brush, whenever behavioral signs of sleep, such as closed eyes or sleep pos-ture, were (seePatti et al., 2010). The animals were sleep-deprived for 6 h immediately before testing in the memory paradigms.

Although this method may allow micro-sleeps, the use of the gen-tle handling method is widely accepted in sleep research and other relatedﬁelds (Fenzl et al., 2007). In fact, this procedure suppresses 97.8% of paradoxical and slow-wave sleeps across 6 h (Fenzl et al., 2007) and induced memory impairment in previous studies (Palchykova et al., 2006; Patti et al., 2010).

2.3. Animal models for memory evaluation

2.3.1. Contextual fear conditioning (CFC)

On theﬁrst day (training session), mice were individually placed

in a dark chamber with a gridﬂoor (22×22×22 cm). After 150 s,

0.4 mA foot shocks lasting 1 s were applied every 30 s for the subse-quent 150 s. Thirty seconds after the last foot shock, the animal was removed from the apparatus.

The CFC test was performed 24 h after the training. Each animal was placed in the same dark chamber, without receiving foot shocks. The freezing duration (defined as complete immobility of the animal, with the absence of vibrissae movements and sniffing) was quantified by observers blind to experimental conditions during a 5 min period.

2.3.2. Passive avoidance task (PAT)

PAT was conducted using methods described byDenti and Epstein (1972). A 2-way shuttle box with a guillotine door placed between the 2 modular testing chambers served as the testing apparatus. One chamber was illuminated by a 40-W light, while the other remained dark. During the conditioning session, the animals were in-dividually placed in the illuminated chamber facing away from the guillotine door. When the mouse entered the dark chamber, the door was quietly lowered and a 0.4 mA foot shock was applied for

1 s through the grid ﬂoor. A test session was performed 10 days

after conditioning using the same procedures, expect for the foot shock. In both sessions (training and testing), the latency to enter the dark chamber was recorded, with a cut-off duration of 300 s.

2.3.3. Plus-maze discriminative avoidance task (PM-DAT)

A modiﬁed elevated plus-maze was used to conduct PM-DAT. The maze was made of wood and consisted of two enclosed arms, with sidewalls and no top (28.5×7×18.5 cm), and 2 open arms with no sidewalls (28.5×7 cm), on opposite sides. A non-illuminated 100-W lamp was placed over the exact center of one of the enclosed arms (aversive enclosed arm). In the training session, each mouse was placed at the center of the apparatus, and, during a 10-min period, every time the animal entered the enclosed arm containing the lamp, an aversive stimulus consisting of the illumination of the 100-W light and a cold wind produced by a hair dryer was administered. The aversive stimulus continued until the animal left the arm. A test session was performed in the same room 10 days after the training session. During the test session, mice were again placed in the center of the ap-paratus and were observed for 3 min; however, the mice did not receive the aversive stimuli when they entered the aversive enclosed arm (al-though the non-illuminated lamp and the hair dryer were still placed in the middle of this arm to help distinguish between the aversive and non-aversive arms).

Total number of entries, deﬁned as the entry of all 4 paws into the arm, in any of the arms, percent time spent in the aversive enclosed arm (time spent in aversive enclosed arm/time spent in both enclosed arms×100), and percent time spent in open arms (time spent in open arms/time spent in both open and enclosed arms×100) were calculated. Learning was evaluated by the de-crease in percent time spent in the aversive enclosed arm through-out the training session. Memory was evaluated by the time spent in the aversive vs. non-aversive enclosed arms in the test session. Percent time spent in the aversive enclosed arm among the groups that showed retention of the task in the test session was compared in order to reveal quantitative differences. Anxiety-like behavior was evaluated by the percent time spent in the open arms of the apparatus. Total number of entries in any of the arms was used to evaluate motor activity. All the measures taken during the PM-DAT were obtained manually by observers blind to experimental conditions.

2.4. Experimental design

2.4.1. Experiments I, II and III: effects of TSD on memory retrieval in mice: inﬂuence of sex

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2.5. Statistical analysis

For the CFC experiment, freezing duration was compared by 2-way ANOVA followed by Duncan's test in the test session. Data were evaluated for baseline sex differences in the PAT and PM-DAT tests using independent-sample t-tests. Latency to enter the dark chamber (PAT) during the test session, total number of entries in any of the arms (PM-DAT), percent time spent in the open arms (PM-DAT), and percent time spent in the aversive enclosed arm (PM-DAT) during the test session were evaluated using 2-way ANO-VAs followed by Duncan's test for effects of TSD, sex, or interactions between the 2 factors. In addition, the decrease in percent time spent in the aversive enclosed arm throughout the training session (PM-DAT) was compared by ANOVA with repeated measures. 2-way ANOVA followed by Duncan's test with sex (M×F) as a between-subject factor and arm type (aversive vs. non-aversive) as a within subject factor was used to analyze time spent in both enclosed arms in the training session, and a 3-way ANOVA was used in the test session to analyze the same parameter. A probability of pb0.05 was considered

signiﬁcant for all comparisons made.

3. Results

3.1. Experiment I: effects of TSD on memory retrieval in mice subjected to the CFC task

In the test session, 2-way ANOVA showed signiﬁcant effects for condition (CTRL or TSD) [F(1,47)=12.49; p=0.001] but not for sex (M or F) [F(1,47)=0.31; p=0.58] or for the interaction, condi-tion× sex [F(1,47)=0.01; p=0.93], indicating that animals that were sleep-deprived, regardless of sex (M-TSD and F-TSD groups), presented a signiﬁcant decrease in freezing duration when com-pared to the respective CTRL groups (M-CTRL and F-CTRL), indicat-ing retrieval impairment (Fig. 1).

3.2. Experiment II: effects of TSD on memory retrieval in mice subjected to the PAT

In the training session, the t-test for independent samples did not show signiﬁcant differences between the sexes in the latency to enter the dark chamber [t (46)=0.66; p=0.51] (Fig. 2A).

In the test session, 2-way ANOVA showed signiﬁcant condition (CTRL or TSD) effects [F(1,44)=10.61; p=0.002] but not signiﬁcant sex (M or F) [F(1,44)=2.42; p =0.13] or interaction condition×sex effects [F(1,44)=0.05; p=0.82], indicating that animals that were

sleep-deprived (M-TSD and F-TSD groups) showed a signiﬁcant de-crease in latency in entering the dark chamber when compared to the CTRL groups (M-CTRL and F-CTRL), indicating impaired memory retrieval (Fig. 2B).

3.3. Experiment III: effects of TSD on memory retrieval in mice subjected to the PM-DAT

In the training session, 2-way ANOVA revealed a signiﬁcant effect

of arm type (aversive×non-aversive) [F(1,96)=195.41; pb0.001]

but not of sex [F(1,96)=0.08; p=0.78] or the interaction arm type×sex [F(1,96)=3.11; p= 0.08]. Post hoc Duncan analysis revealed that both male and female mice spent significantly less time in the aversive enclosed arm than in the non-aversive enclosed arm during the training session (Fig. 3A), demonstrating the effec-tiveness of the aversive stimuli. ANOVA for the percent time spent in the aversive arm with sex as a between-subject factor and time (minutes of observation) as a repeated measure factor revealed sig-nificant effects of time [F(9,432)=3.10; p=0.001] and a trend to-wards sex significant effect [F(1,48)=3.42; p =0.07] (Fig. 3B) but not significant effects of the interaction time×sex [F(9,432)=0.25; p=0.99]. In this session, t-test for independent samples showed that the female mice showed greater total percent time spent in the aversive enclosed arm when compared to male mice [t (48)=2.03; p=0.04] (Fig. 3C).

In the test session, 3-way ANOVA revealed signiﬁcant effects of arm type [F(1,92)=37.60; pb0.001], sex×arm type [F(1,92)=5.88;

p=0.02], and arm type×condition [F(1,92)=5.29; p=0.02] interac-tions but did not reveal signiﬁcant effects of sex [F(1,92)=1.6;

p=0.21], condition [F(1,92)=0.06; p=0.81], sex×condition

[F(1,92)=0.46; p=0.5], or arm type×sex×condition [F(1,92)=0.65; p=0.42] interactions. Post hoc Duncan analysis revealed that male Fig. 1.Effects of total sleep deprivation (TSD) on the retrieval of the contextual fear

conditioning (CFC) task in male (M) or female (F) mice. Male or female mice were trained in the CFC task. 24 h after training, mice were kept in their home cages (CTRL) or subjected to total sleep deprivation (TSD) for 6 h immediately before testing, forming the groups: M-CTRL (n =13); F-CTRL (n =13), M-TSD (n =12) and F-TSD (n =13). Results are presented as the mean±SE freezing duration, in seconds, in the test session.#_{pb0.05 compared to the respective control groups (M-CTRL and F-CTRL,}

2-way ANOVA and Duncan's test).

Fig. 2.Effects of total sleep deprivation (TSD) on retrieval of the passive avoidance task (PAT) in male (M) or female (F) mice. Male or female mice were trained in the PAT. 10 days after training, mice were kept in their home cages (CTRL) or subjected to total sleep deprivation (TSD) for 6 h immediately before testing, forming the groups: M-CTRL (n =12); F-CTRL (n =12), M-TSD (n =12) and F-TSD (n =12). Results are presented as the mean±SE latency to enter the dark chamber, in seconds, in the train-ing (A) and in the test sessions (B).#_pb_{0.05 compared to the respective control groups}

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and female mice kept in CTRL condition spent signiﬁcantly less time in the aversive than in the non-aversive enclosed arm. However, sleep-deprived females (F-TSD) showed similar time spent in both enclosed arms (Fig. 4A), indicating that TSD induced deleterious effects on the re-trieval. Furthermore, comparing the groups that showed retention of the task (M-CTRL, M-TSD and F-CTRL groups), the ANOVA followed by Duncan's test revealed that the F-CTRL and the M-TSD groups presented a higher percent time spent in the aversive enclosed arm when com-pared to the M-CTRL, demonstrating retrieval impairment [F(2,34)= 6.03; p=0.006] (Fig. 5B).

The t-test for independent samples did not reveal signiﬁcant diffe-rences between sexes [t (48)=0.16; p=0.87] for the percent time spent in the open arms during the training session, indicating no sex differences in anxiety. However, the female mice showed an increase in the total number of entries [t (48)=3.73; p=0.001] in this session (Fig. 5A and B, respectively).

A 2-way ANOVA did not reveal significant differences among groups during the test session on the factors of sex [F(1,46)= 0.54; p=0.46], condition [F(1,46)=0.02; p =0.90], or the interaction sex×condition [F(1,46)=0.51; p=0.48] (Fig. 5C). A 2-way ANOVA for the total number of entries revealed significant sex effects [F(1,46)=8.63; p=0.005] but not significant effects of condition [F(1,46)=1.12; p=0.29] or a sex×condition [F(1,46)=0.04; p=0.84] interaction. Duncan's test revealed that female (CTRL and TSD groups) presented higher number of entries when compared to

their respective sex control groups (M-CTRL and M-TSD groups) (Fig. 5D).

4. Discussion

The majorfinding of our study is that TSD negatively modulated emotional memory retrieval in both male and female mice. Impor-tantly, even though TSD induced retrieval impairment with the same magnitude in CFC task and PAT, female mice were more vulne-rable to the deleterious effects of TSD as assessed by PM-DAT. These findings extend our recentfindings on the negative effects of TSD on memory retrieval in male mice (Patti et al., 2010), and are the first report that TSD impacts emotional memory differently according to sex.

The CFC task is a classical conditioning paradigm widely used to investigate brain mechanisms underlying learning and memory (Fendt and Fanselow, 1999; Kim and Fanselow, 1992; Maren et al., 1996) and a substantial literature supports the notion that sleep dep-rivation impairs conditioning fear consolidation (Graves et al., 2003; Hagewoud et al., 2010; Tiba et al., 2008). We veriﬁed the effects of pre-test TSD on both sexes' retrieval of this task and found that, TSD equally decreased the freezing response in both sexes.

We have previously shown that pre-test TSD impaired memory retrieval in the PAT in male mice (Patti et al., 2010). We found no diffe-rences between sexes in the latency to enter the dark chamber of the PAT apparatus during the training session. This may suggest that there Fig. 3.Training performance of male (n =25) or female (n =25) mice subjected to the

plus-maze discriminative avoidance task (PM-DAT). Results are presented as the mean ±SE of time spent in the aversive and non-aversive enclosed arms (A), percent time spent in the aversive enclosed arm minute by minute throughout the training session (B), and percent time spent in the aversive enclosed arm in the training session as a whole (C). *pb0.05 compared to time spent in the non-aversive enclosed arm (2-way ANOVA and Duncan's test);+_{pb0.05 compared to male mice (t-test for independent samples).}

Fig. 4.Effects of total sleep deprivation (TSD) on retrieval of the plus-maze discrimina-tive avoidance task (PM-DAT) in male (M) or female (F) mice. Mice were kept in their home cages (CTRL) or subjected to total sleep deprivation (TSD) for 6 h immediately before the 3-min test session, performed 10 days after training. Groups were: M-CTRL (n=12); F-CTRL (n=12), M-TSD (n=13) and F-TSD (n=13). Results are presented as the mean±SE of time spent in the aversive and non-aversive enclosed arms (A) and percent time spent in the aversive enclosed arm (B) in the test session. *pb0.05 compared to time spent in the non-aversive enclosed arm (2-way ANOVA and Duncan's test).●

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were no basal differences between sexes in the acquisition of the task; however, the PAT does not allow evaluation of learning (opposed to the PM-DAT). Pre-test TSD resulted in clear memory retrieval deﬁcits for both sexes in this task.

Both the CFC task and the PAT are widely employed in memory ﬁeld investigation nevertheless they can be markedly inﬂuenced by other behavioral factors, such as anxiety and motor activity; however, these factors are not explicitly measured during the tasks. Therefore, we examined the effects of TSD using the PM-DAT, which allows con-current but independent evaluation of learning, memory, motor ac-tivity, and innate fear response (to the open arms) (Gulick and Gould, 2011; Ribeiro et al., 2011; Silva and Frussa-Filho, 2000) in order to better understand the possible interaction of sex with sleep deprivation and memory. Furthermore, the PM-DAT may be more sensitive to sex differences, considering that it may have a spatial component in the choice of arm; males may perform better than females, since they often show better spatial learning than females (Baldan Ramsey and Pittenger, 2010; Duvoisin et al., 2010; Ter Horst et al., 2011).

Using the PM-DAT, our group and others have reported that im-paired memory can be identiﬁed as reduced avoidance of the aver-sive enclosed arm in the test session, namely, when the time spent in this arm is the same as that spent in the non-aversive enclosed arm. Alternatively, impaired memory can be demonstrated by an in-crease in the percent time spent in the aversive enclosed arm in the test session, even under conditions in which the animal is able to avoid this arm. This measure is sensitive to many different manipu-lations, including pre-training scopolamine (Claro et al., 1999; Silva et al., 1999), chlordiazepoxide (Silva and Frussa-Filho, 2000), am-phetamine (Silva et al., 2002), and paradoxical sleep deprivation (Alvarenga et al., 2008), among many others. Pre-test TSD induced memory retrieval deﬁcit in both sexes. However, only female mice in the TSD group (F-TSD) failed to discriminate between the 2 arms

during the test session. Furthermore, female mice kept in their home cages already displayed basal discriminative memory impairment com-pared to males (since they displayed increased percent time spent in the aversive enclosed arm; see above), suggesting a synergism between lower basal memory performance and the memory-impairing effects of TSD. Thus, female mice may be more vulnerable than male mice to the deleterious effects of pre-test TSD.

Male mice that were kept in experimental condition (CTRL or TSD) and females in the control condition were capable of discriminating be-tween the 2 enclosed arms (aversive×non-aversive) during the test ses-sion, showing retention of the discriminative task. Among these groups, non-sleep-deprived females (F-CTRL) and sleep-deprived males (M-TSD) performed more poorly than did the male control group (M-CTRL). These results are in line with those which describe poorer per-formance of female rodents in spatial tasks (Berger-Sweeney et al., 1995; Roof and Havens, 1992). However, our results seem to be in contrast to those ofPierard et al. (2011), who found that TSD disrupted contextual but not serial spatial discrimination (such as those tested in the current task). It could be due to the nature of the memory models. Speciﬁcally, whilePierard et al. (2011) employed a non-reinforced task (4-hole board), we used 3 tasks involving aversive components, restraining our ﬁndings to emotional memories.

In the present study, TSD negatively modulated retrieval of both CFC and PAT in a similar manner. In the PM-DAT, female mice already presented a decrement in performance when compared to male ani-mals. This decreased performance was exacerbated by pre-test TSD. In this sense, a prolonged period of TSD could lead to a poorer perfor-mance of female mice in CFC and PAT as well. The investigation of the cognitive effects of prolonged TSD periods warrants further investigation. In the PM-DAT, learning is evaluated by the progressive decrease in the exploration of the aversive enclosed arm where light and air puffs are presented (Frussa-Filho et al., 2010). This measure is sensi-tive to manipulations that produce deﬁcits in learning in humans Fig. 5.Effects of total sleep deprivation (TSD) on retrieval of the plus-maze discriminative avoidance task (PM-DAT) in male (M) or female (F) mice. Male or female mice were trained in the PM-DAT. 10 days after training, mice were kept in their home cages (CTRL) or subjected to total sleep deprivation (TSD) for 6 h immediately before the 3-min test session. Groups were: M-CTRL (n =12); F-CTRL (n =12), M-TSD (n =13) and F-TSD (n =13). Results are presented as the mean ±SE of percent time spent in the open arms in the training (A) and test sessions (C), and total number of entries in all arms of the apparatus in the training (B) and test (D) sessions.+_pb_{0.05 compared to male mice}

(t-test for independent samples);▲

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(e.g.,Alvarenga et al., 2008c.f.Dinges et al., 1997). Within this con-text, female mice displayed a higher exploration of the aversive enclosed arm during the training session, suggesting an acquisition deﬁcit in the discriminative avoidance task when compared to male mice. This in agreement with previous studies suggesting that male and female rodents show differential learning, depending on condi-tions (Ramsey and Pittenger, 2010). For example, many studies sug-gest that males perform better in tests of spatial memory than females (LaBuda et al., 2002).

Female presented an enhanced spontaneous locomotion when compared to male mice in the PM-DAT. It is possible that such hyper-locomotion could have contributed, at least in part, to the observed learning impairment. Nevertheless, there was no signiﬁcant correlation between the total number of entries (locomotion parameter) and per-cent time spent in the aversive enclosed arm entries (indicative of learning) in the training session (Pearson's correlation=−0.15; p=0.48) which suggest that the observed learning impairment was not due to the spontaneous hyperlocomotion presented by females. However, the hyperlocomotion shown by female animals in our study is in agreement with previous studies that showed that females had higher baseline activity when compared to male mice.

Another important issue is the inﬂuence of the estrous cycle phase on females' behavior. Estradiol has been demonstrated to impact

hip-pocampal long-term potentiation (LTP)– a molecular mechanism

proposed for learning and memory–via activation of the estrogen re-ceptorβ(ERβ) (Liu et al., 2008). Thus, theﬂuctuation of estradiol

levels during the estrous cycle could interfere with learning and

memory processes. However,Cardoso et al. (2010) demonstrated

that female rats which were ovariectomized with or without estradiol treatment for either 7 or 21 days presented a similar pattern of explo-ration of the enclosed arms in the PM-DAT when compared to control females in proestrus, indicating similar levels of memory retention. Thus, estrogen itself is unlikely to explain all of the sex differences in memory formation processes. In addition, there are male-speciﬁc signaling processes that contribute to memory formation, such as dif-ferences in the plasticity mechanisms of memory formation. These differences may result from direct effects of sex hormones but could also result from differences in sex chromosome gene expression in the brain (Mizuno and Giese, 2010). Taking into account the studies cited above, in the present study we broadly evaluated the effects of TSD on females irrespective of their estrous cycle phase, although such effects cannot be discounted. Therefore, an evaluation of the ef-fects of TSD on memory during each estrous cycle phase would be an interesting future study.

5. Conclusion

To our knowledge, this is theﬁrst study reporting the effects of TSD on memory retrieval in both sexes in mice. Furthermore, our re-sults agree across three different memory tasks (CFC, PAT, and PM-DAT), except for the basal retention deﬁcit presented by female mice kept in their home cages during the PM-DAT (F-CTRL group). This discrepancy could be due to the qualitative difference between the aversive stimuli applied in these tasks, as has been shown follow-ing ethanol administration (Kameda et al., 2007). Collectively, our re-sults demonstrate that TSD impaired emotional memory retrieval in different memory tasks both in males and females. Of note, female mice seem to be more vulnerable to the cognitive deleterious effects of TSD in a discriminative avoidance task. Additionally, the emotional memory impairment does not seem to be related to changes in other behaviors, such as anxiety and motor activity.

Disclosure statement

All authors disclose any actual or potential conﬂict of interest including any ﬁnancial, personal or other relationships with other

people or organizations within three years of beginning the work that could inappropriately inﬂuence (bias) the present work. All au-thors critically reviewed the content and approvedﬁnal version for publication.

Contributors

LFS, CLP, KAZ, HAF and RF-F were responsible for the study con-cept and design. LFS, CLP, KAZ and HAF contributed to the acquisition of animal data. LFS, CLP, KAZ, MLA and RF-F assisted with data analy-sis and interpretation ofﬁndings. CLP, KAZ, MLA and RF-F drafted the manuscript. ST provided critical revision of the manuscript for impor-tant intellectual content.

Acknowledgments

This research was supported by fellowships from Fundação de Amparo a Pesquisa do Estado de São Paulo (FAPESP/CEPID/SLEEP #1998/14303-3), Conselho Nacional de Desenvolvimento Cientíﬁco e Tecnológico (CNPq), Fundação Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) and Associação Fundo de Pesquisa à Pesquisa (AFIP). The funding resources had no involvement in the study concept, writing the report or in the decision to submit the paper for publication. The English language in this manuscript has been proofread and edited by Dr. Eileen K. Sawyer, but we are entirely responsible for the scientiﬁc content of the paper. The authors would like to thank Ms. Teotila R. R. Amaral, Ms. Claudenice M. Santos, Mr. Cleomar S. Ferreira and Mr. Antonio Rodrigues dos Santos Ferreira for capable technical assistance. S.T and R.F.F. are recipients of the CNPq fellowship.

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