論文の公開元へThe effect of acute social defeat stress on sleep in mice
概要
Stress response and cost of allostasis
Homeostasis is the dynamic state of equilibrium of physiologic systems such as body temperature, oxygen tension, pH, blood glucose level and ion composition of extracellular fluid, which is essential for life and has to be maintained within a certain range by all living organisms. A stressor is a stimulus that challenges homeostasis, and animals respond with physiologic and behavioral changes in an attempt to maintain or restore homeostasis, which is called stress response (Chrousos, 2009). The psychological aspect of the stressor is known to be of great importance in stress response. Uncontrollability and unpredictability of aversive stimuli are generally associated with severe stress (de Boer et al., 1990; Ferrari et al., 2003; Koolhaas et al., 2011). Thus, stress is considered a state where homeostasis is actually threatened or perceived to be so. The stress response is considered to be aimed at adaptation to the changing natural environment for better survival and ultimately, for better reproduction of the individual in natural environment (Korte et al., 2005).
Stress response includes relatively stereotypic and innate reactions which were acquired through evolutional process (Chrousos, 2009). Central nervous system plays the major role in regulation of the stress response. Arousal and vigilance are promoted in acute stress, and the excitations in hypothalamic-pituitary-adrenal (HPA) axis and sympathetic nervous system typically play major physiologic roles in stress response (Chrousos, 2009). Excitation of HPA axis increases the secretion of glucocorticoids, which exerts pleiotropic action throughout the body (Chrousos and Kino, 2005). The reactions prepare the animal to perform behavior required in urgent situations. For example, elevated levels of catecholamines and glucocorticoids promote energy mobilization through stimulation of glucogenesis, glycogenolysis, lipolysis and hepatic glucose secretion, and increased heart rate and arterial blood pressure by sympathetic activity promote distribution of the nutrients and oxygen to active organs such as skeletal muscles, the heart and the brain (Sapolsky et al., 2000). Moreover, shortly nonadaptive behavioral and physiologic functions, such as eating, growth and reproduction, are prevented (Chrousos, 1998, 2008). The glucocorticoids prevent inflammation, which may hamper behavioral performance, through suppression of proinflammatory cytokine secretions (Boumpas, 1993; Chrousos, 1995), although the effect of stress on immune system is rather complex, and increase of proinflammatory cytokines could also occur after acute stress presumably in preparation for possible injury during stress (Steptoe et al., 2007; Chrousos, 2009).
Although the stress response is ideally beneficial to handle the situation, it also costs animals (Chrousos, 2009). This is well explained in the concept of “allostasis” with clear terminology, while the word “stress” is often used in ambiguous ways (McEwen, 1998; McEwen and Wingfield, 2003). Allostasis is defined as the adaptive process where animals actively achieve stability through physiologic, behavioral and psychological changes, i.e., stress response described above in the context of adaptive process. The state with sustained activity of allostasis is called allostatic state. Animals may get into allostatic state in confrontation with social conflict, weather change, natural disasters, predator emergence, etc. Each animal is considered to have a certain capacity to cope with situations, and allostatic state imposes cumulative cost on the animal, which is called allostatic load. If the physiologic and psychological capacity of an individual is put in danger by the intensity and/or frequency of stressor, allostatic load can expand intensely, leading to allostatic overload. In such case, the individual’s health is in danger. The shortly adaptive alterations in allostasis start to damage itself. For example, prolonged stress increases the risk of cardiovascular diseases such as hypertension, atherosclerosis and coronary heart disease (Melin et al., 1999; Kaplan et al., 2009; Khayyam-Nekouei et al., 2013; Rozanski, 2016) and gastric ulcer formation (Weiner, 1996; Grundy et al., 2006; Deding et al., 2016). In the hippocampus, which is important in spatial and episodic memory and mood regulation, dendritic shrinkage and loss of spines are induced by stress, where glucocorticoids play a major role (McEwen, 1999; Popoli et al., 2012; McEwen et al., 2015). Disruptions also occur in immune system or energy metabolism by glucocorticoids, catecholamines and other mediators (Glaser and Kiecolt-Glaser, 2005; Tamashiro et al., 2011; Hirotsu et al., 2015). Furthermore, the behavior may be altered in maladaptive ways, causing psychological disorders, e.g., depression and post-traumatic stress disorder (Charney and Manji, 2004; Huhman, 2006). Uncontrollability and unpredictability of stressors, to which animals have difficulty in adjusting their behavior, are considered especially important for the development of stress-related disorders (Koolhaas et al., 2011).
It is known that coping styles to stressor are different among individuals even in the same species (Koolhaas et al., 1999, 2007; Korte et al., 2005). Differences are observed not only in behavior but also in physiologic stress response. A certain coping style may be beneficial in certain conditions and can be also disadvantageous in other conditions. In feral populations of rodents, for example, their coping styles were characterized between proactive and reactive coping (Koolhaas et al., 1999). Some male animals tend to show strong intermale aggression, whereas others are not aggressive. The individual difference of aggression level is known to be related to the general coping styles in which they react to various environmental challenges. Aggressive animals usually show proactive coping. Proactive animals easily develop routines, in which the behavior is rigid and stereotypic, responding relatively independently of the environmental stimuli. On the other hand, reactive animals, which are not aggressive, are more flexible in their behavior and sensitive to environmental changes. The difference in their coping styles has been studied in various laboratory settings (Van Oortmerssen et al., 1990; Benus et al., 1991; Koolhaas et al., 1999). For example, in the learning task in a maze to get food as reward in mice, when the position of food was changed in the maze, it was revealed that reactive mice were more flexible in changing their behavior than proactive mice. Contrary, in response to a small change in the maze, such as a small piece of tape in the floor or angle change of the maze relative to outside cues, it is reported that proactive mice did not pay attention to the change, and the performance was not influenced, whereas reactive mice showed exploration on the maze again and took much more time and errors on the task (Van Oortmerssen et al., 1990; Coppens et al., 2010). Thus, a proactive coping style may be beneficial in stable environmental conditions, in which quick behavior based on previous experience is effective. The reactive coping animals may perform better in unstable changing environmental conditions because they are more aware of changes. In addition to the behavioral difference, is also reported that proactive or reactive animals show higher responsivity to stressors in sympathetic activity or HPA axis respectively (Koolhaas et al., 1999). Importantly, the variation in coping style is considered to have been developed through natural selection, in which higher performance in reproduction is considered more successful. Thus, the individual’s health is not necessarily the goal of the stress system (Korte et al., 2005).
Then, how is sleep-wake behavior influenced by stress and involved in the adaptation process?
Social defeat stress models
The effect of stress has been studied in various animal models, including restraint/immobilization stress, electric shock, ether exposure and social defeat (SoD) (Sutanto and de Kloet, 1994). The acute physiologic reactions such as HPA axis and sympathetic activation as well as adverse consequences by sever or chronic stress have been studied (Koolhaas et al., 1997, 2011; Willner, 2005; Nestler and Hyman, 2010). Among these stressors, social conflicts often occur in natural contexts in social animals, and SoD stress has great impacts on their behavior and physiology. SoD stress has been used as ethologically relevant models of stress (Toyoda, 2017).
Social animals such as rodents live in social rank, which is usually decided by winning and losing in social conflicts. The rank is a critical determinant of resource allocation like territory, food, shelter and chance of reproduction. Thus, wining in a conflict can be of great importance. Persistent fighting of an individual with difficulty to dominate its opponent, however, may not be adaptive because injury and death could happen. In addition, it is also suggested that the dominant animals with difficulty in maintaining their social rank have an increased risk in a series of cardiovascular disorders, whereas subordinate animals usually do not (Ely, 1981; Koolhaas et al., 1999; Kaplan et al., 2009). To avoid unpreferable consequences, animals show submissive behavior and accept a lower social rank in which available resources are compromised. In humans, social conflicts are a major source of stress, and exposure to SoD such as loss in aggressive social confrontation, being bullied or being abused, is associated with increased risk in depression, anxiety and post-traumatic stress disorder (Agid et al., 2000; Björkqvist, 2001; Heim and Nemeroff, 2001; Charney and Manji, 2004). Due to this interest, the effect of social conflict of animals has been studied intensively in the losers.
Rodent studies of SoD stress are often conducted in a resident-intruder paradigm. The paradigm is based on the aggressive behavior of a resident animal toward an unfamiliar male intruding in its home cage. It is known that territorial aggression of adult male rats and mice is strongly enhanced in the presence of females and/or sexual experience (Goyens and Noirot, 1975; de Catanzaro, 1981; Albert et al., 1988). Thus, resident animals are generally prepared from breeding colonies and/or kept with females (Golden et al., 2011; Koolhaas et al., 2013). In the paradigm, male intruder animals are put in a male resident’s cage, and eventually subordinate themselves to the territorial resident conspecific with submissive behaviors such as submissive posture, escaping or freezing behavior when attacked and defeated. After the physical SoD, the intruder is often subjected to different durations of psychological stress of olfactory, auditory and visual stimulus through wire mesh or perforated partition which is used to prevent further physical contact. The effects of SoD have been studied in the intruder animals. Acute SoD stress stimulates sympathetic system and HPA axis and increase the heart rate, the body temperature and the blood corticosterone level (Koolhaas et al., 1997; Kataoka et al., 2014). These stress responses dissipate and return to baseline levels within hours after the end of the stress period. SoD stress, however, also induces longer-lasting behavioral and physiologic changes. In rats, for example, locomotor activity in the open field test was decreased for one week or more, and the circadian amplitude of the body temperature was decreased for several days mainly due to increased body temperature during the circadian rest phase after a single SoD (Meerlo et al., 1996a, 1996b). Furthermore, body mass growth was decreased for several days. When SoD was repeated for 2 consecutive days, the decrease in the growth became much stronger with decline of food intake (Meerlo et al., 1996c). These observations indicate that SoD is a big life event in social animals, and even a single SoD can have a great impact on their physiology and behavior. Moreover, social animals are known to develop depression-like behavior in response to chronically repeated SoD stress (Von Frijtag et al., 2000; van Kampen et al., 2002; Rygula et al., 2005; Huhman, 2006). In mice, the procedure of chronic SoD stress has been well established, in which a male C57BL6j mouse (intruder) is socially defeated by an unfamiliar male CD-1 mouse (resident) with larger body size in daily basis for 10 days. Each day, the intruder mice are subjected to psychological sensory stress through perforated partition all day after 5-10 min of physical interaction with a CD-1 resident. After the chronic SoD stress, mice show depression-like behavior including anhedonia, anxiety, and social avoidance (Golden et al., 2011; Henriques-Alves and Queiroz, 2015). The anxiety and social avoidance behaviors can last at least 4 weeks (Berton et al., 2006; Tsankova et al., 2006; Krishnan et al., 2007). These long-lasting behavioral changes in animals that experience repeated SoD stress are commonly associated with alterations in gene expression patterns in the brain (Tsankova et al., 2007).
Function and regulation of sleep
Although stress coping of animals and humans is usually explained based on the state of wakefulness, the brain works with three distinct vigilance states, which are wakefulness, slow wave sleep (SWS) and rapid eye movement sleep (REMS) in mammals. Those states are relatively clearly defined primarily based on the electroencephalogram (EEG) signal. Sleep or sleep-like state is observed not only across animals but also in invertebrate such as drosophila (Hendricks et al., 2000; Shaw et al., 2000). Sleep is considered to be a critical process in which the brain actively maintains its functions, which has long been possessed throughout the evolutional process even with the fact that animals need to stay defenseless and cannot work to gain materials for living during sleep (Rial et al., 2010).
Sleep has been reported to have numerous functions, which may help animals to adjust their physiology and behavior on stress exposure. One of the most well-known function is its role in memory. Sleep has critical roles in memory consolidation. Memory consolidation is a process where initially unstable memories created during the awake state are transformed into more stable representations, which involves the integration into the network of pre-existing long-term memories. In human, for example, brief sleep (3h) after learning made emotional memory still detectable even after 4 years (Wagner et al., 2006). It is well-known that neuronal re-activation occurs during SWS after learning tasks or exploration of a novel environment. The re-activation is observed in a similar spatio- temporal patterns of neuronal firing that occur in brain areas including hippocampus and cerebral cortices (Wilson and McNaughton, 1994; Ji and Wilson, 2007). The re-activation is suggested to be important in the memory consolidation during sleep (Rasch et al., 2007; Girardeau et al., 2009; Bendor and Wilson, 2012). REMS is considered to be important in consolidation or processing of emotional memory ( Wagner et al., 2001; Nishida et al., 2009; Goldstein and Walker, 2014). Recently, the theta rhythm during REMS has been shown to be causally related to the contextual memory consolidation (Boyce et al., 2016). The differential functions of SWS and REMS in memory consolidation, however, is still obscure, and both types of sleep are important in normal memory consolidation (Diekelmann and Born, 2010). It is also reported as a structural evidence that dendritic spine formation in the motor cortex after learning tasks is promoted by sleep (Yang et al., 2014).
In addition to the memory function, sleep has a myriad of physiologic functions. For example, sleep enhances clearance of the metabolites such as beta-amyloid, which deposits in the brain tissue during awake period (Kang et al., 2009; Xie et al., 2013). It has also been hypothesized that sleep plays a role in the regulation of synaptic homeostasis to maintain appropriate cognitive performance by downscaling synaptic strength to the baseline level (Tononi and Cirelli, 2003, 2006). Moreover, sleep also influence the endocrine system. Secretions of growth hormone or glucocorticoids are promoted or inhibited respectively (Takahashi et al., 1968; Weitzman et al., 1983). The immune system and the metabolic system of bodily energy are functioning in close association with sleep-wake cycle, where adequate sleep is critical in optimal functioning (Besedovsky et al., 2012; Morselli et al., 2012). Overall, sleep generally functions to maintain the optimal or regular physiologic state, which is contrastive to the allostatic state with homeostatic deviation during stress.
Animals need to adjust sleep/wake behavior to cope with changing environment. Although the vigilance states involve a brain-wide operational change, the switching and the propensity of those states are regulated by specific neural circuits, which integrate the internal states of the body and the brain as well as the external environmental condition through cognitive processes (Saper et al., 2005, 2010; Weber and Dan, 2016). Circadian rhythm and homeostatic regulation of sleep are the major modulators of the sleep/wake cycle (Borbély et al., 2016). The suprachiasmatic nucleus located in the hypothalamus serves the central pacemaker of circadian rhythm (Wurts and Edgar, 2000; Hastings et al., 2018). The suprachiasmatic nucleus is considered to play the regulatory effect on sleep/wake behavior primarily through dorsomedial hypothalamic nucleus, which innervates brain areas promoting sleep or wakefulness (Chou et al., 2002, 2003). In addition to the direct neuronal regulation of sleep/wake cycle, because diverse physiologic systems work along the circadian rhythm, sleep/wake behavior may also be modulated indirectly, for example, through hormonal or metabolic system (Gnocchi and Bruscalupi, 2017; Hastings et al., 2018). Homeostatic regulation of sleep is based on the fact that after a prolonged wakefulness, animals normally sleep longer with higher intensity (Borbély et al., 2016). High sleep intensity is expressed by enhanced slow wave activity (SWA) of SWS-EEG, which gradually dissipates during the recovery sleep period (Suzuki et al., 2013; Dispersyn et al., 2017; Wang et al., 2018). Homeostatic sleep need is considered to accumulate during wakefulness. The primary mechanism of the sleep need, however, remains still obscure in mammals. The endogenous sleep promoting substances, such as adenosine, prostaglandin D2, interleukin-1 and tumor necrosis factor- α, have been discovered (Feldberg and Sherwood, 1954; Huang et al., 2011; Krueger et al., 2011; Urade and Hayaishi, 2011). Among them, adenosine has been believed to play important roles in homeostatic sleep regulation. Adenosine accumulates during wakefulness in some brain areas such as cholinergic basal forebrain, hippocampus and cortex (Huston et al., 1996; Porkka-Heiskanen et al., 1997, 2000). The adenosine exerts its somnogenic effect through adenosine A1 receptors and adenosine A2A receptors in the central nervous system (Bjorness et al., 2009, 2016; Halassa et al., 2009; Huang et al., 2011). On the other hand, recently, the forward-genetics approach revealed that a dominant mutation in the gene of SIK3, which is a serine-threonine protein kinase, causes constitutively high sleep need (Funato et al., 2016). Further phosphoproteomic analyses indicated that mostly synaptic subset of neuronal proteins in the brain of the mutant mice are hyperphosphorylated (Wang et al., 2018). Importantly, the hyperphosphorylated state is also observed in the sleep-deprived mice, and the SIK inhibitor reduced elevated sleep need. Thus, this mechanism may be the central of cellular and molecular regulation of homeostatic sleep need although the mechanism linking the hyperphosphorylated state and the resultant expression of sleep need remains unknown. In addition to circadian and homeostatic sleep regulation, motivational processes also modulate propensity of sleep/wake behavior. For example, humans can stay awake late at night exceeding the normal time to sleep when motivated to work on cognitive or physical activities. Dopaminergic neurons arising from ventral tegmental area in the midbrain is considered to play an important role in the motivational regulation of sleep (Qu et al., 2010; Eban- Rothschild et al., 2016; Oishi et al., 2017a, 2017c).
Interaction between stress and sleep
To cope with external challenges, animals require alertness, and stress is a series of behavioral and physiologic responses to the challenge. Thus, stress commonly promotes wakefulness and inhibits or disrupts sleep. In fact, we often experience difficulties in falling or staying asleep at night when we are exposed to stressful life events, while the insomnia may be mitigated and resolved when individuals overcome or adjust themselves to stressful situations (Basta et al., 2007; Suchecki et al., 2009; Hirotsu et al., 2015). The transient insomnia may be adaptive for wild animals to survive with the prediction of external threat such as predator’s attack that could occur during the normal sleep period. In humans, however, insomnia is generally undesirable and is one of the most common sleep problems. Chronic insomnia can be triggered by stressful life events in vulnerable individuals and is considered to be the state of hyperarousal, which is led by constant emotional arousal often with “fear of sleeplessness” (Basta et al., 2007; Kalmbach et al., 2018). Insomnia often occur simultaneously with the psychiatric disorders such as anxiety and depression, and disrupted sleep, in turn, contributes to the development and deterioration of the psychiatric disorders (Basta et al., 2007; Meerlo et al., 2008; Medina et al., 2014; Kalmbach et al., 2018; Steiger and Pawlowski, 2019).
On the other hand, after the removal from an acute stress exposure in animal studies, alterations in the sleep architecture are often observed (Suchecki et al., 2009, 2012; Sanford et al., 2015). A sleep rebound may be induced in animals to compensate for the sleep loss during stressful situations, but it is believed that sleep alterations are not only a homeostatic response to the sleep loss but also created by the stress. Some stressors appear to induce even more sleep than the sleep actually lost during stress. The type of sleep that is induced and the extent to which sleep stages are enhanced are highly variable between the types of stress or even the study design. For example, acute immobilization or restraint for 1-2 h is followed by a selective increase in REMS (Rampin et al., 1991; Meerlo et al., 2001b), whereas inescapable footshock stress or learned helpless paradigm may not cause even a compensatory rebound of REMS (Adrien et al., 1991; Sanford et al., 2003a, 2003b, 2003c, 2010). In addition to the physical properties of the stressors, the controllability of the stressor also influences the following sleep responses. For example, an experiment was conducted on yoked pairs of mice experiencing the same amounts of footshock. One of the yoked pairs experienced escapable foodshock, where the mice can terminate the footshock by moving to the safe side of the experimental box, whereas the other experiencing inescapable foodshock cannot stop it by its action. Mice that experienced escapable footshock stress showed an increase in REMS, whereas mice with inescapable footshock stress showed a decrease in REMS (Sanford et al., 2010). On the other hand, strong effect is observed on SWS after acute SoD stress. It is consistently reported that acute SoD stress increases SWA in the following sleep in rodents (Meerlo et al., 1997, 2001a; Meerlo and Turek, 2001; Kamphuis et al., 2015; Henderson et al., 2017). This suggests that SoD stress strongly enhances homeostatic sleep need. Moreover, one study reported that acute SoD stress in mice strongly increased the amount of SWS up to the 12 h period (Meerlo and Turek, 2001). The SWA enhancement was dissipated over the 6h period and SWA became even significantly less during the 12-18 period compared to baseline. This indicates that in this study, the extent of SWS increase in amount was much larger than needed to compensate the homeostatic sleep need that was imposed during SoD. Overall, stress can strongly enhance sleep depending on the stress protocols. Stress costs animals allostatic load. Sleep may function as a recovery process of the deviated homeostasis through its myriad of physiologic effects. The memory consolidation by sleep may help animals handle next challenges in better ways by optimizing their behavior for better survival in the natural environments.
Sleep deprivation is often used to experimentally enhance sleep pressure, which is observed in the following period as enhancement of SWA during SWS and/or increase of the amount of both types of sleep. Using sleep deprivation, investigation has been conducted to elucidate the molecular and neuronal mechanisms of accumulation of homeostatic sleep need and sleep promotion after a prolonged wake period, and some relevant mechanisms have been identified (Vyazovskiy et al., 2009; Huang et al., 2011; Zhang et al., 2015; Gent et al., 2018; Wang et al., 2018). Adenosine has been considered to be important in the recovery sleep, and phosphorylation of a series of neuronal proteins in the brain is suggested to be the central molecular mechanism of the accumulation of the homeostatic sleep need. On the other hand, experience during wake period is known to affects the following sleep, and some stress protocols have strong effects (Suchecki et al., 2009, 2012; Sanford et al., 2015). Especially, SoD stress has been reported to enhance the intensity and/or the amount of the following SWS in rodents (Meerlo et al., 1997, 2001a; Kamphuis et al., 2015; Henderson et al., 2017). However, the mechanism of SWS enhancement by SoD stress has not been investigated, and thus it is unknown whether the mechanism of SWS enhancement is largely similar to that by the sleep deprivation or involves other specific mechanisms. In humans, insomnia is often triggered by stress even though stress itself may create the sleep need, and the disrupted sleep contributes to the exacerbation of psychiatric disorders. The investigation of the sleep homeostasis in the brain during and after stress may help understand the interrelation of sleep disruption and psychiatric disorders.
Differences of the SoD effects on sleep among studies in mice
Although SWA is consistently enhanced during SWS after SoD stress in mice and rats (Meerlo et al., 1997, 2001a; Meerlo and Turek, 2001; Kamphuis et al., 2015; Henderson et al., 2017), the extent of SWS promotion in amount varies between SoD studies and protocols. Male C57BL/6j mice when defeated by aggressive mice of the same strain during a 1-h interaction period showed a strong increase of SWS (Meerlo and Turek, 2001), whereas only a small increase of the SWS amount was observed in another study using the same SoD procedure (Vaanholt et al., 2003). This difference may be explained by variations in the aggressive behavior of the resident mice or prior stress experiences of the intruder mice in the laboratory environment. When highly aggressive male CD-1 mice were used in another study for the SoD of C57BL/6j mice during a 5- min interaction period followed by a 20-min period of olfactory, visual and auditory contact between the resident and intruder mice, a SWS increase in the intruder mice was preceded by an increase of wakefulness (Henderson et al., 2017).
In the previous studies about effects of SoD stress on sleep in mice, one of the biggest concerns in interpreting the results is behavioral variations of the aggressive interaction. The intensity and frequency of attacks can vary in studies depending on the levels of aggressiveness of the residents used and the difference in body sizes between residents and intruders. For example, in the SoD protocol with 1-h interaction with a mouse of the same strain described above, aggressive interactions and attacks may occur continuously or intermittently with periods of non-social interaction. The frequency of the aggression may influence the quality of the stress and lead to different results on the subsequent sleep. On the other hand, male CD-1 mice are often used to defeat C57BL/6j mice. Due to the difference of the strain, it is easy to prepare substantially bigger CD-1 mice relative to experimental C57BL/6j mice. In the protocol, the CD-1 mice are screened by a high level of aggression before use. The duration of physical interaction is usually set to 5-10 min. In this protocol, however, it is generally very difficult or impossible to prevent wounding of C57BL/6j mice during 5-10 min physical interaction period (Golden et al., 2011; Toyoda, 2017). A fixed interaction period likely leads to painful attacks of the CD-1 mouse on the intruder mouse and thus, the sleep-wake behavior of the intruder mice may also be affected by pain. It is also reported that wounding is not a required component in the outcome of chronic SoD stress measured by social avoidance (Krishnan et al., 2007). Thus, psychological nature is considered a significant characteristic of the SoD stress although physical contact is necessary for strong SoD. Overall, the extent to which the SoD procedure (e.g. SoD, pain or sleep deprivation) contributes to the post- SoD stress sleep/wake effects is not well established.
Purpose of the study
The purpose of the study is to establish post-SoD stress sleep/wake effects in a novel mouse model in which physical contact was controlled to minimize the behavioral variation during SoD. I developed a mouse model of SoD stress based on a resident- intruder paradigm to evoke sleep alterations in the intruder C57BL/6j mouse after acute SoD stress by the resident CD-1 mouse trained to display persistent aggression against the intruder mouse. In order to minimize behavioral variations and painful attacks to the submissive intruder mouse, the physical contact was interrupted when the intruder was attacked and showed submissive behavior instead of setting a fixed duration of a physical interaction. After the interruption, the intruder was separated by a partition with wire- mesh opening for psychological stress from the resident mouse. In order to maintain a high level of SoD stress in the intruders during the entire 1-h of SoD session, the partition was removed for physical contacts several times in a session. The effect of SoD stress on subsequent sleep was evaluated. By comparisons with several control procedures, the specific effect of SoD stress was investigated.
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