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Abstract

Acute stress has been shown to have facilitating effects on memory tasks while chronic stress can enhance the development of psychiatric disorders such as depression and anxiety disorders. It is currently unknown how much stress is required to create these debilitating effects. This study is the first step in examining the time-course of the effects of stress in rodents. Anxiety-like behavior in the Elevated Plus Maze (EPM) and fear memory in Pavlovian conditioning using cued fear conditioning were examined after one or seven days of restraint stress. After seven days of restraint stress there was a decrease in anxiety-like behavior in the EPM, compared to rats exposed to a single session of stress. These results suggest the importance of the time delay between stress and behavioral testing. No significant results were seen in cued fear conditioning. To assess whether this effect is similar in males and females, anxiety-like behavior was measured in female rats after one day of restraint stress. No difference was found between female control and stress rats. These results suggest that females were not more vulnerable to the effects a single restraint stress on the EPM.

Introduction

Depression and anxiety disorders are extremely common in the general population. Lifetime prevalence of the two disorders has been estimated at 20.8% and 28.8% respectively (Kessler, Bergund, Demler, Jin, Merikangas, & Walters, 2005). The 12-month prevalence for anxiety disorders was found to be 18.1%, while depression was less common at 9.5% (Kessler et al., 2005). It is clear that these disorders are quite common in the general population, yet there is much to be learned about the causes of these disorders. Symptoms of depression include depressed mood, anhedonia, altered appetite, nervousness, and irritability (Gregus, Wintink, Davis, & Kalynchuck, 2005). These psychopathologies develop by a complex interaction between genetic predisposition and an adverse environment (Frank et al. 2006 and Pryce et al., 2005). 

Stressful, or adverse, life experiences enhance the development of these affective disorders (Pryce et al., 2005, Heim & Nemeroff, 2001).  Stress can be real or perceived and is defined as any threat to the homeostasis of an organism (Morilak et al., 2005). This threat can be either physical or psychosocial in nature. In humans, a variety of stressors are experienced daily. Stress can be chronic or acute. Chronic stress is a persistent, long lasting stressor, such as living with a terrible roommate. Acute stress, on the other hand, is a short lasting and one time stressor, such as failing an exam. In addition, stress can be major or minor. This difference will affect the impact that a certain stressor has on an individual. A major stressor would be something like a death in the family, while a minor stressor may be as simple as getting stuck in traffic. The response to a stressor depends on how the individual assesses their environment and the stressor they experience (Kring, Davison, Neale, & Johnson, 2007).

Stress affects the brain both physiologically and chemically (McEwen 2008). When a person decides that a stressor exceeds their ability to handle the situation, homeostasis is thrown off. Allostasis is the term used to refer to how the body responds to stress in order to maintain homeostasis. Allostatic load is the wear and tear produced from the effects of too much stress. As stress increases, wear and tear on the body also increases, producing the behavioral, physiological, and chemical changes in the brain (Figure 1; adapted from Figure 1. McEwen, 2008).

Treatment of psychiatric disorders is aimed at eliminating the debilitating symptoms rather than the cause of the disorder. Common treatments for depression include tricyclic antidepressants, monoamine oxidase inhibitors, and selective serotonin re-uptake inhibitors (SSRIs). Anxiety disorders are often treated with therapy such as cognitive behavioral therapy or SSRIs. Antidepressants may be used in severe cases of anxiety disorders (Kring et al., 2007). Current somatic treatments have numerous side effects such as, memory lapses, difficulty driving, jitteriness, weight gain, and even an interference with sexual functioning (Kring et al., 2007). In response to these side effects, many patients stop taking the medication and therefore relapse. A better understanding the effects of stress on behavior and brain physiology will allow us to determine the factors that lead to these disorders and will lead to the development of more effective treatment (Heim & Nemeroff, 2001).

The effects of stress can be studied using animal models. These models are able to provide useful insights into the behavioral and physiological mechanisms involved in the stress response. The behavioral effects of stress can be studied in rodent models of behavior such as anxiety-like behavior in the elevated plus-maze and fear memory in Pavlovian conditioning. Although useful, these models are not perfect and some inconsistencies exist. As with humans, different stressors in animal models have different effects. In order to eliminate these inconsistencies, further studies are needed. Because of the wide range of effects of stress, behavioral changes as well as physiological and neurobiological changes must be explored (Pryce et al 2005).

Rodent Models of Stress
As with humans, a laboratory rodent can experience many different stressors. Most stressors in humans have both physical and psychological demands and therefore responses to both types of stressors should be examined. Commonly used protocols consist of either physical or psychological stressors. Physical stressors consist of restraint stress, electric footshock, cold swim, or exposure to high intensity noise. Psychological stressors are more social in nature and often consist of social isolation, resident/intruder, maternal separation, or sleep deprivation. Different stressors are selected in an experiment based upon the way previous studies show that they activate the different neurobiological systems involved in the stress response. In addition to the type of stressor used, a non-stress baseline should be achieved in control and experimental animals.

Restraint stress, or immobilization is commonly used because it is less severe than other physical stressors,such as a footshock, but is still capable of activating the stress response.

Figure 1: Individual differences in stress. Stress does not affect each individual the same way. A stimulus that may be stressful to one individual may not be stressful to another. Environment, life events, and genetics play a role in an individual’s tolerance for stress. When an individual perceives a stimulus as stressful a physiological and behavioral response will be displayed.

In this type of stressor, movement is limited by placement in a plexiglass chamber or immobilization bag. Electric footshock is more severe and can be applied using a metal electric grid to shock the foot or applied to the tail. Forced swim at a cold water temperature is another physical stressor. This type of stressor requires physical exertion in order to prevent a passive coping strategy. High intensity noise exposure is also used as a physical stressor. This protocol can be used as a type of environmental stressor to mimic stress in everyday life (Heinrichs & Koob 2005).

Psychological stressors can also be used in the laboratory and are often used to study developmental factors. Social isolation is a common psychological stressor, in which subject is placed in long-term solitary housing. The resident intruder paradigm is a social conflict stress in which an intruder rat is placed in the home cage of a larger territorial rat. Maternal separation is a useful stress model because it mimics an adverse childhood in humans that is commonly associated with the expression of psychiatric disorders later in life (Heinrichs & Koob, 2005). This stressor involves the removal of a pup from the care of its mother for a certain period of time. Sleep deprivation can be used to elicit a stress response as well. This type of stressor consists of denying the subject any opportunity to sleep by placement on a rotating drum. This type of stressor is controversial because it is quite severe (Heinrichs & Koob, 2005).

Stress and the Brain
The endocrine stress response begins with activation of the hypothalamic-pituitary-adrenal axis (HPA axis). Neurons in the hypothalamus release corticotropin-releasing hormone (CRH), which then travels to the anterior pituitary gland, stimulating the release of adrenocorticotropic hormone (ACTH). This hormone then travels through the blood to the adrenal cortex to release glucocorticoids. Corticosterone is released from the adrenal cortex in rats, while cortisol is released in humans (see Figure 2). Glucocorticoids are anti-inflammatory and important for maintaining homeostasis. Cortisol leads to beneficial short-term responses. Long-term exposure to cortisol, however, can cause damage to the hippocampus and is associated with many different psychiatric disorders (Kring et al., 2007). During times of stress the activity of the HPA axis increases resulting in higher glucocorticoid levels, as seen in depression. Several brain regions involved in the stress response, including the amygdala and hippocampus, modify activity of the HPA axis.

The amygdala is important for HPA axis activity. Located just anterior to the hippocampus, the amygdala is activated during fear conditioning as well as in response to emotionally negative pictures, odors and tastes in humans (Shin & Liberzon, 2009). The amygdala is also activated during the coding and retrieval of emotional stimuli (Shin & Liberzon, 2009). Three different regions make up the anatomy of the amygdala. The central amygdala is known to contain CRH neurons (Heim & Nemeroff, 2001; Mikics, 2008) and has extensive connections with the bed nucleus of the stria terminalis (BNST), which then projects to the paraventricular nucleus of the hypothalamus (PVN) and brainstem (Purves et al., 2008). This nucleus of the amygdala is also noted for its relation to the expression of fear. Activation in this area increases after traumatic experiences (Mikics, 2008). The basolateral amygdala (BLA) has been shown to be important during fear conditioning as well. Lesion of the BLA or inactivation with muscimol in rats eliminated fear conditioning (Conrad, Macmillan, Tsekhanov, Wright, Baran, & Fuchs, 2004). The BLA receives inputs from the hippocampal formation as well as the thalamus and sensory cortical areas. The BLA also sends projections to the central amygdala (CeA) and the prefrontal cortex (PFC) (Correll, Rosenkranz, & Grace 2005; Rasia-Filho, 1999).  In this way, the BLA is able to associate fear with various sensory information. In contrast, the medial amygdala has not been found to be important for fear conditioning. The amygdala has strong connections with the olfactory bulb and piriform cortex. The medial amygdala likely plays a role in social behavior and processes related to social learning and memory (Fekete, Zhao, Sabino, Vale, & Zorrilla, 2009). Frank et al. (2006) suggest that the medial amygdala also plays a role in aggression.

The hippocampus and PFC have also been noted for their role in the stress response. The hippocampal formation is known to play a role in the encoding and consolidation of declarative memory. Interestingly, the size of the hippocampus in London cab drivers was found to be larger than age-matched controls. Additionally, this size increase correlated positively with the time spent driving the cab (Maguire et al., 2001, as cited in Purves et al., 2008). The hippocampus is sensitive to stressful experiences (Shors, 2006), but changes in the hippocampus following stress are often reversible (McEwen, 2008). An important function of the hippocampus is its regulation of the negative feedback system in the HPA axis (Gregus et al., 2005). High levels of glucocorticoids in the hippocampus lead to downregulation of receptors, which inhibits the ability of the hippocampus to regulate the HPA axis (Gregus et al., 2005).

The role of the PFC has also been studied because of the human abilities of avoidance and cognition (Shin & Liberzon, 2009). The PFC is essential for a higher processing of stressful and emotional stimuli. A stressful stimulus for one individual may not be stressful for another. This inter-individual difference is dependent upon the PFC, more specifically the medial Prefrontal Cortex (mPFC) (Quirk, Likhtik, Pelletler, & Pare, 2003).  Correll et al., (2005) found that lesions of the PFC enhanced the response to a train of footshocks, an acute stressor, suggesting a regulatory function of the PFC over the central amygdala after acute stress. In addition, this response was diminished after exposure to chronic cold stress, suggesting the regulatory function of the PFC decreases after chronic stress (Correll et al. 2005).

Effects of Stress on Behavior
Stress has a wide arrange of effects on behavior. These effects vary by age and gender. Various models of stresshave been shown to have different effects on behavior. In humans, stress has been shown to enhance Pavlovian conditioning of emotional stimuli, such as that seen in Post Traumatic Stress Disorder (PTSD).

Figure 2: Hypothalamic-Pituitary-Adrenal Axis (HPA). When an individual perceives a stimulus as stressful a physiological response is displayed in the form of the HPA axis. CRH is released from the PVN, which travels to the anterior pituitary gland. From here ACTH is released into the blood stream and reaches the adrenal cortex. Glucocorticoid release is stimulated from the adrenal cortex and travels to the brain. Glucocorticoid receptors can be found in the hippocampus which when activated stimulate inhibitory control to the hypothalamus as a negative feedback system. CRH = Corticotropin-releasing hormone, ACTH = Adrenocorticotropic hormone.

These patients also exhibit an inability to recall old information but the ability to acquire new information remains intact (Shors, 2006). These types of behaviors, as well as those observed in depression and anxiety disorders, have been studied in numerous animal models.

Anxiety-like behavior in rats is most commonly measured by the open field test or the elevated plus maze (EPM). The open field test measures anxiety by comparing the time spent in the middle of the arena to the time spent close to the walls. The floor of the open field test is divided into zones, with exploration of the inner zones, or those that are in the center of the testing field away from the walls, correlated with less anxiety. Increased anxiety has been shown in rats that underwent one session of footshocks for each of 5 consecutive days. These rats spent less time in the inner zones of the open field test and had fewer zones crossed (Daniels, de Klerk Uys, van Vuuren, & Stein, 2008). They also exhibited increased grooming behavior, which may be a possible coping mechanism in response to stress. Similarly, 10 days of chronic immobilization stress (CIS) increased anxiety-like behavior in the open field test (Vyas & Chattarji, 2004). No effect, however, was observed after social defeat, which results from the resident intruder paradigm mentioned previously (Razzoli, Carboni, Guidi, Gerrard, & Arban, 2007). Chronic unpredictable stress (CUS), which consisted of 10 consecutive days of two alternating forms of stress also failed to produce this effect (Vyas & Chattarji, 2004).

Anxiety-like behavior is also measured using the EPM. Reduced exploration of the open arms of the maze is used as an indicator of anxiety. Anxiogenic factors tend to decrease open arm exploration (Walf and Frye, 2007). Twenty-one consecutive days of CIS has been shown to reduce open arm exploration when compared to unstressed controls (Vyas, Jadhav, & Chattarji, 2006). Social defeat can cause a similar effect in rodents. When combined with social isolation, social defeat produced even more profound effects on anxiety-like behavior (Razzoli et al., 2007). Social isolation is used as to enhance to the effects of social defeat stress. Rats that were housed individually after social defeat spent significantly less time in the open arms of the EPM compared to rats that were housed in pairs after social defeat (Nakayasu & Ishii, 2008). This difference between individually and pair housed rats suggests that a supportive social network may play a possible role in attenuating the effects of stress. Interestingly, studies on adolescent rats have found no difference between stressed rats and non-stressed controls in open arm exploration of the EPM in males, but an increased exploration in female adolescent rats (McCormick, Smith, & Matthews, 2007).

Fear behavior in Pavlovian conditioning is also commonly tested in response to stress. Pavlovian conditioning occurs when a conditioned stimulus, a noise or a context, is paired with an unconditioned stimulus, such as a footshock. Stress has enhancing effects on fear behavior. One 2hr session of restraint stress was enough to enhance freezing time in contextual conditioning when these rats were compared to controls (Cordero, Venero, Kruyt, & Sandi, 2003). This same study found no difference in cued conditioning, which uses an auditory tone as a conditioned stimulus rather than a contextual change. Rats exposed to social defeat also exhibit increased contextual fear (Buwalda et al., 2005).

Fear behavior is also measured after social defeat stress by reexposure to the defeat environment. In this case certain behaviors, other than time freezing in fear conditioning, are measured including risk assessment, defined as a scanning head movement by the rat, as well as grooming, rearing, and social avoidance. After social defeat stress, rats show an increase in risk assessment (Razzoli et al., 2007). Interestingly, the opposite is seen after CIS (Vyas & Chattarji, 2004). Social defeat also led to decreased grooming and rearing (Razzoli et al., 2006, 2007). A decrease in rearing was observed after acute stress by footshock (Daniels et al., 2008). In contrast, the same acute stress caused an increase in grooming behavior. A footshock of .8mA was enough to increase social avoidance even 28 days later in rats (Mikics et al., 2008).

Learning and memory as well as depression-like behavior are also examined in response to stress. Eyeblink conditioning is often used to study learning and memory function. In response to stress, female rats exhibited a decreased conditioned response, while this response increased in males. Control, or non-stressed female rats showed more conditioned responses than males (Hodes & Shors, 2005).  The forced swim test is frequently used to examine depression-like behavior. A rat is placed in a tank of water and allowed to swim for 10-15 minutes. A variety of behaviors are measured, such as time spent struggling and time spent immobile to assess depression. The time spent immobile is representative of helplessness, a typical symptom depression in humans. Corticosterone injections increased the time spent immobile during the forced swim test. This effect was not seen after restraint stress (Gregus et al., 2005).

Together, these results highlight a few inconsistencies in the behavioral effects of stress. Different types of stressors elicit different responses in different models of fear and anxiety. Restraint stress increases anxiety-like behavior in the open field test as well as the EPM, but social defeat only increased anxiety in the EPM, not in the open field test. Also, stress enhanced fear conditioning differently in cued and contextual conditioning. This difference is likely due to the brain regions involved in these models. In order to further explore these differences and to eliminate some of the inconsistencies between stress protocol and behavioral model used, more studies must be conducted.

Additionally, a difference in the effects of stress is highlighted between males and females. Few studies examine the effect of stress on female rats. McCormick et al. (2007), found mixed results in female rats depending on their stage in the estrus cycle. Further studies examining the effects on female rats should be carried out.

Effects of Stress on Brain Anatomy
Neuroimaging studies have shown evidence of consistent changes in humans with certain psychopathologies. Amygdala activity is exaggerated in patients with PTSD (Chung et al.,  1996) as well as panic disorder (van den Heuvel et al., 2005).  In PTSD amygdala activity has been directly correlated with the severity of the disorder (Armony, Corbo, Clement, & Brunet, 2005). Results of neuroimaging studies show that the amygdala is activated in social phobias.  The responsiveness of the amygdala increases during public speaking as well as the anticipation of public speaking (Tillfors, Furmark, Marteinsdottir, & Fredrikson, 2002). Similarly, like PTSD, activity of the amygdala is positively correlated with the severity of anxiety as well as increases in self-reported fear (Tillfors et al., 2001). This effect has also been seen in Generalized Anxiety Disorder. Increased knowledge of how stress affects the amygdala and other brain areas is key to understanding the behaviors associated with these different psychopathologies.

Numerous structural changes have been associated with various brain areas in response to stress. The amygdala undergoes growth during adverse experiences. In response to prolonged immobilization stress, dendrites in the BLA were 45% longer than controls. These lengthened dendrites also had increase of spines spines, which are sites of synaptic input (Vyas et al., 2006). Dendrite lengthening was observed in the BNST, although it did not reach significance. Dendritic arborization was observed in both the BLA and BNST, as evidenced by the number of branch points in dendrites, but not the central amygdala (Vyas et al., 2003, 2006). Chronic cold stress decreased spontaneous firing of CeA neurons, but an increase in firing rate after exposure to a train of footshocks (Correll et al., 2005). Cold stress also increased the responsiveness of BLA neurons (Correll et al., 2005). Additionally, chronic cold stress diminished the regulatory function of the PFC. Vyas et al., (2002) showed that repeated immobilization stress also led to dendritic shortening in the PFC.

In contrast to the amygdala, the hippocampus is reduced in response to stress. A reduction was observed in the volume of the hippocampus in tree shrews in response to stress (Czeh et al., 2001). Stress also reduces the volume of the dentate gyrus of the hippocampus (Gould , Tanapat, McEwen, Flugge, & Fuchs, 1998). This area contains adult stem cells and is a site of neurogenesis (Kozorovitskiy & Gould, 2004). Interestingly, acute stress can enhance the excitability of CA1 pyramidal neurons in the hippocampus (Shors, 2001). It is important to note that these changes are reversible. The hippocampus is noted as one of the most malleable brain regions and changes in this region may not be damage per se, but a form of synaptic plasticity (McEwen, 2008).

Similar to the behavioral effects of stress, physiological and neuroanatomical studies show a few discrepancies in the stress response, most notably, the difference in changes to the hippocampus in response to chronic and acute stress.  Chronic stress reduces hippocampal volume, while acute stress has excitatory effects. This difference raises a very important question about the time-course of the effects of stress. How much stress is needed to reach the changes observed in these chronic stress studies? Currently few studies examine this time-course. These studies focus on the time delay between the onset of stress and the time of behavior testing, rather than the length of stress (Razzoli et al., 2007). Additionally, the time-course study was done using social defeat, which as mentioned previously is a psychological stressor. In order to complete the full picture, a physical stressor should be examined as well. It is imperative that the time-course of stress itself be studied. Few studies examine the effects of a single session of stress, but have many consecutive long sessions of stress. It is possible that single stress sessions can be enough to create a behavioral response.

Neurobiological Changes Due to Stress
Many different mechanisms can be studied as underlying the response to stress. This is largely due to the wide range of effects of stress on behavior as well as brain physiology. Certain neurobiological characteristics are descriptive of patients with different psychopathologies. Depressed patients exhibit hyperactivity of the HPA axis as well as exaggerated responses of ACTH and cortisol (Holsboer, 1999). PET scans of patients with panic disorder elucidate decreased binding in serotonin (5-HT) receptors (Nash et al., 2008) as well as in social phobia (Lanzenberger et al., 2007). Altered binding in 5-HT1a receptors is a possible mechanism behind anxiety disorders, such as social phobia and panic disorder (Spindelegger et al., 2009).

Because of its role as the primary modulator of stress, corticotropin-releasing hormone is a major focus of studies on the biological mechanisms of stress. CRH mRNA has been found in the central (Heim & Nemeroff, 2001), medial, and basolateral amygdala (Fekete et al., 2009). Rats that were separated from their mothers, a form of stress, show increased CRH expression in the central amygdala as well as BNST as adults. In addition, early handling, in which the rats are removed from the cage for several minutes, causes an increase in CRH as well as CRH receptor binding in the amygdala (Cratty, Ward, Johnson, Azzaro, & Birkle 1995). Since CRH causes the release of ACTH, which in turn triggers the release of corticosterone, the response of these hormones to stress is studied extensively. Early isolation, or separation from mother and littermates, for 8hrs increased ACTH and corticosterone levels as well as glucocorticoid mRNA levels (Pryce et al., 2005). A single session of restraint stress elevated corticosterone levels, compared to unrestrained controls, 30 minutes after contextual conditioning training (Cordero et al., 2003). Corticosterone levels remain elevated even 60 minutes after restraint stress (Daniels et al., 2008). Social defeat stress has also been shown to affect corticosterone and ACTH levels in rats. Defeated rats and rats that were faced with the threat of a defeat showed elevated corticosterone and ACTH when reexposed to the defeat environment 21 days later (Razzoli et al., 2007). After social defeat, defeated rats have higher levels of both hormones when compared to controls.

The fos protein can also be used to examine the effects of stress. The gene c-fos codes for the protein and is recognized as an immediate early gene. When a cell is stimulated it begins synthesizing the fos protein which then acts as a transcriptional activator. Neuronal growth factors through the ras pathway as well as protein kinase C can activate the fos protein. Activation of these pathways results in neuronal growth and differentiation. Fos was found to be highly colocalized with CRF mRNA in the medial amygdala. In response to stress, fos expression increases in the forebrain, more specifically in the medial amygdala. It is in this brain region that higher fos expression was observed after social defeat stress (Fekete et al., 2009). Expression of c-fos increased in response to psychosocial encounters regardless of stress or no stress experimental conditions (Mikics et al., 2008). A psychosocial encounter is one in which an opponent rat blocked by a partition or in a plexiglass box is placed in the home cage of the subject. Rats bred for high levels of anxiety-like behavior on the EPM also exhibit increased fos expression after social defeat. Fos expression in these rats increased in the central amygdala, parts of the medial amygdala, and paraventricular nucleus of the hypothalamus (Frank et al., 2006). These data are consistent with evidence of increased dendritic arborization in the amygdala as well as increased levels of glucocorticoids in response to stress.

NMDA receptors became an important area of study in regard to the effects of stress through learning ability in eyeblink conditioning experiments. Deactivation of NMDA during a stressful event prevented an increase in conditioned responses to an air puff, but deactivatio after the event did not (Shors, 2001). This suggests that NMDA receptors are activated during periods of stress. In addition, NR1 hypomorphic mice that have reduced expression of NR1, which is an important subunit of the NMDA receptor, show less aggressive responses when used as residents in the resident-intruder paradigm. These rats also exhibited reduced fos expression in the medial amygdala, BLA, and dentate gyrus (Duncan, Inada, Farrington, Koller, & Moy, 2009). Together, these results suggest that NMDA receptors are important for neuronal activity in response to stress.

Serotonin 5-HT receptors are commonly a target of treatment for many psychopathologies. SSRIs inhibit the reuptake of serotonin in order to increase the amount of serotonin available at the synapse. This reuptake inhibition is done to combat desensitized 5-HT receptors, which is hypothesized to underlie some forms of anxiety. Chronic stress decreases binding to 5-HT1 receptors. Social defeat stress decreases 5-HT receptor sensitivity and also may cause decreased functioning of the receptors in the hippocampus (Buwalda et al., 2005). Studies on early maternal care showed that offspring of mothers that exhibited high licking and grooming, which are measures of good maternal care in rats, had increased turnover in 5-HT activity in the hippocampus. When activated, 5-HT receptors increase the expression of nerve-growth-factor-inducible Factor A (NGFI-A). The gene responsible for glucocorticoid receptors has a binding spot for NGFI-A on exon 1, a promoter region. When NGIF-A binds to this site, expression of glucocorticoid receptors increases in the hippocampus (Meaney & Szyf, 2005). This suggests that serotonin activity in pups can increase a long-term expression of glucocorticoid receptors. One hypothesis is that higher numbers of glucocorticoid receptors can make one more able to deal with the effects of stress and increased levels of glucocorticoids. This is plausible because it will help prevent desensitization of receptors in the hippocampus due to excess glucocorticoids. Amygdalar 5-HT activity is less well studied and has yielded mixed results. Agonists of 5-HT receptors, which increase activity, can be anxiogenic in some tests of anxiety, and anxiolytic in others (Davis, 2000). Interestingly, 5-HT can affect the function of NMDA receptors. Excitatory serotonin receptors (5-HT2a) increase the function of NMDA receptors. On the other hand, inhibitory 5-HT (5-HT1a) has the opposite effect (Bennett, 2010). Given this information, altered 5-HT1a binding in amygdala neurons would decrease the inhibition of NMDA receptors, therefore allowing conditioned fear responses and enhancing the effects of stress. 

Other mechanisms have been examined as well. NK1 receptor antagonists have been shown to decrease anxiety and inhibit fear conditioning. In response to a series of footshocks, NK1 receptor mRNA increased. This increase remained significant 28 days later (Mikics et al., 2008). Neuropeptide Y (NPY) has been shown to have anxiolytic effects on the EPM, although after social defeat stress no change in NPY expression was observed (Panksepp, Burgdorf, Beinfeld, Kroes, & Moskal, 2007).

Current Study
As mentioned previously, behavioral and physiological studies examining the effects of stress have highlighted two very important questions. The first is how much stress is needed to observe the debilitating effects that can lead to certain psychopathologies? The second is how are these effects different in males and females? In order to address these questions two different experiments were carried out. Importantly, it should be noted that the models used in these experiments are not models of psychiatric disorders, but a model of stress. These models of stress target the same regions of the brain that are disrupted in various psychiatric disorders.

Experiment I- Duration of Stress
A restraint stress protocol was used to stress rats for either one or seven days. Restraint stress is a well-accepted model of stress that activates the HPA axis and also elicits behavioral effects. Restraint stress was chosen because it is less harmful to the animals than some other models of stress, such as electric footshock or forced swim. Anxiety-like behavior was examined using the EPM. This model is a good assay of levels of anxiety because it has face, construct, and predictive validity (Walf & Frye 2007). Extensive literature exists using the EPM as an assay of anxiety-like behavior in response to stress.  Fear memory was examined through cued fear conditioning. This assay of fear memory was chosen because it is a well-studied and supported model of fear behavior in rodents.

This study seeks to examine the time-course of the effects of stress. Chronic stress can increase anxiety-like behavior and enhance cued fear conditioning in rodents. In contrast, a single session of stress can have minimal or protective effects. It remains unclear as to how much stress is needed to produce the negative effects of stress.