Long-term effects of footshock and social defeat on anxiety-like behaviours in rats: Relationships to pre-stressor plasma corticosterone concentration

We compared the consequences of two stressors, 'unnatural' inescapable footshocks (IFSs) and 'natural' social defeat (SD), on behaviours typically sensitive to stress [sucrose preference, open field (OF), elevated plus maze (EPM) and acoustic startle responses (ASRs)] and the association with pre-stressor plasma corticosterone concentration. After initial blood sampling, rats (n?=?20 per group) were exposed to either 10 IFSs (1?mA intensity, 5?s duration each) or to 1?h SD (defeat by an aggressive resident male rat and further exposure but separated in a small cage) or to control procedures (handling). Rats were tested once for ASR (day 19), while the other behavioural tests were applied once weekly for 3 weeks. Both stress groups showed short-lasting lowered sucrose preference, and in the EPM they showed shorter total distance moved, shorter distance moved on open arms and less time on open arms compared to controls. In the OF test, IFS rats showed shorter total distance moved up to 2 weeks after stress. The SD group showed shorter total distance moved in the OF, which was only significant 2 weeks after stress. Low pre-stressor plasma corticosterone concentration was only associated with defecation (IFS rats) and latency to enter open arms in the EPM (all low corticosterone subgroups, n?=?10 per subgroup). SD rats with high initial plasma corticosterone concentration showed enhanced ASR compared to the other subgroups with high initial plasma corticosterone concentration (n?=?9 per subgroup). The results indicate that footshock and SD, while generally leading to an increase in anxiety behaviours, represent qualitatively different stressors.     

Effects of Stress on Nonassociative Learning Processes in Male and Female Rats

In this study we assessed habituation and sensitization of the acoustic startle response (ASR) to discern whether intense, inescapable stress affects nonassociative learning differently in male and female rats. Rats were inescapably stressed 2 hours per day over 3 consecutive days. ASR magnitudes were measured at several times post-stress (1, 4, 8, and 15 days after cessation). Females generally showed greater ASR magnitudes (compared to males), but both sexes exhibited short and long-term habituation across the testing days. ASR magnitudes were only affected by stress in male subjects. The effect in males was an increase in short-term sensitization of the ASR on post-stress day-4. The results suggest that stressed males and females react differently to ASR testing, in that stress males appear to develop an exaggerated ASR response over repeated test sessions due to short-term sensitization. The source of the short-term sensitization is discussed with regards to possible stress-induced enhanced contextual learning during ASR testing on post-stress day-1.     

Effects of stress on nonassociative learning processes in male and female rats

In this study we assessed habituation and sensitization of the acoustic startle response (ASR) to discern whether intense, inescapable stress affects nonassociative learning differently in male and female rats. Rats were inescapably stressed 2 hours per day over 3 consecutive days. ASR magnitudes were measured at several times post-stress (1, 4, 8, and 15 days after cessation). Females generally showed greater ASR magnitudes (compared to males), but both sexes exhibited short and long-term habituation across the testing days. ASR magnitudes were only affected by stress in male subjects. The effect in males was an increase in short-term sensitization of the ASR on post-stress day-4. The results suggest that stressed males and females react differently to ASR testing, in that stress males appear to develop an exaggerated ASR response over repeated test sessions due to short-term sensitization. The source of the short-term sensitization is discussed with regards to possible stress-induced enhanced contextual learning during ASR testing on post-stress day-1.     

Effect of prior exposure to a lithium- and an amphetamine-paired flavor on the acoustic startle response in rats

The experiments reported here evaluated the hypothesis that an amphetamine-paired flavor elicits conditioned fear-arousal, whereas a lithium-paired flavor elicits conditioned nausea-disgust by examining the effect of prior flavor exposure on an acoustic startle reaction (ASR). Exposure to a lithium-paired flavor by intraoral infusion, either immediately prior to a startle session (Experiment 1) or during a startle session (Experiments 2 and 3), resulted in a blunted ASR. In contrast, intraoral infusion of an amphetamine-paired flavor resulted in a potentiated ASR. The blunted ASR produced by exposure to a lithium-paired flavor dramatically reversed to a potentiated ASR when rats were pretreated with the antiemetic drug ondansetron prior to the saccharin-lithium pairing (Experiment 3). The findings shed light on a mechanism by which rewarding drugs produce conditioned taste avoidance in rats.     

Acute episodes of predator exposure in conjunction with chronic social instability as an animal model of post-traumatic stress disorder

People who are exposed to horrific, life-threatening experiences are at risk for developing post-traumatic stress disorder (PTSD). Some of the symptoms of PTSD include persistent anxiety, exaggerated startle, cognitive impairments and increased sensitivity to yohimbine, an alpha(2)-adrenergic receptor antagonist. We have taken into account the conditions known to induce PTSD, as well as factors responsible for long-term maintenance of the disorder, to develop an animal model of PTSD. Adult male Sprague-Dawley rats were administered a total of 31 days of psychosocial stress, composed of acute and chronic components. The acute component was a 1-h stress session (immobilization during cat exposure), which occurred on Days 1 and 11. The chronic component was that on all 31 days the rats were given unstable housing conditions. We found that psychosocially stressed rats had reduced growth rate, reduced thymus weight, increased adrenal gland weight, increased anxiety, an exaggerated startle response, cognitive impairments, greater cardiovascular and corticosterone reactivity to an acute stressor and heightened responsivity to yohimbine. This work demonstrates the effectiveness of acute inescapable episodes of predator exposure administered in conjunction with daily social instability as an animal model of PTSD.     

Correlation between Changes in Stress-Induced Corticosterone Secretion and GR mRNA Levels

The current study was conducted to determine the potential relationship between stress-induced corticosterone secretion and corticosteroid receptor mRNA levels after 5 days of intermittent stress. In particular, we were interested in the rate at which animals terminate a stress response, and how this termination may be altered by repeated stress. Adult male Sprague-Dawley rats were subjected to either 5 days of restraint stress or 5 days of an unpredictable stress paradigm. Restraint-stress induced corticosterone secretion was measured on Days 1 and 5 in both groups, and animals were killed on Day 6. Glucocorticoid receptor (GR), and mineralocorticoid (MR) mRNA levels were determined using in-situ hybridization techniques. Five days of restraint stress caused an habituation of the plasma corticosterone response to stress measured 60 and 90 min post-stress initiation; this pattern of corticosterone secretion was not observed in the animals subjected to unpredictable stress. Five days of either stress paradigm did not alter MR mRNA levels measured within the hippocampus or GR mRNA levels within the hippocampus or the medial parvocellular division of the paraventricular nucleus of the hypothalamus (mpPVN). However, an individual's GR mRNA levels measured within the CA1/2 region of the hippocampus and the mpPVN were significantly correlated with the degree of habituation of the corticosterone response to stress measured on Day 5. This suggests that an increase in the rate of termination of the stress response and levels of GR within the hippocampus and mpPVN may be functionally related.     

Temporal specificity of fear conditioning: effects of different conditioned stimulus-unconditioned stimulus intervals on the fear-potentiated startle effect

Separate groups of rats were given 30 pairings of a light (conditioned stimulus, CS) and 500-ms shock (unconditioned stimulus, US) at CS-US intervals of 0, 25, 50, 100, 200, 800, 3,200, 12,800, or 51,200 ms. Other groups had lights and shocks inconsistently paired. The startle reflex was elicited 2-4 days later with a noise burst alone or 25-51,200 ms after light onset. After CS-US pairings over a wide range of intervals (25-51,200 ms), startle was potentiated in testing, sometimes as rapidly as 50 ms after light onset. Magnitude of potentiation and resistance to extinction were generally greater with longer CS-US intervals. In several groups, potentiation was maximal at a test interval that matched the CS-US interval used in training. This temporal specificity sharpened with increasing numbers of training trials but even occurred with a single training trial in which a 51,200-ms CS-US interval was used. The data indicate that even during simple fear conditioning, animals rapidly learn a temporal CS-US relationship. This has important implications for understanding the neural mechanisms of fear conditioning.     

An Acute Stressor Enhances Sensitivity to a Chemical Irritant and Increases 51CrEDTA Permeability of the Colon in Adult Rats

We investigated the effect of prior acute stress on colonic permeability induced by a chemical irritant known to induce symptoms similar to inflammatory bowel disease in rodents. Adult male rats (n = 12) were stressed by a single session of ten unpredictable, uncontrollable foot shocks, and half were home cage controls (n = 12). Twenty-nine days later, half of each treatment group was exposed to 4% DSS (dextran sulphate sodium) solution in their drinking water for 48 hours while half received pure water over two periods separated by 17 days. After food deprivation overnight and light isoflurane anaesthesia the following morning, the animals were given a colonic infusion of 2000 nCi (nanocurie) 51CrEDTA (51Cr-labelled ethylenediaminetetraacetic acid) and then placed individually in metabolic cages for a six hours continuous urine collection. Radioactivity in urine was measured by a gamma counter and percentage recovery of 51CrEDTA calculated as an indicator of colonic mucosal permeability. Results concluded that pre-shocked animals exposed to DSS showed significantly higher mucosal permeability than the pre-shocked animals given water, and the non-shocked animals given either DSS or water. Pre-shock in combination with two exposures to a chemical irritant separated by 17 days had a pronounced effect on colonic permeability, indicating that stress should be considered a possible initiating or contributory factor to increased intestinal permeability related to a mucosal challenge.     

Gender differences in the negative affective priming of aggressive behavior

The negative affective priming of aggression was examined across different aversive contexts (general stress exposure and frustration) with a laboratory aggression paradigm that measured the intensity of shocks participants delivered to a putative employee. Participants' emotional responses were gauged via startle eyeblink reactions and self-report mood ratings. Aside from gender differences in overall aggression, men but not women exposed to general stress showed significant increases in aggression across blocks. However, frustration produced increases in aggression in both genders. Although both genders showed robust startle increases during stress, startle activation was related to increases in aggression in men and decreases in aggression in women. These findings suggest that general stress and experiences of negative emotion trigger physical aggressive responses more strongly in men than in women.     

Glucocorticoids are required for extinction of predator stress-induced hyperarousal

Background

The role of glucocorticoids in extinction of traumatic memories has not been fully characterized despite its potential as a therapeutic target for acquired posttraumatic stress disorder (PTSD). The predator stress paradigm allows us to determine whether glucocorticoids mediate the extinction of both context-dependent and context-independent fear memories.

Methods

Male C57BL/6J mice were exposed to a predator (cat) then repeatedly exposed to the predator stress context in the absence of the cat. Context-dependent (associative) fear memory was assessed as suppression of activity during re-exposure to the predator stress context without the cat (extinction trials). Context-independent fear (non-associative) was assessed seven days after extinction trials using measures of hyperarousal and anxiety-like behaviours in environments unlike the predator stress context. To assess the role of glucocorticoids, mice were injected with metyrapone (50 mg/kg) 90 min prior to extinction trials in predator stressed mice and context-dependent and context-independent fear memories were assessed. Finally, metyrapone-treated predator stressed mice were injected with corticosterone (5 or 10 mg/kg) immediately following extinction trials and context-dependent and context-independent fear memories were assessed.

Results

Repeated re-exposure to the predator stress context without the cat present extinguished context-dependent fear memory, and also reduced hyperarousal, a generalized, chronic PTSD-like symptom. We show that extinction of context-independent predator stress-induced hyperarousal is dependent on endogenous glucocorticoids during the extinction trials. Furthermore, the inhibition of extinction by metyrapone on startle amplitude was reduced by exogenous administration of corticosterone following extinction trials. Overall, these data implicate glucocorticoids in the extinction of hyperarousal, a core symptom of PTSD.     

An acute stressor enhances sensitivity to a chemical irritant and increases51CrEDTA permeability of the colon in adult rats

We investigated the effect of prior acute stress on colonic permeability induced by a chemical irritant known to induce symptoms similar to inflammatory bowel disease in rodents. Adult male rats (n=12) were stressed by a single session of ten unpredictable, uncontrollable foot shocks, and half were home cage controls (n=12). Twenty-nine days later, half of each treatment group was exposed to 4% DSS (dextran sulphate sodium) solution in their drinking water for 48 hours while half received pure water over two periods separated by 17 days. After food deprivation overnight and light isoflurane anaesthesia the following morning, the animals were given a colonic infusion of 2000 nCi (nanocurie)⁵¹CrEDTA (⁵¹Cr-labelled ethylenediaminetetraacetic acid) and then placed individually in metabolic cages for a six hours continuous urine collection. Radioactivity in urine was measured by a gamma counter and percentage recovery of⁵¹CrEDTA calculated as an indicator of colonic mucosal permeability. Results concluded that pre-shocked animals exposed to DSS showed significantly higher mucosal permeability than the pre-shocked animals given water, and the non-shocked animals given either DSS or water. Pre-shock in combination with two exposures to a chemical irritant separated by 17 days had a pronounced effect on colonic permeability, indicating that stress should be considered a possible initiating or contributory factor to increased intestinal permeability related to a mucosal challenge.     

Pharmacological suppression of corticosterone secretion in response to a physical stressor does not prevent the delayed persistent increase in circulating basal corticosterone concentration

Elevated basal plasma corticosterone concentrations have been observed for several days after the cessation of severe stress. In the present study, we examined whether or not the acute plasma corticosterone response to stress is necessary to elicit increased basal plasma corticosterone concentrations the following day. Pretreatment with metyrapone (100 m a g , intraperitoneal)1 h before inescapable stress (40 2mA tail shocks delivered over a 1-h period) (IS)blocked the acute plasma corticosterone response to IS. However, elevated basal plasma corticosterone concentrations still emerged the next day. These results suggest that the corticosterone response to stress, and its attendant feedback, are not necessary to produce persistent hypothalamic-pituitary-adrenal axis (HPAA) activation.     

Cholinergic Responsiveness of Rat CA1 Hippocampal Neurons In Vitro: Modulation by Corticosterone and Stress

Corticosterone can activate two corticosteroid receptor types in rat hippocampus: low doses activate mineralocorticoid receptors (MR) while high doses additionally activate glucocorticoid receptors (GR). We found that corticosterone, administered to adrenalectomized rats in vivo, dose-dependently modulates carbachol responsiveness of CA1 hippocampal neurons, recorded subsequently in vitro. Thus, the carbachol (3 μM) induced membrane depolarization in CA1 neurons was relatively large in hippocampal slices where either (almost) no corticosteroid receptors were activated (0-1 μg corticosterone/100g body weight) or where both MRs and GRs were occupied by high corticosterone doses (100-1000 μg/100g). Slices from rats that received intermediate doses of corticosterone (10-30 μg/100g) resulting in predominant MR occupation, displayed significantly suppressed carbachol responses. In adrenally intact rats with MRs and GRs fully activated by a very high dose of corticosterone (1 mg/100g), carbachol responses were increased compared to rats that received only the vehicle or that were untreated. When endogenous corticosterone levels were elevated by ether stress, carbachol responses were not increased. These findings suggest that a shift in the relative occupation of MRs and GRs occurring under physiological conditions is associated with modulation of acetylcholine sensitivity in CA1 neurons. After stress, however, the sensitivity to acetylcholine is rather low, although MRs and GRs are fully activated by endogenous corticosterone; this may point to the involvement of additional stress-induced factors modulating the cholinergic responses.     

Is acoustic startle a viable exposure protocol for posttraumatic stress disorder? A clinical case study

Exaggerated startle is a common symptom (based on Diagnostic and Statistical Manual of Mental Disorders [fourth edition] Criterion D) for many patients with posttraumatic stress disorder (PTSD). Findings from previous studies suggest that exaggerated startle may be due to trauma exposure or pretrauma vulnerability factors for PTSD development. The present clinical case study reports on a patient with PTSD characterized by a very prominent startle response and preference against standard trauma-related exposure strategies. On the basis of recent findings that interoceptive exposure exercises (e.g. shaking head side to side, hyperventilation) elicit trauma-related memories (Wald & Taylor, 2008), the authors sought to determine whether repeated application of an acoustic startle stimulus would serve to diminish the prominent startle response and facilitate exposure and overall symptom reduction by eliciting trauma-related memories. The protocol was successful in eliciting vivid and distressing trauma-related memories. Over the course of seven exposure trials, the patient demonstrated a decrease in distress elicited during the protocol, improved mood, and reduced general anxiety and trauma-related distress. He also reported significantly decreased startle response to loud noises encountered during activities of daily living. Although preliminary, these finding suggest that the acoustic startle protocol may be a viable (interoceptive) exposure strategy for individuals with PTSD, particularly those with exaggerated startle responses and those who are not amenable to standard trauma-related exposure strategies.     

Is Acoustic Startle a Viable Exposure Protocol for Posttraumatic Stress Disorder? A Clinical Case Study

Exaggerated startle is a common symptom (based on Diagnostic and Statistical Manual of Mental Disorders [fourth edition] Criterion D) for many patients with posttraumatic stress disorder (PTSD). Findings from previous studies suggest that exaggerated startle may be due to trauma exposure or pretrauma vulnerability factors for PTSD development. The present clinical case study reports on a patient with PTSD characterized by a very prominent startle response and preference against standard trauma-related exposure strategies. On the basis of recent findings that interoceptive exposure exercises (e.g. shaking head side to side, hyperventilation) elicit trauma-related memories (Wald & Taylor, 2008), the authors sought to determine whether repeated application of an acoustic startle stimulus would serve to diminish the prominent startle response and facilitate exposure and overall symptom reduction by eliciting trauma-related memories. The protocol was successful in eliciting vivid and distressing trauma-related memories. Over the course of seven exposure trials, the patient demonstrated a decrease in distress elicited during the protocol, improved mood, and reduced general anxiety and trauma-related distress. He also reported significantly decreased startle response to loud noises encountered during activities of daily living. Although preliminary, these finding suggest that the acoustic startle protocol may be a viable (interoceptive) exposure strategy for individuals with PTSD, particularly those with exaggerated startle responses and those who are not amenable to standard trauma-related exposure strategies.     

Electrolytic,but not neurotoxic,lesions to the lateral tegmental tract increase acoustic startle amplitude and reduce startle stimulus-induced freezing

Startle amplitude and startle stimulus-induced freezing (an index of fear) were measured in a standard acoustic startle response (ASR) paradigm in male Sprague-Dawley rats. Groups with electrolytic lesions to the lateral tegmental tract (LTG) or with axon-sparing lesions to the area around LTG made with the neurotoxin NMDA were compared with vehicle-injected or sham operated control groups on these response measures. Replicating previous results (Leaton & Brucato, 2001), electrolytic lesions to LTG significantly reduced freezing and produced a persistent 300% increase in ASR amplitude compared with all other groups. The NMDA lesions had no effect on freezing or on ASR amplitude compared with the controls. In additional testing the rats with electrolytic lesions to LTG did not differ from controls in the acquisition or retention of context freezing using a footshock unconditioned stimulus. The data made a small, but necessary, step in further clarifying two pathways that modify ASR. The source of the descending pathway that provides tonic inhibition of the sensory input to the ASR circuitry is not within the LTG. The ascending pathway that carries the fear-inducing dimensions of the acoustic stimulus to the amygdala by way of the medial geniculate nucleus does not have an intermediate synapse in the area within LTG.     

Metyrapone reveals that previous chronic stress differentially impairs hippocampal-dependent memory

Chronic stress facilitates fear conditioning in rats with hippocampal neuronal atrophy and in rats in which the atrophy is prevented with tianeptine, a serotonin re-uptake enhancer. The purpose of this study was to determine whether the lack of dissociation between fear conditioning performance and hippocampal integrity was masked by the presence of endogenous corticosteroids during training. As in previous studies, rats were stressed by daily restraint (6 h/day for 21 days), trained in the conditioning chamber (day 23), and then assessed for conditioned fear (day 25) at a time when hippocampal dendritic atrophy persists. On the training day, half of the control and stressed rats were. injected with metyrapone to reduce corticosterone release. Two hours later, two paired or unpaired presentations of tone and footshock were delivered. Although metyrapone reduced conditioned fear in all rats, only stressed rats showed dissociated fear conditioning (i.e. tone conditioning was reduced while contextual conditioning was eliminated). Chronically stressed rats, regardless of metyrapone treatment displayed more rearing in the open field when tested immediately after the completion of fear conditioning. These data support the hypothesis that increased emotionality and enhanced fear conditioning exhibited by chronically stressed rats maybe due to endogenous corticosterone secretion at the time of fear conditioned training. Moreover,these data suggest that chronic stress impairs hippocampal-dependent processes more robustly than hippocampal-independent processes after metyrapone to reduce corticosterone secretion during aversive training.     

Maternal separation in early life impairs tumor immunity in adulthood in the F344 rat

Neonatal stress alters the hypothalamic-pituitary-adrenal (HPA) axis in rodents, such that, when these animals are exposed to stress as adults they hypersecrete corticosterone. Given that glucocorticoids are immunosuppressive, we examined the impact of maternal separation on HPA axis reactivity, natural killer (NK) cytotoxicity, and tumor growth in Fischer 344 rats following chronic restraint stress in adulthood. Pups underwent a chronic stress protocol whereby they were separated from their dams for 3 h on postnatal days 1-21. In adulthood, corticosterone responses were assessed following exposure to chronic (6 days for 10 h) restraint stress. Rats allocated to the chronic stress condition were inoculated with MADB106 tumor cells on day 4 of the restraint protocol. Blood was assessed for NK cytotoxicity on the final day of the chronic restraint protocol, and tumor colonization was assessed 3 weeks thereafter. Maternal separation impaired developmental weight gain (P < 0.05), depressed NK cytotoxicity (P < 0.05), and increased tumor colonization in the presence of chronic restraint stress in adulthood (P < 0.00 l). These findings occurred independently of circulating plasma corticosterone as only adult stress exposure potentiated corticosterone responses (P < 0.05). Our findings indicate that maternal separation and chronic stress can impair NK cytotoxicity and hence tumor immunity, but these effects are not directly mediated by perturbations in HPA axis function.     

Greater sustained anxiety but not phasic fear in women compared to men

Startle reflex studies in rodents indicate that female are more reactive than rats in experimental models of sustained anxiety but not in models of phasic fear (Toufexis, 2007). This study examined evidence for a similar effect in humans. Participants were exposed to three conditions, (1) predictable aversive shocks signaled by a cue, (2) unpredictable shocks, and (3) no shocks. Acoustic startle stimuli were delivered regularly across conditions. Phasic startle potential to the threat cue in the predictable condition was not affected by sex. In contrast, and consistent with basic research, the sustained increase in startle in the predictable and unpredictable conditions was greater in women compared to men. Animal studies suggest that such an effect may be mediated by the effects of sexual dimorphism in limbic structures, including the bed nucleus of the stria terminalis. However, psychosocial factors may also contribute to this effect.     

Hypothalamic circuit regulating colonic transit following chronic stress in rats

Although acute stress accelerates colonic transit, the effect of chronic stress on colonic transit remains unclear. In this study, rats received repeated restraint stress (chronic homotypic stress) or various types of stress (chronic heterotypic stress) for 5 and 7 days, respectively. Vehicle saline, oxytocin (OXT), OXT receptor antagonist or corticotropin-releasing factor (CRF) receptor antagonists were administered by intracerebroventricular (ICV) injection prior to restraint stress for 90 min. Immediately after the stress exposure, the entire colon was removed and the geometric center (GC) of Na51CrO4 (a nonabsorbable radioactive marker; 0.5 μCi) distribution was calculated to measure the transit. Gene expression of OXT and CRF in the paraventricular nucleus (PVN) was evaluated by in situ hybridization. Accelerated colonic transit with the acute stressor was no longer observed following chronic homotypic stress. This restored colonic transit was reversed by ICV injection of an OXT antagonist. In contrast, chronic heterotypic stress significantly accelerated colonic transit, which was attenuated by ICV injection of OXT and by a CRF receptor 1 antagonist. OXT mRNA expression in the PVN was significantly increased following chronic homotypic stress, but not chronic heterotypic stress. However, CRF mRNA expression in the PVN was significantly increased following acute and chronic heterotypic stress, but not chronic homotypic stress. These results indicate that central OXT and CRF play a pivotal role in mediating the colonic dysmotility following chronic stress in rats.