Inescapable Shock Research Articles

Adrenergic and glucocorticoid modulation of the sterile inflammatory response

Exposure to an intense, acute stressor, in the absence of a pathogen, alters immune function. Exposure to a single bout of inescapable tail shock increases plasma and tissue concentrations of cytokines, chemokines, and the danger associated molecular pattern (DAMP) Hsp72. Although previous studies have demonstrated that adrenergic receptor (ADR) and glucocorticoid receptor (GCR)-mediated pathways alter pathogen or microbial associated molecular pattern (MAMP)-evoked levels of cytokines, chemokines, and Hsp72, far fewer studies have tested the role of these receptors across multiple inflammatory proteins or tissues to elucidate the differences in magnitude of stress-evoked sterile inflammatory responses. The goals of the current study were to (1) compare the sterile inflammatory response in the circulation, liver, spleen, and subcutaneous (SQ) adipose tissue by measuring cytokine, chemokine, and DAMP (Hsp72) responses; and (2) to test the role of alpha-1 (α1), beta-1 (β1), beta-2 (β2), and beta-3 (β3) ADRs, as well as GCRs in signaling the sterile inflammatory response. The data presented indicate plasma and SQ adipose are significantly more stress responsive than the liver and spleen. Further, administration of ADR and GCR-specific antagonists revealed both similarities and differences in the signaling mechanisms of the sterile inflammatory response in the tissues studied. Finally, given the selective increase in the chemokine monocyte chemotactic protein-1 (MCP-1) in SQ tissue, it may be that SQ adipose is an important site of leukocyte migration, possibly in preparation for infection as a consequence of wounding. The current study helps further our understanding of the tissue-specific differences of the stress-induced sterile inflammatory response.

Six weeks of voluntary wheel running modulates inflammatory protein (MCP-1, IL-6, and IL-10) and DAMP (Hsp72) responses to acute stress in white adipose tissue of lean rats

To prime local tissues for dealing with potential infection or injury, exposure to an acute, intense stressor evokes increases in circulating and local tissue inflammatory proteins. Regular physical activity facilitates stress-evoked innate reactivity and modulates the expression of inflammatory proteins in immuno-metabolic tissues such as white adipose tissue (WAT). The impact of regular physical activity on stress-evoked inflammatory protein expression in WAT, however, remains unclear. To investigate this question, lean male F344 rats (150-175g) were allowed voluntary access to a running wheel for 6weeks followed by exposure to an acute stressor (100, 1.5mA-5s inescapable tail shocks). Using ELISAs, corticosterone, heat shock protein 72 (Hsp72), macrophage chemoattractant protein (MCP-1), tumor necrosis factor-alpha (TNF-α), interleukin (IL)-1β, IL-6, and IL-10 concentrations were measured in plasma and subcutaneous, intraperitoneal (epididymal and retroperitoneal WAT depots) and visceral (omental and mesenteric WAT depots) WAT compartments. Acute stress increased plasma concentrations of all proteins except TNF-α and, depending upon the compartment examined, WAT concentrations of MCP-1, IL-1β, IL-6, and IL-10. Exercise ubiquitously increased IL-1β within WAT, potentiated stress-evoked Hsp72 in plasma and WAT, and differentially increased stress-evoked MCP-1, IL-6, and IL-10 within WAT. These data suggest: (a) inflammatory proteins in non-obese WAT may serve compartment-specific immune and metabolic roles important to the acute stress response and; (b) voluntary habitual exercise may optimize stress-induced augmentation of innate immune function through increases in stress-evoked Hsp72, MCP-1, IL-6, and IL-10 and decreases in IL-1β/IL10 and TNF-α/IL10 ratios within white adipose tissue.

Effects of mild TBI from repeated blast overpressure on the expression and extinction of conditioned fear in rats

Mild traumatic brain injury (mTBI) and post-traumatic stress disorder (PTSD) are pressing medical issues for the Warfighter. Symptoms of mTBI can overlap with those of PTSD, suggesting the possibility of a causal or mediating role of mTBI in PTSD. To address whether mTBI can exacerbate the neurobiological processes associated with traumatic stress, we evaluated the impact of mTBI from a blast overpressure (BOP) on the expression of a conditioned fear. In the rat, conditioned fear models are used to evaluate the emotional conditioning processes that are known to become dysfunctional in PTSD. Rats were first trained on a variable interval (VI), food maintained, operant conditioning task that established a general measure of performance. Inescapable electric shock (IES) was paired with an audio-visual conditioned stimulus (CS) and followed 1day later by three daily exposures to BOP (75kPa). Subsequently, the CS alone was presented once every 7days for 2months, beginning 4days following the last BOP. The CS was presented during the VI sessions allowing a concurrent measure of performance. Treatment groups (n=10, each group) received IES+BOP, IES+sham-BOP, sham-IES+BOP or sham-IES+sham-BOP. As expected, pairing the CS with IES produced a robust conditioned fear that was quantified by a suppression of responding on the VI. BOP significantly decreased the expression of the conditioned fear. No systematic short- or long-term performance deficits were observed on the VI from BOP. These results show that mTBI from BOP can affect the expression of a conditioned fear and suggests that BOP caused a decrease in inhibitory behavioral control. Continued presentation of the CS produced progressively less response suppression in both fear conditioned treatments, consistent with extinction of the conditioned fear. Taken together, these results show that mTBI from BOP can affect the expression of a conditioned fear but not necessarily in a manner that increases the conditioned fear or extends the extinction process.

Amygdala Deep Brain Stimulation Is Superior to Paroxetine Treatment in a Rat Model of Posttraumatic Stress Disorder

BackgroundPosttraumatic stress disorder (PTSD) is a very debilitating disease refractory to current treatment with selective serotonin reuptake inhibitors (SSRIs) in up to 30 percent of patients, illustrating the need for new treatments of PTSD. Neuroimaging studies have shown increased activity of the amygdala of patients with PTSD. Objective/hypothesisTo investigate amygdala deep brain stimulation (DBS) as a possible novel treatment for PTSD and compare it to current treatment with a commonly used SSRI, paroxetine, in a rat PTSD model. MethodsA PTSD model was created by subjecting rats to inescapable foot shocks in the presence of a conspicuous ball. Response to treatment was measured as a decreased burying behavior when presented with the same ball 1 and 2 weeks after the shocks. Rats were treated with either daily intraperitoneal paroxetine injections or amygdala DBS via an electrode implanted 1 week prior to shocks. Generalized anxiety was assessed using an elevated plus maze. ResultsAnimals treated with amygdala DBS showed less ball burying at 2 weeks relative to the animals treated with paroxetine. The animals treated with paroxetine, however, had a lower general anxiety level compared to the DBS-treated group. ConclusionsIn this PTSD model, paroxetine was found to decrease the measured general anxiety level of rats that underwent the PTSD protocol, but did not counteract shock-induced hyper-vigilance toward the trauma-associated object (ball). Amygdala DBS, however, did decrease shock-induced hyper-vigilance as measured by a lower burying time, but had no effect on general anxiety assessed in the elevated plus maze. By attenuating amygdala function, DBS may act to treat the cause of PTSD, hyperactive amygdala function, and may be a promising novel alternative in cases of PTSD refractory to current pharmacological treatments.

The effect of perceiving control on glutamatergic function and tolerating stress

Control over stressful experience results in subsequent inhibition of fear responses. We indexed glutamatergic levels in the ventromedial prefrontal cortex (vmPFC) before, and following, exposing humans to electric shocks, half of whom believed that they could control the shocks and subsequently presented with aversive images. Our findings show for the first time that perceived lack of control results in an increase in glutamatergic response in the vmPFC and heightened avoidance of a subsequent stressor. Our perceived capacity to control adverse events is critical for psychological functioning. Believing in the fact that one controls the outcomes, even when one does not actually have control, helps to reduce subsequent fear reactivity.1,2,3 Similarly, rats exposed to escapable (versus inescapable) shock are ‘immunized’ against subsequent stressors, which is reflected in reduced fear responses.4 Although there is evidence of the role of glutamate alterations following control manipulation in rats,5,6 there is no parallel evidence in humans. We investigated (a) the role of glutamate in perceived control over aversive experience in humans and (b) the relationship between glutamate change and subsequent stress response. We focused on the vmPFC because glutamate is activated in the vmPFC during stress, and there are projections from the vmPFC to emotional and decision-making functional regions.7 We hypothesized that lacking perceived control would result in greater glutamate because this would aggravate the experience of stress. We assessed glutamate levels in 29 healthy participants; the voxel of interest (151515mm3) was placed adjacent to the anterior border of the genu of corpus callosum and centered on the interhemispheric fissure in order to include both left and right anterior cingulate cortex primarily (Figure 1). Participants were then subsequently exposed to standardized electric shocks. Half of the participants were (erroneously) informed that they could terminate the shock by pressing a button. Glutamate levels were then re-assessed. Fifteen minutes later, participants were then presented with aversive and neutral images, and were instructed that they could terminate the image by pressing a button; this task indexed the tolerance of distress (Supplementary Methods). Change in glutamate level before and after the shock differed between control conditions (F1,27¼4.31, P¼0.05; (Figure 1). Participants who lacked perceived control increased their glutamate level, following shock (t13¼3.32, P¼0.006), whereas those who perceived control did not (t14¼0.28, P¼0.79; Supplementary Results). Participants who lacked perceived control subsequently terminated aversive images sooner than those who perceived control (t24¼3.13, P¼0.005). There was no difference in viewing time for neutral images (t24¼0.41, P¼0.68). In the Control condition, glutamate decrease was associated with perceived prediction of the shocks (R2¼0.57, P¼0.025), but this was not apparent in the No-Control condition (R2¼0.03, P¼0.91) (see Supplementary Figure S1 and Supplementary Results). The observation that lacking perceived control led to increased glutamate in the vmPFC is consistent with evidence that acute stress leads to activation of glutamatergic transmission in the vmPFC,8 with subsequent activation of dopaminergic stress responses.7 The association of glutamate decrease and perceived prediction of shocks in those who perceived control suggests that perceived mastery over adverse stimuli (including capacity to predict occurrence) may be associated with diminished glutamate activation in the vmPFC. The 15-minute duration between manipulated control perception and measurement of subsequent behavior may have impacted the relationship, and it remains to be determined how glutamatergic activity after perceived control impacts subsequent behavior over different time courses. There are significant differences that are relative to animal research, where (a) only actual control over aversive stimuli can be manipulated and (b) control reduces gamma-aminobutyric acid (GABA)ergic interneurons in the vmPFC, which results in activation of glutamatergic output neurons to the dorsal raphe nucleus—the role of glutamate in the animal vmPFC is unknown9 (Supplementary Discussion). Future research needs to study the compatibility between neurotransmitter response to control over stressors in animals and humans, and also to determine neural responses to actual versus perceived control over aversive events. In summary, this study provides a novel direction for future research to investigate a potential neural modulator of how perceived control may impact behavior. This direction could provide important insights into how humans manage stressful events and their aftermath.

Repeated administration of AC-5216, a ligand for the 18kDa translocator protein, improves behavioral deficits in a mouse model of post-traumatic stress disorder

Post-traumatic stress disorder (PTSD) is a severely disabling anxiety disorder that may occur following exposure to a serious traumatic event. It is a psychiatric condition that can afflict anyone who has experienced a life-threatening or violent event. Previous studies have shown that changes in 18 kDa translocator protein (TSPO) expression (or function), a promising target for treating neurological disorders without benzodiazepine-like side effects, may correlate with PTSD. However, few studies have investigated the anti-PTSD effects of TSPO ligands. AC-5216, a ligand for TSPO, induces anxiolytic- and anti-depressant-like effects in animal models. The present study aimed to determine whether AC-5216 ameliorates PTSD behavior in mice. Following the training session consisting of exposure to inescapable electric foot shocks, animals were administered AC-5216 daily during the behavioral assessments, i.e., situational reminders (SRs), the open field (OF) test, the elevated plus-maze (EPM) test, and the staircase test (ST). The results indicated that exposure to foot shocks induced long-term behavioral deficiencies in the mice, including freezing and anxiety-like behavior, which were significantly ameliorated by repeated treatment with AC-5216 but without any effect on spontaneous locomotor activity or body weight. In summary, this study demonstrated the anti-PTSD effects of AC-5216 treatment, suggesting that TSPO may represent a therapeutic target for anti-PTSD drug discovery and that TSPO ligands may be a promising new class of drugs for the future treatment of PTSD.

The effects of behavioral control over stress on GABAergic spontaneous inhibitory postsynaptic currents in prefrontal cortical pyramidal neurons

Traumatic events may lead to anxiety, depression and post-traumatic stress disorder (PTSD). However, the majority of individuals exposed to trauma do not develop these disorders. The stressor controllability paradigm has been widely used as a model for understanding the neurobiology underlying factors that confer vulnerability and resilience to the outcome of traumatic events. In this paradigm rats receive a series of tail shocks: one group of rats have control over the termination of the shock by means of turning a wheel (escapable shock, ES), while the other “yoked” group of rats receive physically identical shocks but have no control over shock termination (inescapable shock, IS). In subsequent behavioral tests that model components of anxiety and depression, IS rats without control show increased signs of behavioral depression, while ES rats that have control over the shock behave as naïve home caged (HC) rats. We have previously reported that individual deep layer pyramidal neurons from the ventral medial prefrontal cortex (vmPFC) exhibit changes in their intrinsic excitability following ES. To examine if there is a corresponding reduction in synaptic inhibition, we tested IS, ES and HC deep layer pyramidal neurons under identical conditions. Collecting such electrophysiological data from pyramidal neurons after exposure to stress is a technical challenge, yet very useful for conductance-based neural simulations and computational modeling. Here we present a data set of spontaneous inhibitory postsynaptic currents (sIPSCs) gathered from whole-cell patch-clamp recordings of individual prefrontal cortical deep layer neurons from adult rats (60-70 days old) after exposure to ES, IS or HC. In order to analyze the data, we provide our script used for the detection of synaptic events written for the scientific/engineering program Igor Pro that allows users to define their own event detection parameters.

Basolateral Amygdala and the Regulation of Fear-Conditioned Changes in Sleep: Role of Corticotropin-Releasing Factor

To determine whether corticotropin-releasing factor (CRF) in the basolateral amygdala (BLA) modulated sleep and fear-conditioned alterations in sleep. After 2 days of habituation to recording procedures, baseline sleep recordings were obtained. The animals were then habituated to the handling procedure necessary for microinjections over 2 consecutive days. In experiment 1, rats received microinjections of 0.5 μL antalarmin (1.61 or 4.82 mM), a CRF receptor 1 antagonist, or distilled water once a week for 3 wk. In experiment 2, rats received a microinjection of either antalarmin or vehicle prior to inescapable shock training (ST; 20 shocks; 0.8 mA, 0.5 sec; 1 min interstimulus interval). The animals were placed back in the context 7 days later for 30 min without shock (CR; context re-exposure). Sleep was recorded for 8 h after each manipulation. NA. Outbred Wistar rats. The rats were surgically implanted with electrodes for recording the electroencephalogram and electromyogram for determining arousal state and with bilateral guide cannulae directed at BLA. Antalarmin microinjected into BLA did not significantly alter sleep under undisturbed conditions. However, antalarmin microinjected bilaterally into BLA prior to ST blocked reductions in rapid eye movement sleep that ST normally produces. Further, the single microinjection prior to ST blocked the reduction in rapid eye movement typically seen after subsequent CR. Behavioral freezing, an indicator of fear memory, was not altered. CRF in BLA is involved in regulating stress-induced alterations in sleep and it plays a role in modulating how stressful memories influence sleep.

P.2.001 Citalopram given to stressed and control pregnant rats causes sex-dependent changes in behaviour and CRH mRNA expression in their offspring

In this study, we investigated whether stress can enhance the toxicity of oligomer Aβ1–40 in the mouse brain. Stress was applied to the animals, consisting of a 2-day inescapable foot shock followed by 3-weekly situation reminders (SRs). We found that stress significantly affected not only the amygdala-dependent (anxiety) but also the hippocampal-dependent (spatial learning and memory) behaviors through the oxidative damage caused in these two regions. However, oligomer Aβ1–40 treatment alone did not induce behavioral impairment. In addition, combined oligomer Aβ1–40 and stress treatment increased the glucocorticoid receptor (GR)/mineralocorticoid receptor (MR) ratio and the expression of corticotrophin releasing factor 1 (CRF-1) receptor in the hippocampus. Changes in the components of the hypothalamic–pituitary–adrenal (HPA) axis, such as the GR/MR ratio and CRF-1 level, were observed, accompanied by increasing Aβ accumulation, oxidative stress, nuclear transcription factor (NF-κB) hypoactivity, and apoptotic signaling in the hippocampus, and decreasing calbindin D28K and NMDA receptor 2A/2B (NR2A/2B) in the hippocampus, along with alteration of the cholinergic neurons (ChAT) in the medium septum/diagnoid band (MS/DB), noradrenergic neurons (TH) in the locus coeruleus (LC), and serotonergic neurons (5-HT) in the Raphe nucleus. Therefore, apoptosis and synaptic dysfunction in the hippocampus severely induced the impairment of spatial learning and memory. These results suggest that stress may play an important role in the early stages of Alzheimer’s disease (AD), and an antioxidant strategy might be a potential therapeutic approach for stress-mediated disorders.

Acute psychological stress results in the rapid development of insulin resistance

In recent years, the roles of chronic stress and depression as independent risk factors for decreased insulin sensitivity and the development of diabetes have been increasingly recognized. However, an understanding of the mechanisms linking insulin resistance and acute psychological stress are very limited. We hypothesized that acute psychological stress may cause the development of insulin resistance, which may be a risk factor in developing type 2 diabetes. We tested the hypothesis in a well-established mouse model using 180 episodes of inescapable foot shock (IES) followed by a behavioral escape test. In this study, mice that received IES treatment were tested for acute insulin resistance by measuring glucose metabolism and insulin signaling. When compared with normal and sham mice, mice that were exposed to IES resulting in escape failure (defined as IES with behavioral escape failure) displayed elevated blood glucose levels in both glucose tolerance and insulin tolerance tests. Furthermore, mice with IES exposure and behavioral escape failure exhibited impaired hepatic insulin signaling via the insulin-induced insulin receptor/insulin receptor substrate 1/Akt pathway, without affecting similar pathways in skeletal muscle, adipose tissue, and brain. Additionally, a rise in the murine growth-related oncogene KC/GRO was associated with impaired glucose metabolism in IES mice, suggesting a mechanism by which psychological stress by IES may influence glucose metabolism. The present results indicate that psychological stress induced by IES can acutely alter hepatic responsiveness to insulin and affect whole-body glucose metabolism.

Clomipramine reverses hypoalgesia/hypoesthesia and improved depressive-like behaviors induced by inescapable shock in rats

An increased vulnerability to pain complaints, along with a simultaneous increase in experimental pain thresholds, shows the paradoxical phenomenon of pain perception in depressive patients. Clomipramine, a tricyclic antidepressant, could also ameliorate syndromes in chronic pain patients. However, few studies have focused on the effect of antidepressants on experimental pain thresholds. By using a rat model, the learned helplessness paradigm, the present study explored the effect of clomipramine on behavioral deficits and experimental pain thresholds to different stimuli in “helpless” rats. Helpless rats were administered clomipramine (10mg/kg, i.p, b.i.d.) for 5 consecutive days. The depressive-like and anxiety-like behaviors were detected by shuttle box, open field and elevated plus maze test before and after inescapable shock and after medication. The sensitivity to the thermal and mechanical stimuli was also measured by the von Frey hair and Hargreaves test at the indicated time points. Helpless rats displayed shorter total travel distance and fewer rearing times in the open field test and decreased percentage of time spent in the open arms in the elevated plus maze test. In addition, they exhibited significant hypoalgesia/hypoesthesia to mechanical and thermal stimuli. Clomipramine alleviate depressive-like and anxiety-like behaviors and increased the sensitivity to von Frey filament stimuli with no effect on the sensitivity to radiant heat stimuli in helpless rats. These suggested that clomipramine could reverse mechanical but not thermal hypoalgesia/hypoesthesia and simultaneously improved behavioral deficits.

Impact of the time in an animal model of mood disorder

The objective of the study is to evaluate whether intervening and testing in different rest-activity periods of the day would produce different measurements in animal behavior studies. Methodology: Thirty-five, 60-day-old male Wistar rats were submitted to an inescapable foot shock (IFS) stress model and behavioral tests (Light–Dark Box test). The animals received intervention and were tested in both light and dark phases, resulting in the following groups: control L (tested in the light), control D (tested in the dark), LL (IFS and tested in the light), LD (IFS in the light and tested in the dark), DL (IFS in the dark and tested in the light), and DD (IFS and tested in the dark). Results: The Light–Dark Box test showed that control L was not significantly different from other groups in any of the parameters. However, when comparing control D with the intervention groups, we observed a difference in the mean length of time spent in the light compartment (t=2.56; p=0.045). A significant difference in the number of crossings into the light compartment was only observed between the control D and the LL and LD groups (t=−2.608; p=0.028; t=−2.571; p=0.030, respectively). The latency time for the control D group was significantly lower than that of the DD group (t=−2.556; p=0.043). Conclusions: These results show that behavior testing during the animal's period of highest activity (dark period) revealed differences caused by the intervention, whereas no differences were apparent when the control group was observed during the day.

Re-exposure and environmental enrichment reveal NPY-Y1 as a possible target for post-traumatic stress disorder

Exposure-based cognitive behavioral therapy in post-traumatic stress disorder (PTSD) patients relieves symptoms caused by fear association as well as symptoms that are not the result of associative learning. We used the inescapable foot shock model (IFS), an animal model for PTSD, to study the possible involvement of glutamate receptors, the corticotropin-releasing factor (CRF) system, and the neuropeptide Y (NPY) system in the reduction of stress sensitization following repeated re-exposure to the conditioning context. Starting one week after the IFS procedure, the rats were repeatedly re-exposed to the shock environment. Stress sensitivity was measured in a modified open field test (sudden silence was used as a stressor). Selected mRNAs (GluN1, -2A-C, GluA1-4, GluK1-5, CRF, CRF-R1, NPY, NPY-Y1) were quantified in the amygdala.Repeated re-exposure (RE) to the IFS context reduced both trauma-associated anxiety (to the IFS context) and the enhanced stress sensitivity (in the open field). Changes in glutamate receptor subunits (GluN1, GluN2A-B, GluA1, GluA4, GluK3, GluK4) were detected in the amygdala that were normalized by RE. However, infusion of the AMPA/kainate antagonist NBQX in the BLA (basolateral amygdala) did not improve the anxious behavior. RE normalized IFS-induced increases in CRF-R1 mRNA and increased NPY-Y1 mRNA expression in the amygdala. Previously, and repeated here, we showed that environmental enrichment (EE) enhances recovery from IFS. EE led to similar changes in CRF-R1 and NPY-Y1 expression as RE did. Importantly, administration of [Leu31, Pro34]-NPY (Y1 agonist) in the BLA normalized the enhanced sensitivity to stress after IFS.Our data suggest that the NPY-Y1 receptor in the amygdala may serve as a therapeutic target for the treatment of PTSD.

Repeated Forced Swim Stress Induces Learned Helplessness in Rats

The goal of our study was to determine if another form of inescapable, uncontrollable stress, milder than repeated electric shock, could induce learned helpless (LH) behavior in rats and if antidepressant drugs can prevent this behavior. Rats were first exposed to repeated forced swim stress (RFS) and 24 hr after last swim, rats were tested for escape behavior [Escape Latency (EL) and % Escape Failure (EF)] in a shuttle‐box. RFS segregated the stressed rats into two clusters based on EL and EF. One cluster, LH, showed increased EL (27.1 ± 1.4 s) and EF (82.7 ± 8 %) compared to controls (11.1 ± 1.3 s; 11.7 ± 4.3%), showing stress susceptibility. The other cluster, non‐helpless (NH), showed EL (8.8 ± 2.4s) and EF (11.5 ± 5.3%) similar to the controls. All RFS rats performed similar to controls in the open field and elevated plus maze tests, suggesting the absence of locomotion deficits or increased anxiety. In rats pretreated for 10 days with desipramine (DMI), no increased EL or EF were observed in the shuttle‐box after RFS. DMI also prevented a reduction in GRK3 in the locus coeruleus observed after RFS in untreated LH rats. These data suggest that, like inescapable unpredictable shock, RFS can induce LH in rats, which is prevented by DMI pretreatment. Hence, RFS is another inescapable stress paradigm that can identify stress‐susceptible and stress‐resilient populations in rats. (Supported by NARSAD)

MicroRNA expression in rat brain exposed to repeated inescapable shock: differential alterations in learned helplessness vs. non-learned helplessness

MicroRNA (miRNA) expression was measured within frontal cortex of male Holtzman rats subjected to repeated inescapable shocks at days 1 and 7, tested for learned helplessness (LH) at days 2 and 8, and sacrificed at day 15. We compared rats that did vs. did not exhibit LH, as well as rats that were placed in the apparatus and tested for avoidance but not given shocks (tested controls, TC). Non-learned helpless (NLH) rats showed a robust adaptive miRNA response to inescapable shock whereas LH rats showed a markedly blunted response. One set of 12 miRNAs showed particularly large, significant down-regulation in NLH rats relative to tested controls (mir-96, 141, 182, 183, 183*, 298, 200a, 200a*, 200b, 200b*, 200c, 429). These were encoded at a few shared polycistronic loci, suggesting that the down-regulation was coordinately controlled at the level of transcription. Most of these miRNAs are enriched in synaptic fractions. Moreover, almost all of these share 5'-seed motifs with other members of the same set, suggesting that they will hit similar or overlapping sets of target mRNAs. Finally, half of this set is predicted to hit Creb1 as a target. We also identified a core miRNA co-expression module consisting of 36 miRNAs that are highly correlated with each other across individuals of the LH group (but not in the NLH or TC groups). Thus, miRNAs participate in the alterations of gene expression networks that underlie the normal (NLH) as well as aberrant (LH) response to repeated shocks.

Behavioral Stress-Induced Activation of FoxO3a in the Cerebral Cortex of Mice

The transcription factor FoxO3a is highly expressed in brain, but little is known about the response of FoxO3a to behavioral stress and its impact in the associated behavioral changes. We tested the response of brain FoxO3a in the learned helplessness (LH) paradigm and tested signaling pathways that mediate the response of FoxO3a. A single session of inescapable shocks (IES) in mice reduced FoxO3a phosphorylation at the Akt-regulating serine/threonine residues and induced prolonged nuclear accumulation of FoxO3a in the cerebral cortex, both indicating activation of FoxO3a in brain. The response of FoxO3a is accompanied by a transient inactivation of Akt and a prolonged activation of glycogen synthase kinase-3beta (GSK3β). Noticeably, FoxO3a formed a protein complex with GSK3β in the cerebral cortex, and the interaction between the two proteins was stronger in IES-treated mice. Inhibition of glycogen synthase kinase-3 was able to abolish IES-induced LH behavior, disrupt IES-induced GSK3β-FoxO3a interaction, and reduce nuclear FoxO3a accumulation. In vitro approaches further revealed that the interaction between GSK3β and FoxO3a was strongest when both were active; FoxO3a was phosphorylated by recombinant GSK3β; and glycogen synthase kinase-3 inhibitors effectively reduced FoxO3a transcriptional activity. Importantly, IES-induced LH behavior was markedly diminished in FoxO3a-deficient mice that had minimal FoxO3a expression and reduced levels of FoxO3a-inducible genes. FoxO3a is activated in response to IES by interacting with GSK3β, and inhibition of GSK3β or reducing FoxO3a expression promotes resistance to stress-induced behavioral disturbance by disrupting this signaling mechanism.

Effects of Stressor Predictability and Controllability on Sleep, Temperature, and Fear Behavior in Mice

Predictability and controllability are important factors in the persisting effects of stress. We trained mice with signaled, escapable shock (SES) and with signaled, inescapable shock (SIS) to determine whether shock predictability can be a significant factor in the effects of stress on sleep. Male BALB/cJ mice were implanted with transmitters for recording EEG, activity, and temperature via telemetry. After recovery from surgery, baseline sleep recordings were obtained for 2 days. The mice were then randomly assigned to SES (n = 9) and yoked SIS (n = 9) conditions. The mice were presented cues (90 dB, 2 kHz tones) that started 5.0 sec prior to and co-terminated with footshocks (0.5 mA; 5.0 sec maximum duration). SES mice always received shock but could terminate it by moving to the non-occupied chamber in a shuttlebox. SIS mice received identical tones and shocks, but could not alter shock duration. Twenty cue-shock pairings (1.0-min interstimulus intervals) were presented on 2 days (ST1 and ST2). Seven days after ST2, SES and SIS mice, in their home cages, were presented with cues identical to those presented during ST1 and ST2. NA. NA. NA. On each training and test day, EEG, activity and temperature were recorded for 20 hours. Freezing was scored in response to the cue alone. Compared to SIS mice, SES mice showed significantly increased REM after ST1 and ST2. Compared to SES mice, SIS mice showed significantly increased NREM after ST1 and ST2. Both groups showed reduced REM in response to cue presentation alone. Both groups showed similar stress-induced increases in temperature and freezing in response to the cue alone. These findings indicate that predictability (modeled by signaled shock) can play a significant role in the effects of stress on sleep.

Propofol Enhances Memory Formation via an Interaction with the Endocannabinoid System

Propofol is associated with postoperative mood alterations and induces a higher incidence of dreaming compared with other general anesthetics. These effects might be mediated by propofol's inhibitory action on fatty acid amide hydrolase, the enzyme that degrades the endocannabinoid anandamide. Because propofol is also associated with a higher incidence of traumatic memories from perioperative awareness and intensive care unit treatment and the endocannabinoid system is involved in regulating memory consolidation of emotional experiences, the authors investigated whether propofol, at anesthetic doses, modulates memory consolidation via an activation of the endocannabinoid system. Male Sprague-Dawley rats were trained on an inhibitory avoidance task in which they received an inescapable foot shock upon entering the dark compartment of the apparatus. Drugs were administered intraperitoneally immediately or 30, 90, or 180 min after training. On the retention test 48 h later, the latency to reenter the dark compartment was recorded and taken as a measure of memory retention. The anesthetic doses of propofol administered after training significantly increased latencies of 48-h inhibitory avoidance performance (483.4 ± 181.3, 432.89 ± 214.06, 300 and 350 mg/kg, respectively; mean ± SD) compared with the corresponding vehicle group (325.33 ± 221.22, mean ± SD), which is indicative of stronger memory consolidation in propofol treated rats. Administration of a nonimpairing dose of the cannabinoid receptor antagonist rimonabant blocked the memory enhancement induced by propofol (123.39 ± 133.10, mean ± SD). Delayed administration of propofol 90 and 180 min after training or immediate posttraining administration of the benzodiazepine midazolam or the barbiturate pentobarbital did not significantly alter retention. These findings indicate that propofol, in contrast to other commonly used sedatives, enhances emotional memory consolidation when administered immediately after a stressful event by enhancing endocannabinoid signaling.

Toward a Novel Endogenous Anxiolytic Factor, Fibroblast Growth Factor 2

Toward a Novel Endogenous Anxiolytic Factor, Fibroblast Growth Factor 2

Behaviour of a genetic mouse model of depression in the learned helplessness paradigm

H/Rouen (displaying a helpless phenotype in the tail suspension test) mice exhibiting features of depressive disorders and NH/Rouen (displaying non-helpless phenotype) mice were previously created through behavioural screening and selective breeding. Learned helplessness (LH), in which footshock stress induces a coping deficit, models some aspects of depression in rodents, but so far, fewer LH studies have been performed in mice than in rats. To study H/Rouen and NH/Rouen in the LH paradigm. When CD1 mice were submitted to footshock with various training durations and shock intensities, the most suitable parameters to induce a behavioural deficit were 0.3 mA and four training sessions. A significantly longer latency to escape shocks was found in male H/Rouen mice compared to male NH/Rouen mice. On the other hand, once shocked, NH/Rouen mice showed more severe coping deficits than H/Rouen mice. In addition, a sub-chronic treatment with fluoxetine lacked efficacy in NH/Rouen mice, whereas it improved performances in H/Rouen mice. We also found that a shock reminder at day 8, subsequent to inescapable shocks, maintained helplessness for 20 days. Finally, female H/Rouen mice responded to chronic fluoxetine administration after 10 days of treatment, while a 20-day treatment was necessary to improve the behavioural deficit in H/Rouen male mice. H/Rouen and NH/Rouen lines displayed different despair-related behaviour in the LH paradigm. Fluoxetine had beneficial effects after sub-chronic or chronic but not acute treatment of H/Rouen mice, thus providing a pharmacological validation of the protocols.