Inescapable Footshock Stress Research Articles

Molecular signatures of astrocytes and microglia maladaptive responses to acute stress are rescued by a single administration of ketamine in a rodent model of PTSD

Stress affects the brain and alters its neuroarchitecture and function; these changes can be severe and lead to psychiatric disorders. Recent evidence suggests that astrocytes and microglia play an essential role in the stress response by contributing to the maintenance of cerebral homeostasis. These cells respond rapidly to all stimuli that reach the brain, including stressors. Here, we used a recently validated rodent model of post-traumatic stress disorder in which rats can be categorized as resilient or vulnerable after acute inescapable footshock stress. We then investigated the functional, molecular, and morphological determinants of stress resilience and vulnerability in the prefrontal cortex, focusing on glial and neuronal cells. In addition, we examined the effects of a single subanesthetic dose of ketamine, a fast-acting antidepressant recently approved for the treatment of resistant depression and proposed for other stress-related psychiatric disorders. The present results suggest a prompt glial cell response and activation of the NF-κB pathway after acute stress, leading to an increase in specific cytokines such as IL-18 and TNF-α. This response persists in vulnerable individuals and is accompanied by a significant change in the levels of critical glial proteins such as S100B, CD11b, and CX43, brain trophic factors such as BDNF and FGF2, and proteins related to dendritic arborization and synaptic architecture such as MAP2 and PSD95. Administration of ketamine 24 h after the acute stress event rescued many of the changes observed in vulnerable rats, possibly contributing to support brain homeostasis. Overall, our results suggest that pivotal events, including reactive astrogliosis, changes in brain trophic factors, and neuronal damage are critical determinants of vulnerability to acute traumatic stress and confirm the therapeutic effect of acute ketamine against the development of stress-related psychiatric disorders.

Stressor control and regional inflammatory responses in the brain: regulation by the basolateral amygdala

Increasing evidence has connected the development of certain neuropsychiatric disorders, as well as neurodegenerative diseases, to stress-induced dysregulation of the immune system. We have shown that escapable (ES) and inescapable (IS) footshock stress, and memories associated with ES or IS, can differentially alter inflammatory-related gene expression in brain in a region dependent manner. We have also demonstrated that the basolateral amygdala (BLA) regulates stress- and fear memory-induced alterations in sleep, and that differential sleep and immune responses in the brain to ES and IS appear to be integrated during fear conditioning and then reproduced by fear memory recall. In this study, we investigated the role of BLA in influencing regional inflammatory responses within the hippocampus (HPC) and medial prefrontal cortex (mPFC) by optogenetically stimulating or inhibiting BLA in male C57BL/6 mice during footshock stress in our yoked shuttlebox paradigm based on ES and IS. Then, mice were immediately euthanized and RNA extracted from brain regions of interest and loaded into NanoString® Mouse Neuroinflammation Panels for compilation of gene expression profiles. Results showed differential regional effects in gene expression and activated pathways involved in inflammatory-related signaling following ES and IS, and these differences were altered depending on amygdalar excitation or inhibition. These findings demonstrate that the stress-induced immune response, or “parainflammation”, is affected by stressor controllability and that BLA influences regional parainflammation to ES or IS in HPC and mPFC. The study illustrates how stress-induced parainflammation can be regulated at the neurocircuit level and suggests that this approach can be useful for uncovering circuit and immune interactions in mediating differential stress outcomes.

Transcriptional Profiling of Rat Prefrontal Cortex after Acute Inescapable Footshock Stress.

Stress is a primary risk factor for psychiatric disorders such as Major Depressive Disorder (MDD) and Post Traumatic Stress Disorder (PTSD). The response to stress involves the regulation of transcriptional programs, which is supposed to play a role in coping with stress. To evaluate transcriptional processes implemented after exposure to unavoidable traumatic stress, we applied microarray expression analysis to the PFC of rats exposed to acute footshock (FS) stress that were sacrificed immediately after the 40 min session or 2 h or 24 h after. While no substantial changes were observed at the single gene level immediately after the stress session, gene set enrichment analysis showed alterations in neuronal pathways associated with glia development, glia-neuron networking, and synaptic function. Furthermore, we found alterations in the expression of gene sets regulated by specific transcription factors that could represent master regulators of the acute stress response. Of note, these pathways and transcriptional programs are activated during the early stress response (immediately after FS) and are already turned off after 2 h-while at 24 h, the transcriptional profile is largely unaffected. Overall, our analysis provided a transcriptional landscape of the early changes triggered by acute unavoidable FS stress in the PFC of rats, suggesting that the transcriptional wave is fast and mild, but probably enough to activate a cellular response to acute stress.

Connectivity and functional characterization of the mouse contralateral projecting neurons in basolateral amygdala

Amygdala is widely known as a critical neural hub for modulating negative emotions such as fear and anxiety. Recent studies have shown that the basolateral amygdala (BLA) has reciprocal innervation, which plays an important role in regulating social interaction and associative memory. However, the connectivity pattern and functional role of the contralateral projecting BLA neurons in stress-related anxiety are not fully understood. Here, using the virus tracing strategy, we first characterized the connectivity pattern of the contralateral projecting neurons in BLA. Then, we examined the effect of stress on the functional changes of those specific neuronal populations using a mouse model of inescapable footshock stress. The results showed that the fibers of the contralateral projecting BLA neurons labeled by enhanced green fluorescent protein (EGFP) have no collateral projections to the ipsilateral or contralateral hippocampus. Interestingly, they have some collateral projections to the ipsilateral medial prefrontal cortex. Behavioral results showed that footshock stress increases the anxiety-like behavior in mice; however, the excitatory synaptic transmission and intrinsic excitability of the contralateral projecting BLA neurons measured by whole-cell patch clamp recording did not change after stress exposure. In conclusion, our results indicate that contralateral projecting BLA neurons may not contribute to footshock stress-related anxiety-like behavior.

Differential effects of voluntary wheel running and toy rotation on the mRNA expression of neurotrophic factors and FKBP5 in a post-traumatic stress disorder rat model with the shuttle-box task

Life-threatening experiences can result in the development of post-traumatic stress disorder. We have developed an animal model for post-traumatic stress disorder (PTSD) using a shuttle box in rats. In this paradigm, the rats were exposed to inescapable foot-shock stress (IS) in a shuttle box, and then an avoidance/escape task was performed in the same box 2 weeks after IS. A previous study using this paradigm revealed that environmental enrichment (EE) ameliorated avoidance/numbing-like behaviors, but not hyperarousal-like behaviors, and EE also elevated hippocampal brain-derived neurotrophic factor (BDNF) expression. However, the differential effects of EE components, i.e., running wheel (RW) or toy rotation, on PTSD-like behaviors has remained unclear. In this experiment, we demonstrated that RW, toy rotation, and EE (containing RW and toy rotation) ameliorated avoidance/numbing-like behaviors, induced learning of avoidance responses, and improved depressive-like behaviors in traumatized rats. The RW increased the hippocampal mRNA expression of neurotrophic factors, especially BDNF and glial-cell derived neurotrophic factor. Toy rotation influenced FK506 binding protein 5 mRNA expression, which is believed to be a regulator of the hypothalamic-pituitary-adrenal (HPA)-axis system, in the hippocampus and amygdala. This is the first report to elucidate the differential mechanistic effects of RW and toy rotation. The former appears to exert its effects via neurotrophic factors, while the latter exerts its effects via the HPA axis. Further studies will lead to a better understanding of the influence of environmental factors on PTSD.

Metabolomics identifies perturbations in amino acid metabolism in the prefrontal cortex of the learned helplessness rat model of depression

Major depressive disorder is a serious psychiatric condition associated with high rates of suicide and is a leading cause of health burden worldwide. However, the underlying molecular mechanisms of major depression are still essentially unclear. In our study, a non-targeted gas chromatography–mass spectrometry-based metabolomics approach was used to investigate metabolic changes in the prefrontal cortex of the learned helplessness (LH) rat model of depression. Body-weight measurements and behavioral tests including the active escape test, sucrose preference test, forced swimming test, elevated plus-maze and open field test were used to assess changes in the behavioral spectrum after inescapable footshock stress. Rats in the stress group exhibited significant learned helpless and depression-like behaviors, while without any significant change in anxiety-like behaviors. Using multivariate and univariate statistical analysis, a total of 18 differential metabolites were identified after the footshock stress protocol. Ingenuity Pathways Analysis and MetaboAnalyst were applied for predicted pathways and biological functions analysis. “Amino Acid Metabolism, Molecule Transport, Small Molecule Biochemistry” was the most significantly altered network in the LH model. Amino acid metabolism, particularly glutamate metabolism, cysteine and methionine metabolism, arginine and proline metabolism, was significantly perturbed in the prefrontal cortex of LH rats.

Fear-potentiated behaviour is modulated by central amygdala angiotensin II AT1 receptors stimulation.

Central nucleus of the amygdala (CeA) is one of the most important regulatory centres for the emotional processes. Among the different neurotransmitter systems present in this nucleus, AT1 receptors have been also found, but their role in the generation and modulation of emotions is not fully understood. The present work evaluated the effect of intra-amygdalar injection of losartan (AT1 receptor antagonist) and angiotensin II (Ang II) in the anxiety state induced by fear-potentiated plus maze in male Wistar rats. Fear in the elevated plus maze can be potentiated by prior inescapable footshock stress. The decrease in the time spent in the open arms induced by the inescapable footshock was totally prevented by losartan (4 pmol) administration in CeA. It was also found that Ang II (48 fmol) administration decreased the time spent in the open arms in animals with or without previous footshock exposure. The locomotor activity and grooming behaviour were also evaluated. The results obtained from the different parameters analyzed allowed us to conclude that the Ang II AT1 receptors in CeA are involved in the anxiety state induced by stress in the fear-potentiated plus-maze behaviour.

Persistent inhibition of hippocampal long‐term potentiation in vivo by learned helplessness stress

The persistent cognitive disruptive effects of stress have been strongly implicated in the pathophysiology of depression and post-traumatic stress disorder. Here we examined factors influencing the time course of recovery from the inhibitory effect of acute inescapable stressors on the ability to induce long-term potentiation (LTP) in the dorsal hippocampus in vivo. We tested different forms of LTP, different stressors and different inbred strains of rats. Acute elevated platform stress completely, but transiently (<3 h), inhibited induction of both NMDA receptor-dependent LTP induced by a standard high frequency (200 Hz) conditioning stimulus and an additional LTP that required voltage-dependent Ca(2+) channel activation triggered by strong (400 Hz) conditioning stimulation. In contrast, acute inescapable footshock stress, used to study learned helplessness, inhibited LTP for at least 4 weeks. Contrary to expectations, there was no clear relationship between the ability of the footshock to trigger helpless behavior, a model of stress-induced depression, and the magnitude of LTP inhibition. Moreover, LTP did not appear to be affected by genetic susceptibility to learned helplessness, a model of genetic vulnerability to depression. This long-lasting synaptic plasticity disruption may underlie persistent impairment of hippocampus-dependent cognition by excessive acute inescapable stress.

GABAergic Antagonism of the Central Nucleus of the Amygdala Attenuates Reductions in Rapid Eye Movement Sleep After Inescapable Footshock Stress

Rapid eye movement sleep (REM) appears to be especially susceptible to the effects of stress; inescapable footshock stress (IS) can produce reductions in REM that can occur without recovery sleep. The amygdala has well-established roles in stress and emotion; the central nucleus of the amygdala (CNA) projects to REM regulatory regions in the brainstem and has been found to play a key role in the regulation of REM. The objective of this study was to determine whether the reduction in REM induced by IS could be regulated by CNA and brainstem regions. The GABAergic agonist muscimol (MUS) and GABAergic antagonist bicuculline (BIC) were microinjected into CNA before IS, and sleep was recorded for 20 h. In a second experiment using the same manipulations, sleep was recorded for 2 h, after which the rats were killed to evaluate Fos expression (a marker of neuronal activity) in the locus coeruleus (LC), a brainstem REM regulatory region. NA. The subjects were male, outbred Wistar rats. The rats were surgically implanted with standard electrodes or with telemetry transmitters for determining arousal state. IS preceded by control or MUS microinjections selectively reduced REM and increased Fos expression in LC. By comparison, microinjection of BIC into CNA prior to IS attenuated both the reduction in REM and Fos expression in LC to levels seen in non-shocked controls. The results suggest that the effects of IS on REM may involve local GABAergic inhibition in CNA and activation of LC.

Relation between the Hypothalamic-Pituitary-Thyroid (HPT) Axis and the Hypothalamic-Pituitary-Adrenal (HPA) Axis during Repeated Stress

Previous work has indicated that acute and repeated stress can alter thyroid hormone secretion. Corticosterone, the end product of hypothalamic-pituitary-adrenal (HPA) axis activation and strongly regulated by stress, has been suggested to play a role in hypothalamic-pituitary-thyroid (HPT) axis regulation. In the current study, we sought to further characterize HPT axis activity after repeated exposure to inescapable foot-shock stress (FS), and to examine changes in proposed regulators of the HPT axis, including plasma corticosterone and hypothalamic arcuate nucleus agouti-related protein (AGRP) mRNA levels. Adult male Sprague-Dawley rats were subjected to one daily session of inescapable FS for 14 days. Plasma corticosterone levels were determined during and after the stress on days 1 and 14. Animals were killed on day 15, and trunk blood and brains were collected for measurement of hormone and mRNA levels. Repeated exposure to FS led to a significant decrease in serum levels of 3,5,3′-triiodothyronine (T₃) and 3,5,3′,5′-tetraiodothyronine (T₄). Stress-induced plasma corticosterone levels were not altered by repeated exposure to the stress. Despite the decrease in peripheral hormone levels, thyrotropin-releasing hormone (TRH) mRNA levels within the paraventricular nucleus of the hypothalamus were not altered by the stress paradigm. Arcuate nucleus AGRP mRNA levels were significantly increased in the animals exposed to repeated FS. Additionally, we noted significant correlations between stress-induced plasma corticosterone levels and components of the HPT axis, including TRH mRNA levels and free T₄ levels. Additionally, there was a significant correlation between AGRP mRNA levels and total T₃ levels. Changes in body weight were also correlated with peripheral corticosterone and TRH mRNA levels. These results suggest that repeated exposure to mild-electric foot-shock causes a decrease in peripheral thyroid hormone levels, and that components of the HPA axis and hypothalamic AGRP may be involved in stress regulation of the HPT.

Possible neural mechanisms involved in footshock stress-induced enhancement of exploratory behavior in mice.

The effect of inescapable footshock stress on open-field activity, as measured by the number of ambulations, was studied in male mice. Ambulations significantly increased after footshock stress, the most significant effect appeared after 20 min-stimulation and the effect decreased as footshock time lengthened. The footshock stress-induced enhancement of ambulation was inhibited by haloperidol (0.2, 0.5 and 1 mg/kg), phentolamine (5 and 10 mg/kg), mianserin (20 mg/kg), atropine (0.5, 1 and 2 mg/kg), naltrexone (10 mg/kg) and MK-801 (0.05, 0.1 and 0.2 mg/kg), but was not influenced by propranolol (5, 10 and 20 mg/kg) or diazepam (1, 2 and 5 mg/kg). Haloperidol (0.5 and 1 mg/kg) and mianserin (5, 10 and 20 mg/kg) also exerted an inhibitory effect on non-stressed normal mice. These results suggest that dopaminergic, alpha-adrenergic, cholinergic, opioidergic and N-methyl-D-aspartate (NMDA) receptor-mediated neurotransmission systems are involved in the footshock stress-induced ambulatory activation.

Intermittent but Not Continuous Inescapable Footshock Stress Affects Immune Responses and Immunocyte Beta-Endorphin Concentrations in the Rat

It is well known that a variety of stressors influence immune responses. The opioid peptide-beta-endorphin (BE) is deeply involved in stress responses, is synthesized in cells of the immune system, and participates in the modulation of immune function. We analyzed the ability of two different stress paradigms to modulate the beta-endorphin concentrations in the immune cells and the immune response in the rat. Two and 24 h after the exposure to inescapable intermittent footshock (1.6 mA, 60 Hz, 1 s, every 5 s for 20 min) the concentrations of beta-endorphin in splenocytes, peripheral blood mononuclear cells and lymph node cells were significantly increased. In contrast, the exposure to a continuous footshock for 3 min did not affect the concentrations of the opioid peptide. Similarly, phytohemoagglutinin-induced proliferation of splenocytes and natural killer activity were significantly impaired only after the exposure to intermittent footshock stress. On the contrary, plasma corticosterone levels were similarly elevated after both paradigms of stress. The pretreatment with the corticotropin-releasing hormone (CRH) receptor antagonist prevented both the stress-induced increase of immunocyte BE and immunosuppression. In conclusion, our data suggest that intermittent and continuous footshock stressors activate different neuroendocrine responses and that CRH plays a central role in mediating the immune effects of the intermittent footshock stress. The possible relationship between the beta-endorphin changes and immunosuppression is discussed.

Enhanced processing of an aversive memory following inescapable shock in rats

The aim of the present study was to determine the effect of inescapable shock on appetitive and aversive memory retrieval. Rats conditioned in a T-maze with appetitive (10% sucrose) and aversive (2.0 mA) events were subjected, respectively, ti inescapable, escapable, and no-shock stress treatments. A retention test conducted 48 hr following exposure to stress treatments showed an enhanced avoidance behavior to enter a previously shocked goalbox with the absence of such a difference in responding to the nonshocked goalbox by the animals exposed to inescapable footshock stress. This differential response was not observed in two other groups, one given escapable footshock and the other no shock at all. This finding suggests memory processing is selectively enhanced in the helplessness condition and parallel to the retrieval bias that is evidenced in humans in depressive mood and induced helplessness.

Footshock stress-induced supersensitivity to isoprenaline in the isolated pacemaker of the rat: Effects of the compounds RU-38486 and RU-28362

1. 1. Three daily sessions of inescapable footshock stress of 30 min duration each increased the sensitivity of the isolated pacemaker of the rat to the chronotropic effect of isoprenaline. 2. 2. The effect of inescapable footshock stress on the sensitivity of the isolated rat pacemaker to the chronotropic effect of isoprenaline was prevented by the daily administration of the compound RU-38486, a potent antiglucocorticoid which blocks the cytosolic receptor for corticosterone. 3. 3. The administration of the compound RU-28362, a potent agonist of the cytosolic receptor for corticosterone, during 3 days to rats which were not submitted to footshock stress induces supersensitivity to the chronotropic effect of isoprenaline. 4. 4. It is concluded that corticosterone plays an important role in the qualitative control of the rat pacemaker beta-adrenoceptor population during adaptation to repeated footshock stress.

Direct evidence of conditioned fear-elicited enhancement of noradrenaline release in the rat hypothalamus assessed by intracranial microdialysis

Inescapable footshock stress produced marked increases in noradrenaline (NA) release, which was assessed by intracranial microdialysis, in the hypothalamus of conscious rats. Emotional stress, without physical stimuli (replacement to the environment where the rats had received footshock previously), also increased hypothalamic NA release. These results suggest that foodshock stress caused increases in NA release and this activation of NA neurons appears to be reinstated simply by re-exposure to the environment previously associated with shock.

Chronic stress supersensitizes rats to the inhibition of locomotion produced by arecoline

Chronic inescapable footshock and swim stress supersensitize a hypothalamic muscarinic mechanism which regulates core body temperature. We studied the inhibitory effects of arecoline, a muscarinic agonist, on locomotion in rats before and after chronic swim stress in order to examine the effects of stress on an extra-hypothalamic muscarinic mechanism. We counted crossings in an open field during the days before and after six days of twice-daily swim stress (8 minutes/session at 12C). We first examined the effects of eight doses of ip arecoline (0.01 to 2.0 mg/kg) and normal saline (N = 6 to 8 rats/group) on crossings before and after chronic stress. Doses greater than 0.25 mg/kg inhibited pre-stress crossings relative to saline and did not potentiate the inhibition of locomotion post-stress. The 0.125 mg/kg dose reduced crossings post-stress relative to saline. We reexamined the effects of the 0.125 mg/kg dose saline (N = 20 rats/group) on crossings before and after six days of swim stress. Chronic stress potentiated arecoline-induced inhibition of locomotion. Our results suggest that chronic uncontrollable stressors may supersensitize an extra-hypothalamic muscarinic mechanism involved in the stress response and the pathophysiology of stress-related disease such as major depression.

The involvement of endogenous opiate systems in learned helplessness and stress-induced analgesia.

The participation of endogenous opiate systems in the induction and expression of learned helplessness (LH) and stress-induced analgesia (SIA) was investigated in rats exposed to escapable and inescapable footshock. Following an initial footshock, analgesia was observed only in those animals that could not control their stress exposure and this SIA was prevented by the administration of naloxone. Analgesia was no longer evident in this inescapable group after 48 h. However, exposure to a shuttlebox escape task at this time reinstated the SIA but did not produce SIA in animals previously exposed to escapable footshock. Shuttlebox escape deficits indicative of LH were also exhibited by animals that received an inescapable footshock stress 48 h prior to testing. The analgesia and LH observed in the inescapable group were not affected by the administration of naloxone (3 mg/kg, IP) 10 min before shuttlebox exposure but were prevented when the same dose of naloxone was given before the initial stress. Thus, endogenous opiates clearly participate in the initial induction of LH and SIA and, although the degree of endogenous opiate involvement in the subsequent expression of these behaviors is unclear, it seems evident that their expression can occur in the presence of opiate antagonism and may therefore require the participation of additional transmitter systems.

Sister chromatid exchanges induced by behavioral stress.

Rats subjected to swim stress showed a doubling of sister chromatid exchange (SCE) level. In a second experiment, the generality of SCE induction by behavioral stressors was tested by exposing rats to either swim, white noise, or either continuous or intermittent inescapable footshock stress. The induction of SCEs, although to differing degrees, by qualitatively different stressors, demonstrates that this is a general phenomenon of stress. There may be a cellular genetic basis for some of the effects of stress.

Changes in d-amphetamine elicited rotational behavior in rats exposed to uncontrollable footshock stress

Male and female rats, selected on the basis of their rotational behavior in response to d-amphetamine, were exposed to either escapable footshock stress, inescapable footshock stress or no stress and were then given a shuttlebox escape task on the subsequent day. Following testing, the magnitude and direction of the animals' rotational responses to d-amphetamine were determined again. Inescapable footshock stress induced a selective change in the direction and intensity of rotational behavior that was dependent upon the subjects' sex and preexisting rotational bias. Right-rotating males and left-rotating females shifted their directional bias toward the opposite side, while left-rotating males and right-rotating females displayed increased rotation in their pre-stress direction. Significant correlations were also noted between the intensity of pre-stress rotational behavior and performance on the shuttlebox shock escape task. The results are discussed with respect to stress' actions on the mesocortical dopamine system and how this system's sex-dependent asymmetrical organization may subserve part of the organism's general reaction to uncontrollable stress.

Subsensitivity to β-adrenoceptor agonists in right atria isolated from footshock-stressed rats

1. The effects of three daily sessions of inescapable footshock on the sensitivity of rat isolated right atria to the chronotropic effect of norepinephrine, tyramine and soterenol were studied. 2. Inescapable footshock induces subsensitivity to norepinephrine and tyramine. The maximum response to the partial agonist soterenol was reduced. 3. In vitro denervation and addition of cocaine prevented the demonstration of inescapable footshock-induced subsentivity to norepinephrine. 4. It is concluded that repeated inescapable footshock stress reduces the number of atrial β 1-adrenoceptors and increases the efficiency of the neuronal reputake process.