Acute Restraint Stress Research Articles

PACAP Controls Endocrine and Behavioral Stress Responses via Separate Brain Circuits

BackgroundThe neuropeptide PACAP (pituitary adenylate cyclase-activating polypeptide) is a master regulator of central and peripheral stress responses, yet it is not clear how PACAP projections throughout the brain execute endocrine and behavioral stress responses. MethodsWe used AAV (adeno-associated virus) neuronal tracing, an acute restraint stress (ARS) paradigm, and intersectional genetics, in C57BL/6 mice, to identify PACAP-containing circuits controlling stress-induced behavior and endocrine activation. ResultsPACAP deletion from forebrain excitatory neurons, including a projection directly from medial prefrontal cortex to hypothalamus, impairs c-fos activation and corticotropin-releasing hormone (CRH) messenger RNA elevation in the paraventricular nucleus after 2 hours of restraint, without affecting ARS-induced hypophagia, or c-fos elevation in nonhypothalamic brain. Elimination of PACAP within projections from lateral parabrachial nucleus to extended amygdala, on the other hand, attenuates ARS-induced hypophagia, along with extended amygdala fos induction, without affecting ARS-induced CRH messenger RNA elevation in the paraventricular nucleus. PACAP projections to extended amygdala terminate at protein kinase C delta type (PKCδ) neurons in both the central amygdala and the oval bed nucleus of the stria terminalis. Silencing of PKCδ neurons in the central amygdala, but not in the oval bed nucleus of the stria terminalis, attenuates ARS-induced hypophagia. Experiments were carried out in mice of both sexes with n ≥ 3 per group. ConclusionsA frontocortical descending PACAP projection controls paraventricular nucleus CRH messenger RNA production to maintain hypothalamic-pituitary-adrenal axis activation and regulate the endocrine response to stress. An ascending PACAPergic projection from the external lateral parabrachial nucleus to PKCδ neurons in the central amygdala regulates behavioral responses to stress. Defining two separate limbs of the acute stress response provides broader insight into the specific brain circuitry engaged by the psychogenic stress response.

Poststress social isolation exerts anxiolytic effects by activating the ventral dentate gyrus

After aversive stress, people either choose to return to their previously familiar social environment or tend to adopt temporary social withdrawal to buffer negative emotions. However, which behavior intervention is more appropriate and when remain elusive. Here, we unexpectedly found that stressed mice experiencing social isolation exhibited less anxiety than those experiencing social contact. Within the first 24 h after returning to their previous social environment, mice experienced acute restraint stress (ARS) displayed low social interest but simultaneously received excessive social disturbance from their cage mates, indicating a critical time window for social isolation to balance the conflict. To screen brain regions that were differentially activated between the poststress social isolation and poststress social contact groups, we performed ΔFosB immunostaining and found that ΔFosB + signals were remarkably increased in the vDG of poststress social isolation group compared with poststress social contact group. There were no significant differences between the two groups in the other anxiety- and social-related brain regions, such as prelimbic cortex, infralimbic cortex, nucleus accumbens, etc. These data indicate that vDG is closely related to the differential phenotypes between the poststress social isolation and poststress social contact groups. Electrophysiological recording, further, revealed a higher activity of vDG in the poststress social isolation group than the poststress social contact group. Chemogenetically inhibiting vDG excitatory neurons within the first 24 h after ARS completely abolished the anxiolytic effects of poststress social isolation, while stimulating vDG excitatory neurons remarkably reduced anxiety-like behaviors in the poststress social contact group. Together, these data suggest that the activity of vDG excitatory neurons is essential and sufficient to govern the anxiolytic effect of poststress social isolation. To the best of our knowledge, this is the first report to uncover a beneficial role of temporal social isolation in acute stress-induced anxiety. In addition to the critical 24-h time window, activation of vDG is crucial for ameliorating anxiety through poststress social isolation.

High-Fat Diet Exacerbates Stress Responsivity in Juvenile Traumatic Brain Injury.

Abstract Traumatic brain injury (TBI) is one of the leading causes of death for children in the United States. Juveniles are more likely to sustain TBIs than most other age groups, and TBI has been shown to result in increased anxiety and stress behaviors. In addition, the hypothalamic-pituitary-adrenal (HPA) axis has previously been shown to become dysregulated after a TBI. Further, many children consume diets high in saturated fats and refined sugars, which are also connected to alterations in HPA axis function and behavior disorders. The goal of the current study was to identify a potential relationship between high-fat diet (HFD) consumption and TBI on HPA axis function in juvenile rats. In the present study, male juvenile Long-Evans rats were fed either a combination of an HFD with a high-fructose corn syrup solution or a standard chow diet. On post-natal Day 30, subjects sustained either a sham TBI or a TBI via the Closed-Head Injury Model of Engineered Rotational Acceleration (CHIMERA). Subjects participated in a trial of the open field test (OFT) following injury. In addition, some rats performed in an acute restraint stress test. All subjects were euthanized 7 days post-injury. Brain and blood plasma samples were collected for use in real-time polymerase chain reaction (RT-PCR), immunohistochemistry, and corticosterone or adrenocorticotropic hormone (ACTH) assays. Immediately following TBI, injured juveniles had increased time to righting and walking, with HFD-fed TBI rats having increased time to walking over Chow-fed TBI rats. HFD-fed TBI rats had a reduced number of entries to the center of the OFT, in addition to reduced time spent in the center compared with HFD Sham controls and Chow TBI rats. During the acute restraint stress test, HFD-fed TBI rats had elevated pre-stress ACTH and corticosterone and post-stress ACTH levels. Pre-stress ACTH levels were significantly elevated in HFD TBI compared with Chow TBI. Further, pre-stress ACTH:corticosterone ratios were elevated in HFD TBI compared with Chow TBI. cFos immunoreactivity in the paraventricular nucleus (PVN) of the hypothalamus following the acute restraint stress test was elevated in HFD-fed TBI rats. HFD TBI rats had greater activation of cFos in the PVN compared with Chow TBI. In addition, RT-PCR showed significantly reduced expression of relevant HPA axis genes, NR3C1, NR3C2, and CRHR2, in the hypothalamus of TBI subjects compared with Sham subjects. Further, AVP and CRHR2 in the hypothalamus were significantly reduced in HFD TBI compared with Chow TBI. These results offer evidence that TBI paired with high-fat diet consumption can cause HPA axis dysfunction, resulting in more anxiety-like behaviors.

Synthetic corticotropins and the GABA-receptor system: Direct and delayed effects.

The central effectors of the stress system are greatly interconnected and include, among others, a large group of peptides derived from proopiomelanocortin. In addition to natural corticotropins, a number of artificial molecules that contain some ACTH fragments in their structure are also referred to members of this family. Some of them possess a wide range of biological activity. The molecular mechanism underlying the biological activity of such peptides is partly based on allosteric modulation of various receptors. We analyzed the ability of some biologically active synthetic corticotropins (ACTH(4-7)PGP, ACTH(6-9)PGP, ACTH(7-10)PGP), and glyproline PGPL to affect the GABA-receptor system of rat brain. The effects of the peptides were studied in the isolated plasma membranes of brain cells, as well as after systemic peptide administration in the rat model of acute restraint stress. The delayed effect of stress or preadministration of each of the studied peptides on [3 H]GABA binding was different for its high- and low-affinity-specific sites. The studied peptides individually affected the binding of [3 H]GABA in their own way. Acute restraint stress caused a decrease in [3 H]GABA binding at its low-affine site and did not affected the high-affine site. Preliminary peptide administration did not influence this effect of stress.

Body surface temperature of rats reveals both magnitude and sex differences in the acute stress response

Understanding how biological markers of stress relate to stressor magnitude is much needed and can be used in welfare assessment. Changes in body surface temperature can be measured using infrared thermography (IRT) as a marker of a physiological response to acute stress. While an avian study has shown that changes in body surface temperature can reflect the intensity of acute stress, little is known about surface temperature responses to stressors of different magnitudes and its sex-specificity in mammals, and how they correlate with hormonal and behavioural responses. We used IRT to collect continuous surface temperature measurements of tail and eye of adult male and female rats (Rattus norvegicus), for 30 minutes after exposure to one of three stressors (small cage, encircling handling or rodent restraint cone) for one minute, and cross-validated the thermal response with plasma corticosterone (CORT) and behavioural assessment. To obtain individual baseline temperatures and thermal responses to stress, rats were imaged in a test arena (to which they were habituated) for 30 seconds before and 30 minutes after being exposed to the stressor. In response to the three stressors, tail temperature initially decreased and then recovered to, or overshot the baseline temperature. Tail temperature dynamics differed between stressors; being restrained in the small cage was associated with the smallest drop in temperature, in male rats, and the fastest thermal recovery, in both sexes. Increases in eye temperature only distinguished between stressors early in the response and only in females. The post stressor increase in eye temperature was greater in the right eye of males and the left eye of females. In both sexes encircling may have been associated with the fastest increase in CORT. These results were in line with observed behavioural changes, with greater movement in rats exposed to the small cage and higher immobility after encircling. The female tail and eye temperature, as well as the CORT concentrations did not return to pre-stressor levels in the observation period, in conjunction with the greater occurrence of escape-related behaviours in female rats. These results suggest that female rats are more vulnerable to acute restraint stress compared to male rats and emphasise the importance of using both sexes in future investigations of stressor magnitude. This study demonstrates that acute stress induced changes in mammalian surface temperature measured with IRT relate to the magnitude of restraint stress, indicate sex differences and correlate with hormonal and behavioural responses. Thus, IRT has the potential to become a non-invasive method of continuous welfare assessment in unrestrained mammals.

Chronic stress induced loudness hyperacusis, sound avoidance and auditory cortex hyperactivity

Hyperacusis, a debilitating loudness intolerance disorder, has been linked to chronic stress and adrenal insufficiency. To investigate the role of chronic stress, rats were chronically treated with corticosterone (CORT) stress hormone. Chronic CORT produced behavioral evidence of loudness hyperacusis, sound avoidance hyperacusis, and abnormal temporal integration of loudness. CORT treatment did not disrupt cochlear or brainstem function as reflected by normal distortion product otoacoustic emissions, compound action potentials, acoustic startle reflexex, and auditory brainstem responses. In contrast, the evoked response from the auditory cortex was enhanced up to three fold after CORT treatment. This hyperactivity was associated with a significant increase in glucocorticoid receptors in auditory cortex layers II/III and VI. Basal serum CORT levels remained normal after chronic CORT stress whereas reactive serum CORT levels evoked by acute restraint stress were blunted (reduced) after chronic CORT stress; similar changes were observed after chronic, intense noise stress. Taken together, our results show for the first time that chronic stress can induce hyperacusis and sound avoidance. A model is proposed in which chronic stress creates a subclinical state of adrenal insufficiency that establishes the necessary conditions for inducing hyperacusis.

Bed nucleus of the stria terminalis CB1 receptors and the FAAH enzyme modulate anxiety behavior depending on previous stress exposure

The endocannabinoid (eCB) anandamide (AEA) is synthesized on-demand in the post-synaptic terminal and can act on presynaptic cannabinoid type 1 (CB1) receptors, decreasing the release of neurotransmitters, including glutamate. AEA action is ended through enzymatic hydrolysis via FAAH (fatty acid amid hydrolase) in the post-synaptic neuron. eCB system molecules are widely expressed in brain areas involved in the modulation of fear and anxiety responses, including the Bed Nucleus of the Stria Terminalis (BNST), which is involved in the integration of autonomic, neuroendocrine, and behavioral regulation. The presence of the CB1 and FAAH was described in the BNST; however, their role in the modulation of defensive reactions is not fully comprehended. In the present work we aimed at investigating the role of AEA and CB1 receptors in the BNST in modulating anxiety-related behaviors. Adult male Wistar rats received local BNST injections of the CB1 receptor antagonist AM251 (0.1–0.6 nmol) and/or the FAAH inhibitor (URB597; 0.001–0.1 nmol) and were evaluated in the elevated plus maze (EPM) test, with or without previous acute restraint stress (2 h) exposure, or in the contextual fear conditioning. We observed that although AM251 and URB597 had no effects on the EPM, they increased and decreased, respectively, the conditioned fear response. Supporting a possible influence of stress in these differences, URB597 was able to prevent the restraint stress-induced anxiogenic effect in the EPM. The present data, therefore, suggest that eCB signaling in the BNST is recruited during more aversive situations to counteract the stress effect.

Restraint stress induced the antinociceptive responses via the dopamine receptors within the hippocampal CA1 area in animal model of persistent inflammatory pain

It has been declared that dopamine receptors within the hippocampal formation are involved in emotion, memory, and pain processing. Remarkably, both CA1 and dentate gyrus (DG) areas of the hippocampal formation are involved in persistent peripheral nociceptive perception. A prior study showed that dopamine receptors within the hippocampal DG have a critical role in antinociception induced by forced swim stress (FSS), as a physical stressor, in the presence of formalin irritation. The present experiments were designed to assess whether dopaminergic receptors within the CA1 have any role in antinociceptive responses induced by restraint stress (RS) as a psychological stressor after applying the formalin test as an animal model of persistent inflammatory pain. The D1- and D2-like dopamine receptor antagonists, SCH23390 and Sulpiride (0.25, 1, and 4 μg/0.5 μl), were injected into the CA1 areas of ninety-six male albino Wistar rats 5 min before a 3-h period of restraint stress. Ten min after stress termination, a 50-μl formalin 2.5 % was subcutaneously injected into the plantar surface of the rat’s hind paw to induce persistent inflammatory pain. Nociceptive behaviors in both phases of the formalin test were analyzed in the 5-min blocks for a 60-min period. The obtained results demonstrate that although RS could induce an antinociceptive response in both phases of the formalin test, microinjection of D1- and D2-like dopamine receptors, antagonists attenuated RS-induced analgesia. These results support the hypothesis that acute restraint stress could induce analgesia via dopaminergic projection to the CA1 region of the hippocampal formation.

Angiotensinergic neurotransmission in the bed nucleus of the stria terminalis is involved in cardiovascular responses to acute restraint stress in rats.

The brain angiotensin II acting via AT1 receptors is a prominent mechanism involved in physiological and behavioral responses during aversive situations. The AT2 receptor has also been implicated in stress responses, but its role was less explored. Despite these pieces of evidence, the brain sites related to control of the changes during aversive threats by the brain renin-angiotensin system (RAS) are poorly understood. The bed nucleus of the stria terminalis (BNST) is a limbic structure related to the cardiovascular responses by stress, and components of the RAS system were identified in this forebrain region. Therefore, we investigated the role of angiotensinergic neurotransmission present within the BNST acting via local AT1 and AT2 receptors in cardiovascular responses evoked by an acute session of restraint stress in rats. For this, rats were subjected to bilateral microinjection of either the angiotensin-converting enzyme inhibitor captopril, the selective AT1 receptor antagonist losartan, or the selective AT2 receptor antagonist PD123319 before they underwent the restraint stress session. We observed that BNST treatment with captopril reduced the decrease in tail skin temperature evoked by restraint stress, without affecting the pressor and tachycardic responses. Local AT2 receptor antagonism within the BNST reduced both the tachycardia and the drop in tail skin temperature during restraint. Bilateral microinjection of losartan into the BNST did not affect the restraint-evoked cardiovascular changes. Taken together, these data indicate an involvement of BNST angiotensinergic neurotransmission acting via local AT2 receptors in cardiovascular responses during stressful situations.

Nanoparticulate MgH2 ameliorates anxiety/depression-like behaviors in a mouse model of multiple sclerosis by regulating microglial polarization and oxidative stress

Multiple sclerosis (MS) is an autoimmune demyelinating disease of the central nervous system (CNS). Anxiety and depression are the most common psychiatric comorbidities of MS, which seriously affect patients’ quality of life, treatment compliance, and prognosis. However, current treatments for anxiety and depression in MS show low therapeutic efficacy and significant side effects. In the present study, we explored the therapeutic effects of a novel low-toxic anti-inflammatory drug, nanoparticulate magnesium hydride (MgH2), on mood disorders of MS. We observed that anxiety/depression-like behaviors in experimental autoimmune encephalomyelitis (EAE) mice were alleviated by MgH2 treatment. In addition, disease severity and inflammatory demyelination were also diminished. Furthermore, we confirmed the suppressive effect of MgH2 on depression in the acute restraint stress model. Mechanistically, MgH2 may play a therapeutic role by promoting microglial M2 polarization, inhibiting microglial M1 polarization, and reducing oxidative stress and mitochondrial damage. Therefore, nanoparticulate MgH2 may be a promising therapeutic drug for psychiatric comorbidities of MS.

Regulation of Cortico-Thalamic JNK1/2 and ERK1/2 MAPKs and Apoptosis-Related Signaling Pathways in PDYN Gene-Deficient Mice Following Acute and Chronic Mild Stress.

The crosstalk between the opioidergic system and mitogen-activated protein kinases (MAPKs) has a critical role in mediating stress-induced behaviors related to the pathophysiology of anxiety. The present study evaluated the basal status and stress-induced alterations of cortico-thalamic MAPKs and other cell fate-related signaling pathways potentially underlying the anxiogenic endophenotype of PDYN gene-deficient mice. Compared to littermates, PDYN knockout (KO) mice had lower cortical and or thalamic amounts of the phospho-activated MAPKs c-Jun N-terminal kinase (JNK1/2) and extracellular signal-regulated kinase (ERK1/2). Similarly, PDYN-KO animals displayed reduced cortico-thalamic densities of total and phosphorylated (at Ser191) species of the cell fate regulator Fas-associated protein with death domain (FADD) without alterations in the Fas receptor. Exposure to acute restraint and chronic mild stress stimuli induced the robust stimulation of JNK1/2 and ERK1/2 MAPKs, FADD, and Akt-mTOR pathways, without apparent increases in apoptotic rates. Interestingly, PDYN deficiency prevented stress-induced JNK1/2 and FADD but not ERK1/2 or Akt-mTOR hyperactivations. These findings suggest that cortico-thalamic MAPK- and FADD-dependent neuroplasticity might be altered in PDYN-KO mice. In addition, the results also indicate that the PDYN gene (and hence dynorphin release) may be required to stimulate JNK1/2 and FADD (but not ERK1/2 or Akt/mTOR) pathways under environmental stress conditions.

Multiple brain regions are involved in reaction to acute restraint stress in CYLD-knockout mice

The lysine 63 deubiquitinase cylindromatosis (CYLD) is expressed at high levels in the brain and is considered to be involved in anxious and depressive behavior, cognitive inflexibility, and autism disorders. Previous research was limited in some brain regions, including the hippocampus, striatum, and amygdala. To better understand whether CYLD plays a role in adaptation to stress and which brain regions are involved, we analyzed the behavior of CYLD-knockout mice in the elevated plus maze (EPM) and light-dark box test (LDT) after acute restraint stress (ARS) and mapped their c-Fos immunoreactivity in brain sections. Here we report that CYLD deficiency leads to an unexpected reaction to ARS in mice, and is accompanied by significant neuronal activation of brain regions including the medial prefrontal cortex (mPFC), dorsal striatum (DS), nucleus accumbens (NAc), and basal lateral amygdala (BLA), but not ventral hippocampus (vHPC). Our findings show that CYLD participates in ARS-induced anxious behavior and that this involves multiple brain regions.

Sleep restriction during opioid abstinence affects the hypothalamic-pituitary-adrenal (HPA) axis in male and female rats

Hypothalamic-pituitary-adrenal (HPA) axis dynamics are disrupted by opioids and may be involved in substance abuse; this persists during withdrawal and abstinence and is associated with co-morbid sleep disruption leading to vulnerability to relapse. We hypothesized that chronic sleep restriction (SR) alters the HPA axis diurnal rhythm and the sexually dimorphic response to acute stressor during opioid abstinence. We developed a rat model to evaluate the effect of persistent sleep loss during opioid abstinence on HPA axis dynamics in male and female rats. Plasma ACTH and corticosterone were measured diurnally and in response to acute restraint stress in rats Before (control) compared to During subsequent opioid abstinence without or with SR. Abstinence, regardless of sleep state, led to an increase in plasma ACTH and corticosterone in the morning in males. There was a tendency for higher PM plasma ACTH during abstinence in SR males (p = 0.076). ACTH and corticosterone responses to restraint were reduced in male SR rats whereas there was a failure to achieve the post-restraint nadir in female SR rats. There was no effect of the treatments or interventions on adrenal weight normalized to body weight. SR resulted in a dramatic increase in hypothalamic PVN AVP mRNA and plasma copeptin in male but not female rats. This corresponded to the attenuation of the HPA axis stress response in SR males during opioid abstinence. We have identified a potentially unique, sexually dimorphic role for magnocellular vasopressin in the control of the HPA axis during opioid abstinence and sleep restriction.

The resilience of adolescent male rats to acute stress-induced delayed anxiety is age-related and glucocorticoid release-dependent

Studies investigated how stressful experiences modulate physiological and behavioral responses and the consequences of stress-induced corticosterone release in anxiety-like behavior. Adolescence is crucial to brain maturation, and several neurobiological changes in this period lead individuals to increased susceptibility or resilience to aversive situations. Despite the effects of stress in adults, information about adolescents' responses to acute stress is lacking. We aimed to understand how adolescence affects acute stress responses. Male adolescent rats (30 days old) were 2 h restrained, and anxiety-like behaviors were measured immediately or 10 days after stress in the elevated plus-maze (EPM) and the light-dark box (LDB) tests. To verify the importance of CORT modulation in stress-induced anxiety, another group of rats was treated, 30 min before restraint, with metyrapone to blunt the stress-induced CORT peak and tested immediately after stress. To show that stress effects on behavior were age-dependent, another set of rats was tested in two different periods – early adolescence (30 days old) and mid-adolescence (40 days old) and were treated or not with metyrapone before the stress session and tested immediately or ten days later in the LDB test. Only early adolescent male rats were resilient to delayed anxiety-like behavior in EPM and LDB tests. Metyrapone treatment increased the rats' exploration immediately and ten days after stress. These data suggest a specific age at which adolescent rats are resilient to the delayed effects of acute restraint stress and that the metyrapone treatment has long-term behavioral consequences.

Pre-clinical Investigations of Therapeutic Markers Associated with Acute and Chronic Restraint Stress: A Nuclear Magnetic Resonance Based Contrast Metabolic Approach

Stress can be defined by two parameters, first the psychological sensing of pressure and second is the body's response. However, the exposure time to stress depicts the biological response produced against it. The effect of acute and chronic restraint stress on anxiety and the production of systemic metabolites were investigated in male Sprague-Dawley (SD) rats. Behavioural test was performed on elevated plus maze (EPM) in conjunction with the statistical analysis that exhibited the habituation during long term exposure to stress when compared with the short-term stress. These behaviour-based changes resulted in interpolated concentration of some serum metabolites like carbohydrates, amino acids and lipids as analysed by NMR. Metabolic analysis along with the multivariate analysis demonstrated that the expression of concentration of metabolites including glutamate, proline, succinate, citrate, and tyrosine is higher in the acute stress than the chronic stress, while glucose and lipids i.e., LDL and VLDL changed in the opposite trends. Thus, the aforesaid study provides an analytical strategy for the characterization of perturbed metabolites induced due to the behavioural modifications in an organism. It may further aid in developing potential therapeutic markers at the metabolic levels which may broaden the treatment options for stress and anxiety related disorders.

A 2-hit model of early life stress and later life restraint stress: Susceptibility or resilience to anxiety and alcohol drinking?

Early life stress adversely influences neurodevelopment and has profound long-term effects on brain function and behavior. Here we measure endogenous stress hormone and determine locomotor, anxiogenic, and operant binge drinking behavior of rats exposed to a 2-hit model of maternal deprivation (MD) stress in infancy followed by acute (ARS) or chronic restraint stress (CRS) paradigms during adolescence/adulthood to determine resiliency or susceptibility to the second stress exposure. Adolescents (n=40) exhibited higher baseline corticosterone (CORT) levels than adults (n=40) and with CRS exposure, showed significantly elevated circulating CORT irrespective of MD exposure status. In adults, MD females had lower baseline CORT levels than controls, whereas MD and control male CORT were indistinguishable. For behavior, N=40 was used in total. In the open field, locomotion was significantly decreased upon ARS, with MD having a significant sustained impact on ambulatory measures with CRS. When tested for anxiety-like behavior on an elevated zero maze, ARS interacted with MD to induce protracted and more profound anxiogenic behavior. Upon CRS, MD anxiogenic behavior was reversed to baseline levels whereas controls, especially males, exceeded baseline levels to spend significantly more time in the open arms of the EZM. Finally, in testing for binge drinking behavior, MD rats lever-pressed significantly more for 10% ethanol on an operant apparatus and exhibited a 3-fold greater drinking baseline than controls. Binge drinking behavior was further elevated with ARS, but quickly returned to the MD baseline even prior to reaching CRS. By contrast, controls had much lower baseline but significantly elevated binge drinking behavior by 3–5-fold in response to ARS which remained elevated through CRS, stabilizing at MD baseline levels. Sex differences were most evident in controls where males exhibited accelerated binge drinking behavior at consistently higher rates than females until Day 21 of CRS where their binge drinking behaviors were similar. There was also a main effect of alcohol binge behavior for MD males, and not females. This study illustrates that, depending on the biobehavioral output, males and females differ in reactivity to stressors where MD stress may confer sex-dependent resilience to subsequent stressors, and males are differently impacted than females.

GluN2D expression is regulated by restraint stress and supports active stress coping bouts

Stress coping strategies represent critical responses to environmental challenges, and active coping has been linked to stress resilience in humans. Understanding the neuroadaptations that support these strategies may provide insights into adaptive and maladaptive stress responses. NMDA receptors (NMDARs) play key roles in neuroadaptation, and NMDARs have been specifically implicated in stress responsiveness. Constitutive knockout mice have been used to implicate the GluN2D NMDAR subunit in regulation of stress-sensitive and affective behavior, but the brain regions in which GluN2D expression changes drive these effects remain unknown. Here we report that following an acute restraint stressor, GluN2D subunit expression is specifically decreased in the bed nucleus of the stria terminalis (BNST), a key region involved in stress processing, in male but not female mice, with no differences found in the thalamus or ventral hippocampus in either sex. Rodents engage in active struggling events during restraint stress that may represent active coping strategies to stress. Thus, we assessed active coping bouts during acute and chronic restraint stress sessions in GluN2D knockout mice. During the first restraint session, GluN2D knockout mice exhibited a pronounced decrease in struggling bouts during restraint stress relative to wild-type littermates, consistent with a role of GluN2D in active coping responses to stress. Repeated, daily restraint sessions revealed a sex-specific role of GluN2D expression on certain aspects of active coping behaviors, with male GluN2D KO mice exhibiting a decrease in total coping bouts measured across five sessions. However, BNST-specific knockdown of GluN2D in male mice did not alter active coping bouts, suggesting either a multi-synaptic role of GluN2D and/or a developmental role of GluN2D in this behavior. Altogether, these data are consistent with a growing literature suggesting that exploration of GluN2D control of stress circuit actions may lead to a novel therapeutic target to consider for stress-related mood disorders.

Medial prefrontal cortex input to basolateral amygdala controls acute stress-induced short-term anxiety-like behavior in mice.

Anxiety is a normal and transitory emotional state that allows the organisms to cope well with the real or perceived threats, while excessive or prolonged anxiety is a key characteristic of anxiety disorders. We have recently revealed that prolonged anxiety induced by chronic stress is associated with the circuit-varying dysfunction of basolateral amygdala projection neurons (BLA PNs). However, it is not yet known whether similar mechanisms also emerge for acute stress-induced, short-lasting increase of anxiety. Here, using a mouse model of acute restraint stress (ARS), we found that ARS mice showed increased anxiety-like behavior at 2 h but not 24 h after stress, and this effect was accompanied by a transient increase of the activity of BLA PNs. Specifically, ex vivo patch-clamp recordings revealed that the increased BLA neuronal activity did not differ among the distinct BLA neuronal populations, regardless of their projection targets being the dorsomedial prefrontal cortex (dmPFC) or elsewhere. We further demonstrated that such effects were mainly mediated by the enhanced presynaptic glutamate release in dmPFC-to-BLA synapses but not lateral amygdala-to-BLA ones. Furthermore, while optogenetically weakening the presynaptic glutamate release in dmPFC-to-BLA synapses ameliorated ARS-induced anxiety-like behavior, strengthening the release increased in unstressed mice. Together, these findings suggest that acute stress causes short-lasting increase in anxiety-like behavior by facilitating synaptic transmission from the prefrontal cortex to the amygdala in a circuit-independent fashion.

Mild acute stress prevents the memory impairment induced by long-term isoflurane anesthesia.

Long-term isoflurane anesthesia exposure could result in postoperative cognitive dysfunction (POCD). Preoperative stress is also reported to be a risk factor of POCD. However, it is unknown whether acute stress could impair memory after long-term isoflurane anesthesia. In this study, we categorized the mice with acute stress into mild (30 min restraint stress), moderate (60 min restraint stress), and severe (120 min restraint stress) stress groups and then we used Open-Field Test (OFT) to detect whether different scales of acute restraint stress successfully induced acute stress in mice. The memory performance of mice was measured using contextual and cued memory test, and the brain-derived neurotrophic factor protein levels of hippocampus was detected by Western blot. We verified that mild stress has pro-cognitive effect, but severe stress has amnestic effect. Moreover, we found that mild and moderate other than severe acute stress could partially attenuate the memory impairment induced by long-term isoflurane anesthesia. Mild and moderate acute stress could partially attenuate the memory impairment induced by long-term isoflurane anesthesia.

Acute restraint stress impairs histamine type 2 receptor ability to increase the excitability of medium spiny neurons in the nucleus accumbens

Histamine, a monoamine implicated in stress-related arousal states, is synthesized in neurons exclusively located in the hypothalamic tuberomammillary nucleus (TMN) from where they diffusely innervate striatal and mesolimbic networks including the nucleus accumbens (NAc), a vital node in the limbic loop. Since histamine-containing TMN neuron output increases during stress, we hypothesized that exposure of mice to acute restrain stress (ARS) recruits endogenous histamine type 2 receptor (H2R) signaling in the NAc, whose activation increases medium spiny neurons (MSNs) intrinsic excitability via downregulation of A-type K+ currents. We employed an ARS paradigm in which mice were restrained for 120 min, followed by a 20-min recovery period, after which brain slices were prepared for ex vivo electrophysiology. Using whole-cell patch-clamp recordings, we found that pharmacological activation of H2R failed to affect MSN excitability and A-type K+ currents in mice that underwent ARS. Interestingly, in mice treated with H2R-antagonist prior to ARS paradigm, H2R activation increased evoked firing and decreased A-type K+ currents similarly to what observed in control mice. Furthermore, H2R-antagonist treatment ameliorated anxiety-like behavior in ARS mice. Together, our findings indicate that ARS paradigm recruits endogenous H2R signaling in MSNs and suggest the involvement of H2R signaling in stress-related motivational states.