Unstressed Rats Research Articles

Status of inflammatory markers and growth factor in gastric ulcer protective effects of Punica granatum L. peel extract in rat

Background: Inflammatory markers, namely, myeloperoxidase (MPO), cytokines (tumor necrosis factor-alpha [TNF-α], and interleukin-1 beta [IL-1β]) and vascular endothelial growth factor (VEGF) have been reported to play an important role in ulcerogenesis and healing. Recently, we reported that the gastric ulcer (GU) protective and healing effects of 50% ethanol (EtOH) extract of Punica granatum peel (PGE) in rats were predominantly due to strengthening of mucosal defense and antioxidants status. Aims and Objectives: The present study incorporates the effects of PGE on rat gastric mucosal inflammatory markers (MPO, TNF-α, and IL-1β) and angiogenic factor (VEGF) in GU induced by cold-restraint stress (CRS) and EtOH in rats. Materials and Methods: PGE (100 mg/kg) was administered orally once daily to rats for 7 days before induction of CRS and EtOH GU. Ulcer index (UI), gastric mucosal MPO, TNF-α, and IL-1β, and VEGF were estimated. Results: CRS rats showed increase in UI and MPO (419.5, P < 0.001) compared with unstressed rats while PGE caused decrease in UI (46.1%, P < 0.05) and MPO (51.1%, P < 0.001) compared with CRS rats. EtOH rats showed increase in UI, TNF-α (936.5%, P < 0.001), IL-1β (37.2%, P < 0.05), and VEGF (13.0%, P < 0.05) compared with control normal saline-treated rats. PGE-treated EtOH rats showed decrease in UI (68.9%, P < 0.05), TNF-α (77.9%, P < 0.01), and IL-1β (24.8%, P < 0.05) but tended to decrease VEGF (7.3%, P < 0.2) compared with EtOH rats. Conclusion: Both the inflammatory markers and VEGF were found to increase in GU rats while PGE EtOH extract decreased the status of inflammatory markers with little change in VEGF level with concomitant decrease in ulceration indicating their role in ulcer healing and repair.

Prior stress promotes the generalization of contextual fear memories: Involvement of the gabaergic signaling within the basolateral amygdala complex

Fear generalization occurs when a response, previously acquired with a threatening stimulus, is transferred to a similar one. However, it could be maladaptive when stimuli that do not represent a real threat are appraised as dangerous, which is a hallmark of several anxiety disorders. Stress exposure is a major risk factor for the occurrence of anxiety disorders and it is well established that it influences different phases of fear memory; nevertheless, its impact on the generalization of contextual fear memories has been less studied. In the present work, we have characterized the impact of acute restraint stress prior to contextual fear conditioning on the generalization of this fear memory, and the role of the GABAergic signaling within the basolateral amygdala complex (BLA) on the stress modulatory effects. We have found that a single stress exposure promoted the generalization of this memory trace to a different context that was well discriminated in unstressed conditioned animals. Moreover, this effect was dependent on the formation of a contextual associative memory and on the testing order (i.e., conditioning context first vs generalization context first). Furthermore, we observed that increasing GABA-A signaling by intra-BLA midazolam administration prior to the stressful session exposure prevented the generalization of fear memory, whereas intra-BLA administration of the GABA-A antagonist (Bicuculline), prior to fear conditioning, induced the generalization of fear memory in unstressed rats. We concluded that stress exposure, prior to contextual fear conditioning, promotes the generalization of fear memory and that the GABAergic transmission within the BLA has a critical role in this phenomenon.

Exercise protects myelinated fibers of white matter in a rat model of depression.

The antidepressive effects of exercise have been a focus of research and are hypothesized to remodel the brain networks constructed by myelinated fibers. However, whether the antidepressant effects of exercise are dependent on changes in white matter myelination are unknown. Therefore, we chose chronic unpredictable stress (CUS) as a model of depression and designed an experiment. After a 4-week CUS period, 40 animals were tested using the sucrose preference test (SPT) and the open field test (OFT). The depressed rats then underwent 4-week running exercise. Next, electron microscopy and unbiased stereological methods were used to investigate white matter changes in the rats. After the 4-week CUS stimulation, body weight, sucrose preference and scores on the OFT were significantly lower in the depression rats than in the unstressed rats (p < .05). After undergoing a 4-week running exercise, the depression rats showed a significantly greater sucrose preference than the depression control rats without running exercise (p < .05). Furthermore, the white matter parameters of the depression rats (including the white matter volumes, the length and volumes of myelinated fibers, and the volumes and thickness of the myelin sheaths) were significantly reduced after the CUS period (p < .05). However, these white matter parameters were significantly increased after running exercise (p < .05). The present study is the first to provide evidence that running exercise has positive effects on white matter and the myelinated fibers of white matter in depressed rats, and this evidence might provide an important theoretical basis for the exercise-mediated treatment of depression.

Inhibition of hormonal and behavioral effects of stress by tryptophan in rats

Objectives: Stress in known to alter hormonal systems. Pharmacological doses of tryptophan, the essential amino acid precursor of serotonin, increase circulating leptin and decrease ghrelin in normal healthy adults. Because systemically injected leptin inhibits stress-induced behavioral deficits and systemically injected serotonin modulates leptin release from the adipocytes, we used tryptophan as a pharmacological tool to modulate hormonal and behavioral responses in unstressed and stressed rats.Methods: Leptin, ghrelin, serotonin, tryptophan, and behavior were studied in unstressed and stressed rats following oral administration of 0, 100, 200, and 300 mg/kg of tryptophan.Results: Following oral administration of tryptophan at a dose of 300 mg/kg, circulating levels of serotonin and leptin increased and those of ghrelin decreased in unstressed animals. No effect occurred on 24-hours cumulative food intake and elevated plus maze performance. Exposure to 2 hours immobilization stress decreased 24 hours cumulative food intake and impaired performance in elevated plus maze monitored next day. Serum serotonin decreased, leptin increased, and no effect occurred on ghrelin. Stress effects on serotonin, leptin, food intake, and elevated plus maze performance did not occur in tryptophan-pretreated animals. Tryptophan-induced decreases of ghrelin also did not occur in stressed animals.Conclusion: The findings show an important role of serum serotonin, leptin, and ghrelin in responses to stress and suggest that the essential amino acid tryptophan can improve therapeutics in stress-induced hormonal and behavioral disorders.

Effects of acute restraint and unpredictable chronic mild stress on brain corticotrophin releasing factor mRNA in the elevated T-maze

Corticotrophin releasing factor (CRF) modulates stress/anxiety-related responses. Previous studies showed that exposure to acute restraint and unpredictable chronic mild stress (UCMS) facilitates elevated T-maze (ETM) avoidance responses, an anxiogenic-like effect. This study verified the role of CRF in the modulation of ETM avoidance and escape reactions, in unstressed rats and in animals exposed to acute restraint or to UCMS, by quantifying CRF mRNA concentrations in stress/anxiety-related brain regions, through semiquantitative in situ hybridization. Results showed that stress exposure altered CRF mRNA in regions related to the modulation of stress/anxiety: the cingulate cortex, the hippocampus, the paraventricular and dorsomedial hypothalamus, the medial and central amygdalas, the dorsal region of the dorsal raphe (dDR) and the ventrolateral periaqueductal gray. A regression analysis showed that the anxiogenic-like effects observed in acute restraint animals were particularly associated to increases in CRF mRNA in the paraventricular hypothalamus, medial and central amygdalas and dDR. On the other hand, anxiogenic-like effects observed after UCMS exposure are associated to increases in CRF mRNA in the medial and central amygdalas, in the BNST and in the ventrolateral periaqueductal grey. This observation suggests important differences in the neurocircuitry that mediates responses to acute and chronic stress exposure. CRF mRNA in regions traditionally related to the modulation of panic reactions (the dorsal periaqueductal grey and the lateral wings of the dorsal raphe) were not observed, what might explain the absence of panicogenic-like effects of stress exposure. These results contribute to a better understanding of the role played by CRF in stress/anxiety-related responses.

Abstract P389: Post-traumatic Stress-induced Sensitization of Angiotensin II Hypertension is Reversed by Blockade of Angiotensin-converting Enzyme or Tumor Necrosis Factor-alpha

Using an Induction-Delay-Expression experimental paradigm, our previous studies demonstrated that post-traumatic stress disorder (PTSD) sensitizes angiotensin (ANG) II-elicited hypertension, which was associated with upregulation of central renin-angiotensin system (RAS) components and proinflammatory cytokines (PICs). The present study investigated whether inhibition of angiotensin-converting enzyme (ACE) or tumor necrosis factor-α (TNF-α) prior to PTSD blocks sensitization of ANG II-elicited hypertensive response. The resident-intruder paradigm was used to model PTSD. Each intruder rat (male Sprague-Dawley) was pretreated with ACE inhibitor (captopril, 0.5 mg/Ml) or with TNF-α inhibitor (pentoxifylline, PTX, 100 mg/kg/day) in the drinking water for two weeks and then exposed to a different resident (male Long-Evans) for 2 hours on three days with each session separated by 1 day. Beginning 3 days after the last exposure, the intruder (PTSD) rats and unstressed control rats received a subcutaneous infusion of ANG II (120 ng/kg/min) for 2 weeks. The PTSD rats had a significantly enhanced hypertensive response to the ANG II infusion and an upregulation of mRNA expression of RAS and PIC components and of a microglial marker in the lamina terminalis (LT) when compared to control rats. Both the sensitized hypertensive response and enhanced gene expression were blocked by pre-treatment with either ACE inhibitor or TNF-α antagonist. These results suggest that upregulation of the brain RAS and PICs produced by a severe stress contribute to PTSD-induced sensitization of the hypertensive response to ANG II.

Neuroadaptations of presynaptic and postsynaptic GABAB receptor function in the paraventricular nucleus in response to chronic unpredictable stress.

Chronic stress impairs GABAA (GABA type A) receptor-mediated inhibition in the hypothalamic paraventricular nucleus (PVN). It is not clear whether GABAB receptor function is also altered. We hypothesize that chronic stress alters GABAB receptor function in PVN corticotrophin-releasing hormone (CRH) neurons to control hypothalamus-pituitary-adrenal axis activity. Whole-cell patch clamp recordings were made of PVN-CRH neurons expressing eGFP driven by CRH promoter in brain slices from unstressed rats and rats exposed to chronic unpredictable mild stress (CUMS). CUMS elevated the basal circulating corticosterone levels and increased the basal firing activity of PVN-CRH neurons. Microinjection of GABAB receptor agonist baclofen into the PVN suppressed the increased corticosterone levels in CUMS rats compared with unstressed rats. CUMS blunted the baclofen-induced inhibition on PVN-CRH neurons and outward currents in these neurons. Furthermore, CUMS reduced expression of GABAB1 (GABAB R1) protein in the PVN. Blocking NMDA receptors with AP5 restored the reduced baclofen-induced currents in CUMS rats but had no effect on GABAB1 expression. Furthermore, CUMS treatment augmented the baclofen-induced decrease in the frequency of glutamatergic excitatory postsynaptic currents (EPSCs) and GABAergic inhibitor postsynaptic currents in PVN-CRH neurons. The GABAB receptor antagonist CGP55845 increased the firing activity of PVN-CRH neurons only in CUMS-treated rats and not in unstressed rats. These findings suggest that chronic stress impairs postsynaptic GABAB receptor function but augments presynaptic GABAB receptor function in controlling glutamatergic and GABAergic synaptic inputs in PVN-CRH neurons.

Vesicular Glutamate Transporter 1 Knockdown in Infralimbic Prefrontal Cortex Augments Neuroendocrine Responses to Chronic Stress in Male Rats.

Chronic stress-associated pathologies frequently associate with alterations in the structure and activity of the medial prefrontal cortex (mPFC). However, the influence of infralimbic cortex (IL) projection neurons on hypothalamic-pituitary-adrenal (HPA) axis activity is unknown, as is the involvement of these cells in chronic stress-induced endocrine alterations. In the current study, a lentiviral-packaged vector coding for a small interfering RNA (siRNA) targeting vesicular glutamate transporter (vGluT) 1 messenger RNA (mRNA) was microinjected into the IL of male rats. vGluT1 is responsible for presynaptic vesicular glutamate packaging in cortical neurons, and knockdown reduces the amount of glutamate available for synaptic release. After injection, rats were either exposed to chronic variable stress (CVS) or remained in the home cage as unstressed controls. Fifteen days after the initiation of CVS, all animals were exposed to a novel acute stressor (30-minute restraint) with blood collection for the analysis of adrenocorticotropic hormone (ACTH) and corticosterone. Additionally, brains were collected for in situ hybridization of corticotrophin-releasing hormone mRNA. In previously unstressed rats, vGluT1 siRNA significantly enhanced ACTH and corticosterone secretion. Compared with CVS animals receiving the green fluorescent protein control vector, the vGluT1 siRNA further increased basal and stress-induced corticosterone release. Further analysis revealed enhanced adrenal responsiveness in CVS rats treated with vGluT1 siRNA. Collectively, our results suggest that IL glutamate output inhibits HPA responses to acute stress and restrains corticosterone secretion during chronic stress, possibly at the level of the adrenal. Together, these findings pinpoint a neurochemical mechanism linking mPFC dysfunction with aberrant neuroendocrine responses to chronic stress.

β-carotene ameliorates CUS-induced circadian alternations of locomotor activity and melatonin patterns in rats

Circadian and stress systems have a crucial role in adaptation of organisms to environmental challenges. This study investigates the ability of Oscillatoria brevis (O. brevis) β-carotene extract (βC) in modulating the circadian alternations of locomotor activity (LA) and serum melatonin (M) rhythms under chronic unpredictable stress (CUS) in rats. Twenty rats (5 rats/group) were used in monitoring LA using running wheels. Eighty rats (20 rats/group) were used in observing circadian serum M profile. Rats were randomly divided into four groups, viz. control, βC-treated, CUS-exposed, and “βC-treated&CUS-exposed” groups. CUS-exposure was applied for 21 days. One hour before exposure, βC was daily administered (10 mg/kg), intraperitoneally (IP). Blood was sampled at 6-h intervals for 5 rats/time point at Zeitgeber (ZT) 3, 9, 15, and 21. Results demonstrated that unstressed rats exhibited circadian M pattern and nocturnal LA rhythm with acrophase around ZT 21 and ZT 15, respectively. CUS-exposure revealed a disturbance in these patterns. Phase shifting of M and LA profiles was recorded. A decrease M acrophase and a significant decrease in LA (p < 0.05) were recorded at ZT 9. Daily βC administration in stressed rats modulates the CRs alternation induced by CUS. It may be concluded that βC ameliorated the induced alternations in circadian rhythms.

Differential dendritic remodeling in prelimbic cortex of male and female rats during recovery from chronic stress

Chronic stress produces differential dendritic remodeling of pyramidal neurons in medial prefrontal cortex of male and female rats. In males, this dendritic remodeling is reversible. However, the timeline of recovery, as well as the potential for reversibility in females, is unknown. Here, we examined dendritic recovery of pyramidal neurons in layer II-II of prelimbic cortex in male and female rats following chronic restraint stress (3h/day for 10days). Dendritic morphology and spine density were analyzed immediately following the cessation of stress, or following a 7- or 10-day recovery period. Chronic stress produced apical dendritic retraction in males, which was coupled with a decrease in the density of stubby spine on apical dendrites. Further, following a 10-day recovery period, the morphology of neurons from stressed rats resembled that of unstressed rats. Male rats given a 7-day recovery period had apical dendritic outgrowth compared to unstressed rats. Immediately after cessation of stress, females showed only minimal dendritic remodeling. The morphology of neurons in stressed females resembled those of unstressed rats following only 7days of recovery, at which time there was also a significant increase in stubby spine density. Males and females also showed different changes in baseline corticosterone concentrations during recovery. These findings not only indicate that dendritic remodeling in prelimbic cortex following chronic stress is different between males and females, but also suggest chronic stress induces differential hypothalamic–pituitary–adrenal axis dysregulation in males and females. These differences may have important implications for responses to subsequent stressors.

Ketamine induces brain-derived neurotrophic factor expression via phosphorylation of histone deacetylase 5 in rats

Ketamine shows promise as a therapeutic agent for the treatment of depression. The increased expression of brain-derived neurotrophic factor (BDNF) has been associated with the antidepressant-like effects of ketamine, but the mechanism of BDNF induction is not well understood. In the current study, we demonstrate that the treatment of rats with ketamine results in the dose-dependent rapid upregulation of Bdnf promoter IV activity and expression of Bdnf exon IV mRNAs in rat hippocampal neurons. Transfection of histone deacetylase 5 (HDAC5) into rat hippocampal neurons similarly induces Bdnf mRNA expression in response to ketamine, whereas transfection of a HDAC5 phosphorylation-defective mutant (Ser259 and Ser498 replaced by Ala259 and Ala498), results in the suppression of ketamine-mediated BDNF promoter IV transcriptional activity. Viral-mediated hippocampal knockdown of HDAC5 induces Bdnf mRNA and protein expression, and blocks the enhancing effects of ketamine on BDNF expression in both unstressed and stressed rats, and thereby providing evidence for the role of HDAC5 in the regulation of Bdnf expression. Taken together, our findings implicate HDAC5 in the ketamine-induced transcriptional regulation of Bdnf, and suggest that the phosphorylation of HDAC5 regulates the therapeutic actions of ketamine.

Long-Term Sertraline Intake Reverses the Behavioral Changes Induced by Prenatal Stress in Rats in a Sex-Dependent Way.

Early life stress is a major factor underlying the vulnerability to respond to stressful events later in life. The present study attempted to evaluate the role of prenatal stress affecting the development of stress-related disorders and their reversion by postnatal exposure to Sertraline (SERT), a front-line medication for medication for posttraumatic stress disorder (PTSD) in humans. To achieve this, adult male and female prenatally stressed (PS) or unstressed (Controls) offspring rats, following oral chronic treatment with SERT (5 mg/kg/day; from 1 month to 4 months old), or not, were studied prior to and after a traumatic event. First, anxiety-like behavior during the prepulse inhibition (PPI) test, a modulation of the startle reflex, was examined in all animals. Subsequently, the animals were subjected to a session of mild inescapable footshocks (IS; 0.35 mA, 5 s) in a shuttle box that was followed by 4 days of situational reminders in the aversive context. Prior to the footshocks no effects of PS or SERT were shown, and no changes in PPI and the habituation to the shuttle box were found. After them, PS led animals to exhibit behavioral alterations. When compared to the Controls, PS animals of both sexes displayed less rearing activity in the aversive environment. PS males responded less to footshock delivery and, in most of the animals, fear extinction was impaired. Moreover, the early postnatal exposure to SERT lessened the behavioral impact of PS in females, while in males it had no effect. Current results extend previous data from our laboratory, showing that PS heightened vulnerability to stress later on, and that SERT acts differently in males and females.

GASTRIC ANTIULCER AND ULCER HEALING EFFECTS OF PUNICA GRANATUM L. PEEL EXTRACT IN RATS: ROLE OF OFFENSIVE AND DEFENSIVE MUCOSAL FACTORS AND OXIDATIVE STRESS

Objective: The present work incorporates the study of gastric antiulcer and ulcer healing effects of dried Punica granatum (PG) peel 50% ethanol extract (PGE) in rats.Methods: PGE (100 mg/kg) was administered orally once daily to rats either before or after induction of gastric ulcers (GU) for 7 d. Antiulcer effects of PGE were seen against acute GU, induced by pylorus ligation (PL), cold restraint stress (CRS), aspirin and ethanol while, ulcer healing in acetic acid (AA)-induced chronic GU in rats. Ulcer index (UI), gastric juice volume, acid-pepsin and mucin secretions and gastric mucosal glycoproteins, free radicals (LPO and NO) and antioxidants (SOD and GSH) were estimated.Results: PGE showed a decrease in UI in all GU models (45.6 to 79.7%, P&lt;0.05 to P&lt;0.001) indicating both protective and healing effects. PGE showed little or no effects on volume, acid-pepsin concentration and output but increased mucin secretion (55.1%, P&lt;0.05) and mucosal glycoproteins (35.7%, P&lt;0.05) in PL rats. CRS rats showed an increase in LPO and NO (48.4 to 58.3%, P&lt;0.01) and SOD (21.8%, P&lt;0.01) but decrease in GSH and CAT (33.1 to 44.8%, P&lt;0.01 to P&lt;0.001) compared with unstressed rats. PGE-treated CRS rats showed a decrease in LPO and NO (44.1 to 61.2, P&lt;0.01 to P&lt;0.001) and SOD (13.2%, P&lt;0.01) and increase in GSH and CAT (43.8 to 48.7%, P&lt;0.01 to P&lt;0.001) compared with CRS rats.Conclusion: PGE seemed to have ulcer cytoprotective effects due to enhanced mucosal resistance and reduction in oxidative mucosal damage possibly via high antioxidant activity.

Effects of berberine on a rat model of chronic stress and depression via gastrointestinal tract pathology and gastrointestinal flora profile assays

Chronic stress and depression are challenging conditions to treat, owing to their complexity and lack of clinically available and effective therapeutic agents. The aim of the present study was to investigate the mechanism by which berberine acts, by examining alterations to gastrointestinal tract histopathology and flora profile in a rat model, following the induction of stress. Research associating gastrointestinal flora and depression has increased, thus, the present study hypothesized that stress induces depression and changes in the gastrointestinal system. The chronic mild stress rat model was previously established based on a set of 10 chronic unpredictable stress methods. In the present study, the measurements of body weight, behavior, gastrointestinal tract histopathology and gastrointestinal flora profile were collected in order to elucidate understanding of chronic stress and depression in this region. In the present study, induced stress and the resulting depression was demonstrated to significantly decrease the body weight and sucrose preference of rats, aswellas significantly increasing traverse time, vertical movement time, grooming time and motionless time in an open‑field test. Following modeling and subsequent treatment with low or high doses of berberine, the measurements were significantly different when compared with unstressed rats. Berberine appears to reverse the physical damage brought about by stress within the gastric mucosa and intestinal microvilli of the stomach, ileum, cecum and colon. Using enterobacterial repetitive intergenic consensus sequence‑based polymerase chain reaction analysis, several distinctive bands disappeared following modeling; however, novel distinctive bands appeared in response to the graded berberine treatment. In conclusion, the present study identified that high concentrations of berberine markedly protects rats from various symptoms of chronic stress and depression, with the potential of facilitating treatment within clinical practice.

Exploring a post-traumatic stress disorder paradigm in Flinders sensitive line rats to model treatment-resistant depression I: bio-behavioural validation and response to imipramine.

Co-morbid depression with post-traumatic stress disorder (PTSD) is often treatment resistant. In developing a preclinical model of treatment-resistant depression (TRD), we combined animal models of depression and PTSD to produce an animal with more severe as well as treatment-resistant depressive-like behaviours. Male Flinders sensitive line (FSL) rats, a genetic animal model of depression, were exposed to a stress re-stress model of PTSD [time-dependent sensitisation (TDS)] and compared with stress-naive controls. Seven days after TDS stress, depressive-like and coping behaviours as well as hippocampal and cortical noradrenaline (NA) and 5-hydroxyindoleacetic acid (5HIAA) levels were analysed. Response to sub-chronic imipramine treatment (IMI; 10 mg/kg s.c.×7 days) was subsequently studied. FSL rats demonstrated bio-behavioural characteristics of depression. Exposure to TDS stress in FSL rats correlated negatively with weight gain, while demonstrating reduced swimming behaviour and increased immobility versus unstressed FSL rats. IMI significantly reversed depressive-like (immobility) behaviour and enhanced active coping behaviour (swimming and climbing) in FSL rats. The latter was significantly attenuated in FSL rats exposed to TDS versus unstressed FSL rats. IMI reversed reduced 5HIAA levels in unstressed FSL rats, whereas exposure to TDS negated this effect. Lowered NA levels in FSL rats were sustained after TDS with IMI significantly reversing this in the hippocampus. Combining a gene-X-environment model of depression with a PTSD paradigm produces exaggerated depressive-like symptoms that display an attenuated response to antidepressant treatment. This work confirms combining FSL rats with TDS exposure as a putative animal model of TRD.

Stress-induced resistance to the fear memory labilization/reconsolidation process. Involvement of the basolateral amygdala complex

Consolidated memories can enter into a labile state after reactivation followed by a restabilization process defined as reconsolidation. This process can be interfered with Midazolam (MDZ), a positive allosteric modulator of the GABA-A receptor. The present study has evaluated the influence of prior stress on MDZ’s interfering effect. We also assessed the influence of both systemic and intra-basolateral amygdala (BLA) infusion of d-cycloserine (DCS), a partial agonist of the NMDA receptors, on the MDZ effect in previously stressed rats. Furthermore, we analyzed the effect of stress on the expression of Zif-268 and the GluN2B sites, two molecular markers of the labilization/reconsolidation process, following reactivation. The results revealed that prior stress resulted into a memory trace that was insensitive to the MDZ impairing effect. Both systemic and intra-BLA DCS administration previous to reactivation restored MDZ’s disruptive effect on memory reconsolidation in stressed animals. Further, reactivation enhanced Zif-268 expression in the BLA in control unstressed rats, whereas no elevation was observed in stressed animals. In agreement with the behavioral findings, DCS restored the increased level of Zif-268 expression in the BLA in stressed animals. Moreover, memory reactivation in unstressed animals elevated GluN2B expression in the BLA, thus suggesting that this effect is involved in memory destabilization, whereas stressed animals did not reveal any changes. These findings are consistent with resistance to the MDZ effect in these rats, indicating that stress exposure prevents the onset of destabilization following reactivation.In summary, prior stress limited both the occurrence of the reactivation-induced destabilization and restabilization.

Chronic Unpredictable Mild Stress Induces Loss of GABA Inhibition in Corticotrophin-Releasing Hormone-Expressing Neurons through NKCC1 Upregulation

Introduction: Prolonged and repeated stresses cause hyperactivity of the hypothalamic-pituitary-adrenal (HPA) axis. The corticotrophin-releasing hormone (CRH)-expressing neurons in the hypothalamic paraventricular nucleus (PVN) are an essential component of the HPA axis. Materials and Methods: Chronic unpredictable mild stress (CUMS) was induced in Sprague-Dawley rats. GABA reversal potentials (E_GABA) were determined by using gramicidin-perforated recordings in identified PVN-CRH neurons through expressing enhanced green fluorescent protein driven by the CRH promoter. Plasma corticosterone (CORT) levels were measured in rats implanted with a cannula targeting the lateral ventricles and PVN. Results: Blocking the GABA_A receptor in the PVN with gabazine significantly increased plasma CORT levels in unstressed rats but did not change CORT levels in CUMS rats. CUMS caused a depolarizing shift in E_GABA in PVN-CRH neurons compared with E_GABA in PVN-CRH neurons in unstressed rats. Furthermore, CUMS induced a long-lasting increase in expression levels of the cation chloride cotransporter Na⁺-K⁺-Cl^--Cl^- (NKCC1) in the PVN but a transient decrease in expression levels of K⁺-Cl^--Cl^- in the PVN, which returned to the basal level 5 days after CUMS treatment. The NKCC1 inhibitor bumetanide decreased the basal firing activity of PVN-CRH neurons and normalized E_GABA and the gabazine-induced excitatory effect on PVN-CRH neurons in CUMS rats. In addition, central administration of bumetanide decreased basal circulating CORT levels in CUMS rats. Conclusions: These data suggest that chronic stress impairs GABAergic inhibition, resulting in HPA axis hyperactivity through upregulation of NKCC1.

GABAB receptor plasticity in corticotrophin releasing hormone neurons in the paraventricular nucleus during chronic unpredictable stress

GABAB (γ‐aminobutytic acid type B) receptor is important in the regulation of neuronal excitability. Chronic stress leads to hyperactivity of corticotrophin releasing hormone (CRH) neurons. However, the role of GABAB receptors in the regulation of CRH neurons in paraventricular nucleus (PVN) of the hypothalamus in chronic stress is not known. Here, we determined pre‐ and postsynaptic GABAB receptor function in identified PVN‐CRH neurons expressing eGFP driven by crh premotor‐driven eGFP. Whole cell patch‐clamp recording were performed on PVN‐CRH neurons in brain slices from unstressed rats and rats with chronic unpredictable mild stress (CUMS). Bath application of the GABAB receptor agonist baclofen induced a significant lesser reduction of firing activity in PVN‐CRH neurons in CUMS than in unstressed rats. Furthermore, baclofen induced lesser outward currents in PVN‐CRH neurons in CUMS than in unstressed rats. Baclofen‐induced currents were eliminated by inclusion of GDP‐β‐s in internal recording solution or blocking G protein‐coupled inwardly‐rectifying K (GIRK) channels with tertiapin. GABAB1 receptor expression level in the PVN was significantly decreased in CUMS than in unstressed rats. Baclofen produced significant greater inhibition of the frequency of glutamatergic excitatory postsynaptic currents (EPSCs) and GABAergic inhibitor postsynaptic currents (IPSCs) without affecting the amplitude of EPSCs and IPSCs in PVN‐CRH neurons in CUMS and unstressed rats. These data suggest that down‐regulated postsynaptic GABAB receptor contributes to suppressing GABAB‐mediated inhibition of PVN‐CRH neurons in chronic stress.Support or Funding InformationNIMH grants MH096086.

Acute Stress Diminishes M‐Current in Corticotrophin‐Releasing Hormone Neurons through AMPK Activation

Acute stress activate hypothalamus‐pituitary‐adrenal gland axis and corticotrophin‐releasing hormone (CRH) neurons in the paraventricular nucleus (PVN) of hypothalamus. However, the molecular and cellular mechanisms underlying hyperactivity of PVN‐CRH neurons in acute stress are not clear. M‐channel is regulated by AMPK activity and plays important role in the regulation of neuron activity. We hypothesized that M‐channel function is impaired in PVN‐CRH neurons during acute stress. Whole‐cell patch‐clamp recording were performed on PVN‐CRH neurons identified by specific expression of eGFP driven by the rat crh premotor. Acute stress (2‐hr restraint) profoundly increased firing rate of PVN‐CRH neurons while decreased M‐current in these neurons. M‐channel blocker XE991 induced lesser increased in firing activity of PVN‐CRH neurons in acutely stressed rats compared with unstressed rats. Microinjection of XE991 into the PVN produced significantly lesser increases in plasma corticosterone concentration in acute stress rats than in unstressed rats. Furthermore, acute stress decreased KV7.3 subunit membrane‐fraction protein expression levels while increased phosphorylated AMPK level in the PVN tissues compared with unstressed rats. However, the Kv7.3 cytoplasma‐fraction protein level and Kv7.2 subunit expression level in the PVN tissue. Treatment with AMPK inhibitor compound C not only recovered the Kv7.3 membrance‐fraction expression level but also reinstalled the reduced M‐current and XE991‐induced excitatory effect on PVN‐CRH neruons in acute stressed rats. Finally, microinjection of compound C into the PVN reinstalled XE991‐induced increase in plasma corticosterone levels. Collectively, these results suggested that acute stress diminishes M‐current in PVN‐CRH neurons, which is related to AMPK activation.Support or Funding InformationThis study was supported by NIMH grants MH096086.

Traumatic Stress Promotes Hyperalgesia via Corticotropin-Releasing Factor-1 Receptor (CRFR1) Signaling in Central Amygdala.

Hyperalgesia is an exaggerated response to noxious stimuli produced by peripheral or central plasticity. Stress modifies nociception, and humans with post-traumatic stress disorder (PTSD) exhibit co-morbid chronic pain and amygdala dysregulation. Predator odor stress produces hyperalgesia in rodents. Systemic blockade of corticotropin-releasing factor (CRF) type 1 receptors (CRFR1s) reduces stress-induced thermal hyperalgesia. We hypothesized that CRF-CRFR1 signaling in central amygdala (CeA) mediates stress-induced hyperalgesia in rats with high stress reactivity. Adult male Wistar rats were exposed to predator odor stress in a conditioned place avoidance paradigm and indexed for high (Avoiders) and low (Non-Avoiders) avoidance of predator odor-paired context, or were unstressed Controls. Rats were tested for the latency to withdraw hindpaws from thermal stimuli (Hargreaves test). We used pharmacological, molecular, and immunohistochemical techniques to assess the role of CRF-CRFR1 signaling in CeA in stress-induced hyperalgesia. Avoiders exhibited higher CRF peptide levels in CeA that did not appear to be locally synthesized. Intra-CeA CRF infusion mimicked stress-induced hyperalgesia. Avoiders exhibited thermal hyperalgesia that was reversed by systemic or intra-CeA injection of a CRFR1 antagonist. Finally, intra-CeA infusion of tetrodotoxin produced thermal hyperalgesia in unstressed rats and blocked the anti-hyperalgesic effect of systemic CRFR1 antagonist in stressed rats. These data suggest that rats with high stress reactivity exhibit hyperalgesia that is mediated by CRF-CRFR1 signaling in CeA.