Sedative-Hypnotic Effects of Glycine max Merr. Extract and Its Active Ingredient Genistein on Electric-Shock-Induced Sleep Disturbances in Rats.

Glycine max Merr. (GM) is a functional food that provides many beneficial phytochemicals. However, scientific evidence of its antidepressive and sedative activities is scarce. The present study was designed to investigate the antidepressive and calmative effects of GM and its biologically active compound, genistein (GE), using electroencephalography (EEG) analysis in an electric foot shock (EFS)-stressed rat. The underlying neural mechanisms of their beneficial effects were determined by assessing corticotropin-releasing factor (CRF), serotonin (5-HT), and c-Fos immunoreactivity in the brain using immunohistochemical methods. In addition, the 5-HT2C receptor binding assay was performed because it is considered a major target of antidepressants and sleep aids. In the binding assay, GM displayed binding affinity to the 5-HT2C receptor (IC50 value of 14.25 ± 11.02 µg/mL). GE exhibited concentration-dependent binding affinity, resulting in the binding of GE to the 5-HT2C receptor (IC50, 77.28 ± 26.57 mg/mL). Administration of GM (400 mg/kg) increased non-rapid eye movement (NREM) sleep time. Administration of GE (30 mg/kg) decreased wake time and increased rapid eye movement (REM) and NREM sleep in EPS-stressed rats. In addition, treatment with GM and GE significantly decreased c-Fos and CRF expression in the paraventricular nucleus (PVN) and increased 5-HT levels in the dorsal raphe in the brain. Overall, these results suggest that GM and GE have antidepressant-like effects and are effective in sleep maintenance. These results will benefit researchers in developing alternatives to decrease depression and prevent sleep disorders.

Investigating the role of nitric oxide in stress adaptive process in electric foot shock stress-subjected mice.

AIM: The present study was designed to investigate the role of nitric oxide (NO) in the non-development of stress adaptation in high-intensity foot-shock stress (HIFS) subjected mice. METHODS: Mice were subjected to low-intensity shocks (LIFS i.e. 0.5 mA) or HIFS (1.5 mA) for 5 days. Stress-induced behavioral changes were assessed by actophotometer, hole board, open field and social interaction tests. Biochemically, the serum corticosterone levels were measured as a marker of stress. L-arginine (100 mg/kg and 300 mg/kg), as NO donor, and L-NAME (10 mg/kg and 30 mg/kg), as nitric oxide synthase (NOS) inhibitor, were employed as pharmacological agents. RESULTS: A single exposure of LIFS and HIFS produced behavioral and biochemical alterations. However, there was the restoration of behavioral and biochemical alterations on 5th day in response to repeated LIFS exposure suggesting the development of stress adaptation. However, no stress adaptation was observed in HIFS subjected mice. Administration of L-arginine (300 mg/kg) abolished the stress adaptive response in LIFS-subjected mice, while L-NAME (30 mg/kg) induced the development of stress adaptation in HIFS subjected mice. CONCLUSION: It is concluded that an increase in the NO release may possibly impede the process of stress adaptation in HIFS-subjected mice.

Antioxidant and Anti-Stress Effects of Taurine Against Electric Foot-Shock-Induced Acute Stress in Rats.

In the present study, we evaluated the antioxidant and anti-stress activities of taurine in electric foot-shock stress model rats. Taurine supplementation markedly increased the hepatic glutathione (GSH) levels, compared to the levels in the stress group. In addition, activities of antioxidant enzymes such as catalase (CAT), glutathione peroxidase (GPx) and glutathione-S-transferase (GST) were improved in the taurine-treated group. Plasma cortisol and dehydroepiandrosterone-sulfate (DHEA-S) levels were significantly reduced in the taurine-supplemented group compared to those in the stress group. In contrast, the levels of 5-hydroxytryptamine (5-HT) and 5-hydroxyindoleacetic acid (5-HIAA) were markedly increased in the taurine or betaine-treated group compared to those in the stress group. It may be concluded that taurine produces beneficial effects in the form of antioxidant status and biochemical alterations in foot-shock-induced acute stress in rats.

Modified Aloe Polysaccharide Restores Chronic Stress-Induced Immunosuppression in Mice.

Chronic stress generally experienced in our daily lives; is known to augment disease vulnerability by suppressing the host immune system. In the present study; the effect of modified Aloe polysaccharide (MAP) on chronic stress-induced immunosuppression was studied; this Aloe compound was characterized in our earlier study. Mice were orally administered with MAP for 24 days and exposed to electric foot shock (EFS; duration; 3 min; interval; 10 s; intensity; 2 mA) for 17 days. The stress-related immunosuppression and restorative effect of MAP were then analyzed by measuring various immunological parameters. MAP treatment alleviated lymphoid atrophy and body weight loss. The numbers of lymphocyte subsets were significantly normalized in MAP-treated mice. Oral administration of MAP also restored the proliferative activities of lymphocytes; ovalbumin (OVA)-specific T cell proliferation; antibody production; and the cell killing activity of cytotoxic T lymphocytes. In summary; oral administration of MAP ameliorated chronic EFS stress-induced immunosuppression.

Electric foot-shock induces neurobehavioral aberrations due to imbalance in oxidative status, stress hormone, neurochemical profile, and irregular cortical-beta wave pattern in rats: A validated animal model of anxiety.

Neuropsychiatric disorders can be modeled on animals to investigate the neural mechanism underlying these disorders. Models of neuropsychiatric disorders, such as anxiety, basically aim to produce the signs and symptoms of human anxiety disorders in laboratory animals. Electric foot-shock is recommended to induce anxiety-like symptoms in rodents. For this purpose, however, a range of current intensities is available in the literature. The present study aims to modify the existing practices of generating anxiety-like symptoms through electric foot-shock by identifying an optimum current intensity and combining it with behavioral paradigms to produce a rat model of anxiety. Furthermore, the validity of the model was confirmed by checking the fulfillment of three validity criteria necessary for the development of any disease model including face validity, construct validity, and predictive validity. In the current study, after pre-testing, 1.0 mA electric intensity was selected to produce the model of anxiety. The results showed that the induction of 1.0 mA electric foot-shock induces abnormal behavioral effects which were similar to anxiety-like effects as evident by social interaction test, light-dark transition test, and open field test. Moreover, aberrations in the levels of the stress hormone, oxidative stress parameters, hippocampal neurotransmitter levels, and cortical-EEG wave pattern were also observed in the rat model of anxiety which were successfully overcome using diazepam. In conclusion, the outcome of our study suggests that electric foot-shock can be an adequate stressor to produce a validated animal model of anxiety and this model can be confidently used to identify and screen new and/or novel anxiolytics.

Effect of chronically electric foot shock stress on spatial memory and hippocampal blood brain barrier permeability.

Maintaining blood-brain barrier (BBB) contributes critically to preserving normal brain functions. According to the available evidence, intense or chronic exposure to stress would potentially affect different brain structures, such as the hippocampus, negatively. The purpose of this study was to define the relationship between the BBB permeability of the hippocampus and the performance of spatial learning and memory under chronically electric foot shock stress. Sixteen rats were divided into the control and stress groups equally. Animals in the stress group were exposed to foot shock (1 mA, 1 Hz) for 10-s duration every 60 s (1 h/day) for 10 consecutive days. The anxiety-related behavior, spatial learning, and memory were assessed by an Open Field (OF) and the Morris Water Maze (MWM) respectively. The hippocampal BBB permeability was determined by Evans blue penetration assay. Our results demonstrated that the stress model not only increased locomotor activities in the OF test but reduced spatial learning and memory in MWM. Moreover, these effects coincided with a significant increase in hippocampal BBB permeability. In sum, the stress model can be used in future studies focusing on the relationship between stress and BBB permeability of the hippocampus.

Mice subjected to uncontrollable electric shocks show depression-like behaviors irrespective of their state of helplessness.

The unpredictable and inescapable electric shock-induced "learned helplessness" paradigm has long been used to produce an animal model of depression to identify the molecules associated with depressive symptoms or to assess the efficacy of pharmacological treatments for depression. After exposure to unpredictable and inescapable shocks (uncontrollable stress), most of mice showed defect in escape behavior in active avoidance test (learned helplessness, LH), while others did not (non-learned helplessness, NLH). Here, we investigated whether mice with LH or NLH exhibited depressive symptoms, including anhedonia, anxiety, and despair. We found that compared with control naïve mice, both uncontrollable shocks-induced LH and NLH mice showed increased anhedonia- and anxiety- but not despair-like behaviors. Notably, mice subjected to uncontrollable shocks showed similar behaviors, irrespective of whether they also showed LH or NLH. Furthermore, since both LH and NLH mice showed only anhedonia- and anxiety- but not despair-like behaviors, this model may be generally inadequate for classic depression-like behavior assessment. In conclusion, uncontrollable electric shock induces depression-like behavior, irrespective of the state of helplessness.

Investigating the role of nisoldipine in foot-shock-induced post-traumatic stress disorder in mice.

This study was designed to investigate the effectiveness of nisoldipine, an L-type voltage-sensitive calcium channel blocker, to ameliorate anxiety and fear response in a mouse model of post-traumatic stress disorder (PTSD). Acute trauma was induced in Swiss albino mice in a 2-day electric foot-shock paradigm consisting of 15 intermittent foot-shocks of 0.8 mA intensity, 10-s duration and 10-s intershock interval, during 5 min, followed by 3 weekly situational reminders, that is, once per week in the same context on three successive weeks. PTSD-induced behavioral changes were assessed using actophotometer, open-field, social interaction test, and freezing behavior. Biochemically, the serum corticosterone levels were estimated. Electric foot-shock and situational reminders produced behavioral alterations and decreased corticosterone levels, assessed on the 21st day following the traumatic event. Administration of sertraline (Ser 15 mg/kg), a selective serotonin reuptake inhibitor (SSRI) and nisoldipine (20 and 40 mg/kg), significantly attenuated the foot-shock-trauma-induced behavioral changes along with normalization of the corticosterone levels. It may be concluded that nisoldipine produces beneficial effects in re-establishing behavioral alterations, which may be due to normalization of reduced corticosterone levels in PTSD in mice.

Anxiolytic effects of GLYX-13 in animal models of posttraumatic stress disorder-like behavior.

In the present study, we investigated the effectiveness of GLYX-13, an NMDA receptor glycine site functional partial agonist, to alleviate the enhanced anxiety and fear response in both a mouse and rat model of stress-induced behavioral changes that might be relevant to posttraumatic stress disorder (PTSD). Studies over the last decades have suggested that the hyperactivity of hypothalamic-pituitary-adrenal (HPA) axis is one of the most consistent findings in stress-related disease. Herein, we used these animal models to further investigate the effect of GLYX-13 on the stress hormone levels and glucocorticoid receptor (GR) expression. We found that exposure to foot shock induced long-lasting behavioral deficiencies in mice, including freezing and anxiety-like behaviors, that were significantly ameliorated by the long-term administration of GLYX-13 (5 or 10 mg/kg). Our enzyme-linked immunosorbent assay results showed that long-term administration of GLYX-13 at behaviorally effective doses (5 or 10 mg/kg) significantly decreased the elevated serum levels of both corticosterone and its upstream stress hormone adrenocorticotropic hormone in rats subjected to the TDS procedure. These results suggest that GLYX-13 exerts a therapeutic effect on PTSD-like stress responding that is accompanied by (or associated with) modulation of the HPA axis, including inhibition of stress hormone levels and upregulation of hippocampal GR expression.

Investigations on GSK-3ß/NF-kB signaling in stress and stress adaptive behavior in electric foot shock subjected mice.

The present study was designed to explore the role of GSK-3ß and NF-kB signaling in electric foot shock-induced stress and stress adaptation. Mice were subjected to foot shocks of 0.5mA intensity and 1s duration of 1h to produce acute stress. Animals were exposed to the same stressor for 5 days to induce stress adaptation. The behavioral alterations were assessed using the actophotometer, hole board, open field and social interaction tests. The serum corticosterone levels were assessed as a marker of the HPA axis. The levels of total GSK-3ß, p-GSK-3ß-S9 and p-NF-kB were determined in the hippocampus, frontal cortex and amygdala. Acute electric foot shock stress produced behavioral and biochemical changes; decreased the levels of p-GSK-3ß-S9, produced no change in total GSK-3ß levels and increased p-NF-kB levels in the brain. However, repeated exposure of foot shock stress restored the behavioral and biochemical changes along with normalization of p-GSK-3ß-S9 and p-NF-kB levels. Administration of AR-A01, a selective GSK-3ß inhibitor, or diethyldithiocarbamic acid (DDTC), a selective NF-kB inhibitor, diminished acute stress-induced behavioral and biochemical changes. Furthermore, AR-A014418 normalized acute stress-induced alterations in p-GSK-3ß-S9 and p-NF-kB levels, however, DDTC selectively restored NF-kB levels without any change in p-GSK-3ß-S9 levels. It probably suggests that NF-kB is a downstream mediator of the GSK-3 signaling cascade. It may conclude that acute stress associated decrease in p-GSK-3ß-S9 and increase in p-NF-kB levels in the brain contribute in the development of behavioral and biochemical alterations and normalization of GSK-3ß/NF-kB signaling may contribute in stress adaptive behavior in response to repeated electric foot shock-subjected mice.

Pair exposure with conspecific during fear conditioning induces the link between freezing and passive avoidance behaviors in rats.

Social factor plays an important role in dealing with posttraumatic stress disorder related to excessive physiological fear response and insufficient fear memory extinction of the brain. However, although social circumstances occurred not only during contextual retrieval but also during fear conditioning, most previous studies focused on the advantageous aspects of social buffering in fear retrieval period. To demonstrate the association between fear responses and fear memory from social stimuli during fear conditioning, pair exposed rats with conspecific as social buffering were subjected to a fear conditioning of passive avoidance test to evaluate memory function and freezing behavior. Whereas single exposed rats showed the significant increase of freezing behaviors and passive avoidance behaviors compared to control rats, pair exposed rats showed significant alleviation of the freezing behaviors and passive avoidance behaviors compared to single exposed rats. Furthermore, we determined a significant correlation between freezing and passive avoidance behavioral alteration in pair exposed rats. Taken together, we suggest that pair exposure with conspecific during fear conditioning helps to cope with both freezing response and fear memory systems and their reciprocal interaction has a crucial potential as a resource for the relief of unreasonable stress responses in posttraumatic stress disorder.

Electric foot shock stress adaptation: Does it exist or not?

Stress adaptation is a protective phenomenon against repeated stress exposure and is characterized by a decreased responsiveness to a repeated stress stimulus. The adaptation is associated with a complex cascade of events, including the changes in behavior, neurotransmitter and gene expression levels. The non-adaptation or maladaptation to stress may underlie the affective disorders, such as anxiety, depression and post-traumatic stress disorder (PTSD). Electric foot shock is a complex stressor, which includes both physical and emotional components. Unlike immobilization, restraint and cold immersion stress, the phenomenon of stress adaptation is not very well defined in response to electric foot shock. A number of preclinical studies have reported the development of adaptation to electric foot shock stress. However, evidence also reveals the non-adaptive behavior in response to foot shocks. The distinct adaptive/non-adaptive responses may be possibly influenced by the type, intensity, and duration of the stress. The present review discusses the existence or non-existence of adaptation to electric foot shock stress along with possible mechanism.

Preclinical experimental stress studies: protocols, assessment and comparison.

Stress is a state of threatened homeostasis during which a variety of adaptive processes are activated to produce physiological and behavioral changes. Preclinical models are pivotal for understanding these physiological or pathophysiological changes in the body in response to stress. Furthermore, these models are also important for the development of novel pharmacological agents for stress management. The well described preclinical stress models include immobilization, restraint, electric foot shock and social isolation stress. Stress assessment in animals is done at the behavioral level using open field, social interaction, hole board test; at the biochemical level by measuring plasma corticosterone and ACTH; at the physiological level by measuring food intake, body weight, adrenal gland weight and gastric ulceration. Furthermore the comparison between different stressors including electric foot shock, immobilization and cold stressor is described in terms of intensity, hormonal release, protein changes in brain, adaptation and sleep pattern. This present review describes these preclinical stress protocols, and stress assessment at different levels.

Investigations in foot shock stress of variable intensity in mice: Adaptation and role of angiotensin II.

The present study investigated the stress adaptation and role of angiotensin in response to repeated exposures of electric foot shocks of varying intensity. Mice were subjected to moderate (0.5mA) or severe (1.5mA) electric foot shocks for 1h for 5 days. Stress-induced behavioral changes were assessed by actophotometer, hole board, open field and social interaction tests. The serum corticosterone levels were measured as an index of HPA axis. Telmisartan (a selective AT1 receptor blocker) was employed as a pharmacological tool. A single exposure of moderate and severe stress produced behavioral deficits and increased the corticosterone levels. The restoration of these alterations was observed in response to repeated exposures of moderate stress, while no adaptation was observed in severe foot shock stress. A single administration of telmisartan (5mg/kg) exacerbated the moderate stress-induced decrease in behavioral activity and increase in corticosterone levels on the first day of stress exposure, suggesting the anti-stress role of angiotensin. In contrast, telmisartan normalized severe stress-induced behavioral and biochemical alterations suggesting the stress inducing actions of angiotensin. Furthermore, treatment with telmisartan abolished the stress adaptive response following repeated exposures of moderate stress suggesting that angiotensin has an adaptive role. It is concluded that there is a differential adaptive response in foot shock stress depending upon the severity of stress. Angiotensin II may act as an anti-stress agent and helps to promote the adaptation during medium stress, whereas it may promote stress response during severe stress.

Pharmacological investigations on cross adaptation in mice subjected to stress immobilization.

AIMS: The present study was designed to investigate the existence of cross adaptation between immobilization and foot shock stress, and to identify the role of endogenous opioids in cross adaptation. METHODS: Mice were subjected to acute stress using a single episode of immobilization of 2h duration; while stress adaptation was induced by repeated exposures to homotypic stressor for 5 days. To explore the existence of cross adaptation, homotypic stressor was replaced with a heterotypic stressor on the 6th day and foot shocks of 0.5 mA intensity, 1s duration with an interval of 2 min were delivered for 1h. Stress-related behavioral alterations were assessed using the actophotometer, hole board, open field and social interaction tests. KEY FINDINGS: A single exposure of immobilization produced the significant behavioral alterations that were restored on the 5th day following repeated applications of immobilization stress, indicating the development of stress adaptation. Furthermore, acute exposure of foot shock (heterotypic stressor) did not produce the behavioral alterations in immobilization stress adapted animals, indicating the development of cross adaptation. Administration of naloxone abolished the restoration of behavioral changes as a part of adaptive/cross adaptive process in repeated immobilization stress-subjected mice. SIGNIFICANCE: It may be concluded that immobilization stress adapted mice exhibit cross adaptation to foot shock stress, with the possible involvement of opioids as endogenous adaptogenic/cross adaptogenic factors.

Investigations into mild electric foot shock stress-induced cognitive enhancement: possible role of angiotensin neuropeptides.

This study was designed to investigate the role of angiotensin neuropeptides in mild electric foot shock stress-induced cognitive enhancement in mice. Mild stress was induced by applying mild electric foot shocks of 0.15 mA intensity for 0.5 s. The stress-induced alteration in cognition was assessed using a Morris water maze test. The animals were subjected to mild electric foot shocks 5 min before we recorded escape latency time (ELT), an index of learning, during the first 4 days of a 5-day trial in the Morris water maze. The time spent in target quadrant (TSTQ), an index of retrieval, was noted on the fifth day without prior administration of electric foot shock. The angiotensin-converting enzyme inhibitor lisinopril (5, 10 and 20 mg/kg), and telmisartan (1, 2 and 5 mg/kg), an angiotensin II receptor blocker, were employed to assess the role of angiotensin neuropeptides. The application of mild electric shocks significantly decreased ELT and increased TSTQ, indicating enhancement in stress-induced learning and memory. However, administration of lisinopril and telmisartan significantly attenuated the stress-induced decrease in ELT and increase in TSTQ. It may be concluded that mild electric foot shock-induced stress triggers the release of angiotensin neuropeptides that may be responsible for memory enhancement.