Effect of restraint and injection methods on heart rate and body temperature in mice

Routine procedures in the laboratory, inducing acute stress, will have an impact on the animals and might thereby influence scientific results. In an attempt to gain more insight into quantifying this acute stress by means of the parameters heart rate (HR) and body temperature (BT), we subjected mice to different restraint and injection methods. We first compared the treatment response of HR and BT, measured by means of radiotelemetry, with the treatment response of plasma corticosterone (pCORT), a common and well-validated parameter for measuring acute stress responses. It was found that HR, and to a lesser extent also BT, parallels pCORT values after subjecting the animals to different methods of restraint. Secondly, the acute stress response caused by different injection methods was evaluated. Again, HR was found to be a more sensitive parameter than BT. We found that, in case of sham injections, the acute stress response after an intraperitoneal (i.p.) injection was more pronounced than after intramuscular (i.m.) or subcutaneous (s.c.) injections, but this difference was found to be inconsistent when saline was used as injection fluid. In a third experiment we investigated if the level of experience of the animal technician influenced the stress response after s.c. injections, but no differences were found. Overall, the results have indicated that HR might be considered as a useful parameter for measuring acute stress responses to routine procedures, but the value of BT seems to be of limited value in this respect.     

Repeated handling, restraint, or chronic crowding impair the hypothalamic-pituitary-adrenocortical response to acute restraint stress.

The purpose of the present study was to assess whether, and to what extent prior handling, restraint or social crowding stress during 3-10 days affects the hypothalamic-pituitary-adrenocortical (HPA) response to an acute short-lasting restraint stress. Also the effect of a feedback inhibitory mechanism of corticosterone in the impairment of HPA axis by these stressors was investigated. Male Wistar rats were pretreated with handling 1 min/day for 3-10 days, restraint 2 times daily for 3-7 days and crowding stress for 7 days before exposure to acute restraint stress in metal tubes for 10 min. Some group of rats received exogenous s.c. corticosterone either once 25 mg/kg or 2 times daily 10 mg/kg for 3-10 days before restraint stress. After the last restraint the rats were decapitated and their trunk blood was collected for the measurement of plasma ACTH and serum corticosterone levels. Handling for 3-7 days, restraint for 3-7 days, and crowding for 7 days and a single pretreatment with corticosterone--all significantly and to a similar extent inhibited the restraint stress-induced increase in ACTH and corticosterone secretion. Chronic pretreatment with corticosterone blunted the restraint stress-induced increase in HPA axis activity. These results indicate that repeated short-lasting stress induced by handling, restraint, or crowding potently attenuates the acute restraint stress-induced stimulatory action of the HPA axis. They also indicate adaptive action of moderate stress on the HPA axis response to acute stress. The results also suggest that a short-lasting hypersecretion of corticosterone during psychological stress may induce a prolonged feedback inhibition of the HPA axis activity. The attenuation of HPA axis response by prior handling has also obvious methodological implications.     

Restraint increases afebrile body temperature but attenuates fever in Pekin ducks (Anas platyrhynchos)

In mammals, procedures such as handling, restraint, or exposure to open spaces induces an increase in body temperature (T(b)). The increase in temperature shares some characteristics with pyrogen-induced fever and so is often called "stress fever." Birds also respond to acute handling with a stress fever, which may confound thermoregulatory studies that involve animal restraint. We have measured the T(b) responses of Pekin ducks on days when they were restrained and compared them to days when the birds remained unrestrained. Restraint induced a 0.5 degrees C increase in T(b) that was sustained for the entire 8 h of restraint. To determine whether the restraint-induced increase in T(b) is mediated by prostaglandins (PGs) we compared the T(b) responses during restraint after intraperitoneal injection with saline to the responses during restraint after injection with diclofenac sodium (15 mg/kg). There was no difference in response, suggesting that restraint affects T(b) by a PG-independent mechanism. We also compared the T(b) response to intramuscular injection of lipopolysaccharide (LPS; 100 microg/kg), a bacterial pyrogen, when the ducks were restrained or unrestrained. Despite T(b) being higher at the time of LPS injection when the ducks were restrained, the maximum temperature reached after LPS injection was higher, and the period that T(b) remained elevated was longer when the ducks were unrestrained. We conclude that restraint should be considered as a potential confounder in thermoregulatory studies in birds and presumably other species too.     

Role of hypothalamic inputs in maintaining pituitary-adrenal responsiveness in repeated restraint

The role of hypothalamic structures in the regulation of chronic stress responses was studied by lesioning the mediobasal hypothalamus or the paraventricular nucleus of hypothalamus (PVH). Rats were acutely (60 min) and/or repeatedly (for 7 days) restrained. In controls, a single restraint elevated the plasma adrenocorticotropin (ACTH), corticosterone, and prolactin levels. Repeated restraint produced all signs of chronic stress, including decreased body and thymus weights, increased adrenal weight, basal corticosterone levels, and proopiomelanocortin (POMC) mRNA expression in the anterior pituitary. Some adaptation to repeated restraint of the ACTH response, but not of other hormonal responses, was seen. Lesioning of the mediobasal hypothalamus abolished the hormonal response and POMC mRNA activation to acute and/or repeated restraint, suggesting that the hypothalamo-pituitary-adrenal axis activation during repeated restraint is centrally driven. PVH lesion inhibited the ACTH and corticosterone rise to the first restraint by approximately 50%. In repeatedly restrained rats with PVH lesion, the ACTH response to the last restraint was reduced almost to basal control levels, and the elevation of POMC mRNA level was prevented. PVH seems to be important for the repeated restraint-induced ACTH and POMC mRNA stimulation, but it appears to partially mediate other restraint-induced hormonal changes.     

Restraint increases prolactin and REM sleep in C57BL/6J mice but not in BALB/cJ mice

Sleep is generally considered to be a recovery from prior wakefulness. The architecture of sleep not only depends on the duration of wakefulness but also on its quality in terms of specific experiences. In the present experiment, we studied the effects of restraint stress on sleep architecture and sleep electroencephalography (EEG) in different strains of mice (C57BL/6J and BALB/cJ). One objective was to determine if the rapid eye movement (REM) sleep-promoting effects of restraint stress previously reported for rats would also occur in mice. In addition, we examined whether the effects of restraint stress on sleep are different from effects of social defeat stress, which was found to have a non-REM (NREM) sleep-promoting effect. We further measured corticosterone and prolactin levels as possible mediators of restraint stress-induced changes in sleep. Adult male C57BL/6J and BALB/cJ mice were subjected to 1 h of restraint stress in the middle of the light phase. To control for possible effects of sleep loss per se, the animals were also kept awake for 1 h by gentle handling. Restraint stress resulted in a mild increase in NREM sleep compared with baseline, but, overall, this effect was not significantly different from sleep deprivation by gentle handling. In contrast, restraint stress caused a significant increase in REM sleep compared with handling in the C57BL/6J mice but not in BALB/cJ mice. Corticosterone levels were significantly and similarly elevated after restraint in both strains, but prolactin was increased only in the C57BL/6J mice. In conclusion, this study shows that the restraint stress-induced increase in REM sleep in mice is strongly strain dependent. The concomitant increases in prolactin and REM sleep in the C57BL/6J mice, but not in BALB/cJ mice, suggest prolactin may be involved in the mechanism underlying restraint stress-induced REM sleep. Furthermore, this study confirms that different stressors differentially affect NREM and REM sleep. Whereas restraint stress promotes REM sleep in C57BL/6J mice, we previously found that in the same strain, social defeat stress promotes NREM sleep. As such, studying the consequences of specific stressful stimuli may be an important tool to unravel both the mechanism and function of different sleep stages.     

Effects of repeated investigator handling of Leach's Storm‐Petrel chicks on growth rates and the acute stress response

Disturbance during development may have lasting effects on the growth rates and stress physiology of birds. Although repeated handling by researchers is often necessary, the possible effects of such handling on the development of semi‐altricial young are unclear. We examined the effect of daily handling on growth rates and plasma corticosterone levels of Leach's Storm‐Petrel (Oceanodroma leucorhoa) chicks on Kent Island in the Bay of Fundy, New Brunswick, Canada, during the 2011 nesting season. From post‐hatch day 7 to post‐hatch days 14–36, birds in the experimental group were extracted from burrows and measured (wing, tarsus, and mass) for ～3 min every day, whereas birds in the control group were left undisturbed. After the treatment period, blood was collected from birds in both groups within 3 min of initially reaching into burrows (baseline) and after a 30‐min restraint stress test to assess the effect of early life disturbance on programming of the hypothalamic‐pituitary‐adrenal (HPA) axis. A second acute restraint stress test was conducted three weeks after the end of the treatment period to investigate possible longer term effects of early life disturbance. Growth rates of wings and tarsi were similar for handled chicks (N = 18) and non‐handled control chicks (N = 21), as were baseline and 30‐min acute restraint stress‐induced corticosterone levels. As also reported in previous studies of other altricial and semi‐altricial species, older chicks (42–64 d old) had higher plasma corticosterone levels than younger chicks (21–43 d old) after acute restraint stress tests, reflecting delayed development of the HPA axis. The age‐related increase in HPA axis sensitivity observed prior to fledging could facilitate foraging and predator avoidance behaviors while minimizing exposure to high levels of corticosterone earlier in development. Overall, we found no evidence that repeated disturbance influenced either growth rates or HPA axis programming of Leach's Storm‐Petrel chicks.     

Mouse strain differences in autonomic responses to stress

In humans, anxiety disorders are often accompanied by an overactive autonomic nervous system, reflected in increased body temperature (BT) and heart rate (HR). In rodents, comparable effects are found after exposure to stress. These autonomic parameters can give important information on stress and anxiety responses in mice. In the present experiments, stress reactivity of three frequently used mouse strains [129 Sv/Ev, Swiss Webster (SW) and C57 BL/6] was assessed using their autonomic stress responses. BT, HR and activity were telemetrically measured. Undisturbed circadian rhythms already showed clear differences between the mouse strains. Hereafter, autonomic responses to stressors with increasing intensity were measured. Strain differences were found in magnitude and duration of the stress responses, especially after high-intensity stressors. Generally, C57BL/6 mice showed the largest autonomic response, SW the lowest and the 129Sv/Ev the intermediate response. Interestingly, the observed ranking in autonomic stress response does not match the behavioral stress responsivity of these strains. Finally, sensitivity to the anxiolytic diazepam (0, 1, 2, 4 and 8 mg/kg) was tested using the stress-induced hyperthermia paradigm. Pharmacological sensitivity to diazepam differed between the strains with the 129Sv/Ev being most sensitive. These studies show that simultaneous measurement of behavioral and autonomic parameters under stressful conditions contributes considerably to a better interpretation of anxiety and stress levels in mice.     

Effect of myiasis and acute restraint stress on plasma levels of immunoreactive beta-endorphin, adrenocorticotrophin (ACTH) and cortisol in the sheep

Cutaneous myiasis in sheep arising from the activity of Lucilia cuprina larvae can result in significant physiological changes in susceptible animals. The stress imposed on the pituitary-adrenal axis of the sheep in response to myiasis and acute restraint is the subject of this investigation. Merino wethers were exposed to handling restraint, and blood sampling, during examination for blowfly strike; where necessary, they were treated for cutaneous myiasis. Significant changes in the plasma concentrations of immunoreactive beta-endorphin (beta-EP), ACTH and cortisol were found in sheep with extensive myiasis, as compared with unstruck sheep or those with only localized myiasis. In five susceptible sheep with extensive cutaneous myiasis, mean plasma levels of beta-EP, ACTH and cortisol were 307 +/- 71 pg ml-1, 953 +/- 58 pg ml-1 and 232 +/- 46 nmol l-1 respectively, compared with 818 +/- 89 pg ml-1, 641 +/- 41 pg ml-1 and 107 +/- 17 nmol l-1 in six unstruck sheep handled similarly. Whereas significant increases in plasma ACTH and cortisol can result from pituitary-adrenal responses to acute emotional or surgical stress, and are usually accompanied by a concomitant release of beta-EP from the pituitary, the present findings indicate a marked reduction in beta-EP levels and a significant increase in ACTH and cortisol in sheep following blowfly strike and acute handling restraint. This result suggests that cutaneous myiasis in susceptible sheep can alter the pituitary-adrenal response to acute restraint stress, and this could occur either by an alteration of precursor processing in the pituitary or by the selective release of ACTH.     

Comparison of Endocrine Response to Stress Between Captive-Raised and Wild-Caught Bighorn Sheep

ABSTRACT Stress hormones in Rocky Mountain bighorn sheep (Ovis canadensis canadensis), produced in response to environmental changes, road development, or high population density, may impact their immune systems to a threshold level that predisposes them to periodic, large-scale mortality. We compared the stress response to a novel environmental situation and repeated handling between bighorn sheep born and raised in captivity (CR) and bighorn sheep born in the wild (WC) and brought into captivity. We measured plasma epinephrine, norepinephrine, cortisol, and fecal glucocorticoid metabolites (FGM). Three weeks after each group's arrival we used a one-time drop-net event to elicit an acute stress response, and we collected blood samples from each sheep over 35 minutes, as well as one fecal sample. We collected blood and fecal samples from both groups on 7 other occasions over the subsequent 6 months. We also collected fecal samples from the pen at approximately 24-hour intervals for 3 days following every handling event to monitor the stress response to handling. We found that CR sheep had a stronger autonomic nervous system response than WC sheep, as measured by epinephrine and norepinephrine levels, but we found a very similar hypothalamic-pituitary–adrenal axis (HPA) response, measured by cortisol levels, to the acute stress event of a drop-net restraint. We also found that once the WC sheep had acclimated, as indicated by the return to the initial baseline FGM levels within 12 weeks, the CR and WC groups' HPA responses to sampling events were not significantly different from one another. Fecal samples can provide a noninvasive mechanism for managers to monitor baseline FGM for a given herd. Using long-term monitoring of FGM rather than values from a single point in time may allow managers to correlate these levels to outside influences on the herd and better understand the impacts of management changes, population density, or increased human developments on the health of the sheep population.     

Prolactin response to stress in neonatally estrogenized male rats

Male Wistar rats were injected either 500 micrograms of estradiol benzoate or olive oil on their first day of life. Blood samples were obtained from the adult by decapitation, by decapitation after 15 min of restraint, by decapitation 10 min after a 5 min period of ether exposure or by jugular venipuncture after 60 s of ether exposure. Prolactin (PRL) plasma levels were measured by RIA. The PRL levels in samples obtained by decapitation were similar to control and estrogenized groups. A similar response to restraint was also found in both groups. Sixty s of exposure to ether stress stimulates PRL secretion only in the estrogenized males, this effect being blocked by treatment with Normifensine (5 mg/kg two hours prior to blood sampling). These results suggested that estrogenized male rats show greater sensitivity to acute ether stress than the controls, and that changes in the dopaminergic system could be involved in this response.     

The effects of handling and anesthetic agents on the stress response and carbohydrate metabolism in northern elephant seals

Free-ranging animals often cope with fluctuating environmental conditions such as weather, food availability, predation risk, the requirements of breeding, and the influence of anthropogenic factors. Consequently, researchers are increasingly measuring stress markers, especially glucocorticoids, to understand stress, disturbance, and population health. Studying free-ranging animals, however, comes with numerous difficulties posed by environmental conditions and the particular characteristics of study species. Performing measurements under either physical restraint or chemical sedation may affect the physiological variable under investigation and lead to values that may not reflect the standard functional state of the animal. This study measured the stress response resulting from different handling conditions in northern elephant seals and any ensuing influences on carbohydrate metabolism. Endogenous glucose production (EGP) was measured using [6-(3)H]glucose and plasma cortisol concentration was measured from blood samples drawn during three-hour measurement intervals. These measurements were conducted in weanlings and yearlings with and without the use of chemical sedatives--under chemical sedation, physical restraint, or unrestrained. We compared these findings with measurements in adult seals sedated in the field. The method of handling had a significant influence on the stress response and carbohydrate metabolism. Physically restrained weanlings and yearlings transported to the lab had increased concentrations of circulating cortisol (F(11, 46)?=?25.2, p<0.01) and epinephrine (F(3, 12)?=?5.8, p?=?0.01). Physical restraint led to increased EGP (t?=?3.1, p?=?0.04) and elevated plasma glucose levels (t?=?8.2, p<0.01). Animals chemically sedated in the field typically did not exhibit a cortisol stress response. The combination of anesthetic agents (Telazol, ketamine, and diazepam) used in this study appeared to alleviate a cortisol stress response due to handling in the field without altering carbohydrate metabolism. Measures of hormone concentrations and metabolism made under these conditions are more likely to reflect basal values.     

The use of radiotelemetry to assess the time needed to acclimatize guineapigs following several hours of ground transport

The objective of this study was to investigate the effect of ground transportation on guineapigs. Physiological parameters, i.e. heart rate (HR), body temperature (BT) and activity (ACT), were measured before and after transport, using previously implanted radiotelemetry transmitters. Body weight was measured before and after transport. After a postsurgical recovery period and data recording at the breeder's facility, the animals were transported for 2.25 h (Group 1) and for 7.5 h (Group 2) to a different animal facility. Data collection started immediately after arrival at the second animal facility. All parameters measured changed significantly after transport. These results suggest that a 10- to 12-day period is required for guineapigs to return to pre-transport levels of HR, BT and ACT.     

Higher heart rate of laboratory mice housed individually vs in pairs

Many studies have shown that housing mice individually over a long period significantly alters their physiology, but in most cases measurement has required human interference and restraint for sampling. Using a radio-telemetry system with implantable transmitters, we recorded heart rate (HR), motor activity (ACT) and body temperature (BT) of freely moving male mice (NMRI) housed either individually or in pairs with an ovarectomized female. Data for each parameter were collected at 5 min intervals for two consecutive 24 h periods. Even after several weeks of habituation to the social conditions, HR was increased in mice housed individually compared with mice housed in pairs, although their measured ACT did not differ. Additionally, BT tended to be reduced in individually-housed mice. When the data were analysed according to different ACT levels, HR was increased in individually-housed mice during phases of low and high, but not intermediate, motor activity. Furthermore, individually-housed mice had more, but shorter, resting bouts, indicating disruption of the normal circadian sleep pattern. Enhanced HR in individually-housed mice does not necessarily indicate stress, but might be an important physiological indicator of discomfort. The fact that individual housing alters basic physiological parameters in laboratory mice highlights the need to control for housing-dependent variation, especially in experiments that are sensitive to changes in these parameters.     

The effects on heart rate and temperature of mice and vas deferens responses to noradrenaline when their cage mates are subjected to daily restraint stress

Performing stressful procedures in view of cage mates may cause stress in observer animals. However, it is not known if stressful procedures performed in close proximity to, but not in view of cage mates are stressful for the (observer) cage mates. Radiotelemetry and postmortem in vitro studies of the vas deferens were used to determine the effects of stress on observers. Heart rate (HR) and core body temperature (cBT) were recorded for 1 h following weighing of a cage mate or 1 h during restraint of a cage mate and the hour following return of the restrained mouse to the cage. This procedure was repeated daily for 15 days. HR and cBT were increased in observers during both restraint and weighing of cage mates. Analysis of the area under the curve showed that HR and cBT in observers were significantly higher during restraint of a cage mate than after weighing of a cage mate. When mice were returned to the cage after weighing or restraint, HR and cBT were significantly higher in the cage mates of restrained animals. Comparison between days 1, 3, 7 and 14 found that, as the experiment progressed, HR and cBT were significantly reduced in the observer mice during the hour following return of the cage mates after restraint. Results from previous studies have shown that chronic stress causes the vas deferens to become hypersensitive to exogenous application of noradrenaline (NAd). In this study, vas deferens from observers of restraint had a significantly increased response to NAd. These results indicate that stressful procedures should be conducted in isolation from other mice.     

Dissociation between adrenocorticotropin and corticosterone responses to restraint after previous chronic exposure to stress

The effect on emotional reactivity produced by a model for chronic stress in which different types of acute stresses were randomly combined for 29 days was studied in adult male rats. Chronically stressed rats showed a slight decrease in body weight gain and an increase in relative adrenal weight. Chronic stress did not modify defecation rate but reduced exploratory activity in the holeboard. Neither basal nor acute-stress induced levels of adrenocorticotropin (ACTH) were modified by previous chronic stress. Likewise, basal corticosterone levels were similar in both groups. However, corticosterone response to acute restraint stress was higher in chronically stressed than in control rats. The mechanisms underlying the dissociation between ACTH and corticosterone as well as its possible implications are discussed.     

Effects of wire-bottom caging on heart rate, activity and body temperature in telemetry-implanted rats

Some experimental procedures are associated with placement of animals in wire-bottom cages. The goal of this study was to evaluate stress-related physiological parameters (heart rate [HR], body temperature [BT], locomotor activity [LA], body weight [BW] and food consumption) in rats under two housing conditions, namely in wire-bottom cages and in bedding-bottom cages. Telemetry devices were surgically implanted in male Sprague-Dawley rats. HR, BT and LA were recorded at 5 min intervals. Analysis under each housing condition was performed from 16:00 to 08:00 h of the following day (4 h light, 12 h dark). During almost all of the light phase, the HR of rats housed in wire-bottom cages remained high (371 ± 35 bpm; mean ± SD; n = 6) and was significantly different from that of rats housed in bedding-bottom cages (340 ± 29 bpm; n = 6; P < 0.001; Student's t-test). In general, BT was similar under the two housing conditions. However, when rats were in wire-bottom cages, BT tended to fluctuate more widely during the dark phase. LA decreased when animals were housed in wire-bottom cages, in particular during the dark phase. Moreover, there was a significant difference with respect to the gain in BW: BW of rats housed in bedding-bottom cages increased 12 ± 2 g, whereas that of rats in wire-bottom cages decreased by 2 ± 3 g (P < 0.001). Our results demonstrate that housing rats in wire-bottom cages overnight leads to immediate alterations of HR, BW and LA, which might be related to a stress response.     

Comparative analysis and physiological impact of different tissue biopsy methodologies used for the genotyping of laboratory mice

Genotyping of genetically modified mice and control of authenticity of the genetic background of congenic or coisogenic strains by polymerase chain reaction (PCR) is a routine procedure that can be performed with different tissue biopsies causing variable grades of trauma. In this study, some invasive and non-invasive sampling methods were compared, with the main focus on the impact on animal physiology. We compared ear punch, tail biopsy, hair plugging, mouth and rectum swabs and the simple restraint of the animals, scoring for the impact on heart rate (HR), core body temperature (BT) and motor activity by telemetry, during biopsy and for the following 6 h. Furthermore, in order to correlate the physiological impact with the practicability and reliability of the genotyping results, we performed a PCR analysis of the biopsy samples obtained by using the same collection procedures analysed by telemetry. All sampling methods and restraint induced significant increase in HR and BT and a slight increase in motor activity for 1 h, independent of the invasiveness of the method used. Genotyping of all biopsies allowed the proper identification of transgenic animals, tail biopsies, ear punches and hair follicles giving clear signals, the last method being fast, but also susceptible to cross contaminations during sampling by large numbers of animals. Restraint and all biopsy methods provoked similar physiological changes, indicating that the handling of the animals is of major importance and that the sampling procedure does not strongly influence the physiological parameters.     

Sympathetic hyperreactivity to air-jet stress in the chromosome 1 blood pressure quantitative trait locus congenic rats

A chromosome 1 blood pressure quantitative trait locus (QTL) was introgressed from the stroke-prone spontaneously hypertensive rats (SHRSP) to Wistar-Kyoto (WKY) rats. This congenic strain (WKYpch1.0) showed an exaggerated pressor response to both restraint and cold stress. In this study, we evaluated cardiovascular and sympathetic response to an air-jet stress and also examined the role of the brain renin-angiotensin system (RAS) in the stress response of WKYpch1.0. We measured mean arterial pressure (MAP), heart rate (HR), and renal sympathetic nerve activity (RSNA) responses to air-jet stress in WKYpch1.0, WKY, and SHRSP. We also examined effects of intracerebroventricular administration of candesartan, an ANG II type 1 receptor blocker, on MAP and HR responses to air-jet stress. Baseline MAP in the WKYpch1.0 and WKY rats were comparable, while it was lower than that in SHRSP rats. Baseline HR did not differ among the strains. In WKYpch1.0, air-jet stress caused greater increase in MAP and RSNA than in WKY. The increase in RSNA was as large as that in SHRSP, whereas the increase in MAP was smaller than in SHRSP. Intracerebroventricular injection of a nondepressor dose of candesartan inhibited the stress-induced pressor response to a greater extent in WKYpch1.0 than in WKY. Intravenous injection of phenylephrine caused a presser effect comparable between WKYpch1.0 and WKY. These results suggest that the chromosome 1 blood pressure QTL congenic rat has a sympathetic hyperreactivity to an air-jet stress, which causes exaggerated pressor responses. The exaggerated response is at least partly mediated by the brain RAS.     

Bupleurum falcatum prevents depression and anxiety-like behaviors in rats exposed to repeated restraint stress

Previous studies have demonstrated that repeated restraint stress in rodents produces increases in depression and anxietylike behaviors and alters the expression of corticotrophinreleasing factor (CRF) in the hypothalamus. The current study focused on the impact of Bupleurum falcatum (BF) extract administration on repeated restraint stress-induced behavioral responses using the forced swimming test (FST) and elevated plus maze (EPM) test. Immunohistochemical examinations of tyrosine hydroxylase (TH) expression in rat brain were also conducted. Male rats received daily doses of 20, 50, or 100 mg/kg (i.p.) BF extract for 15 days, 30 min prior to restraint stress (4 h/day). Hypothalamicpituitary- adrenal axis activation in response to repeated restraint stress was confirmed base on serum corticosterone levels and CRF expression in the hypothalamus. Animals that were pre-treated with BF extract displayed significantly reduced immobility in the FST and increased open-arm exploration in the EPM test in comparison with controls. BF also blocked the increase in TH expression in the locus coeruleus of treated rats that experienced restraint stress. Together, these results demonstrate that BF extract administration prior to restraint stress significantly reduces depression and anxiety-like behaviors, possibly through central adrenergic mechanisms, and they suggest a role for BF extract in the treatment of depression and anxiety disorders.