Validation of a behavioral recording automated system in the elevated plus-maze test

The elevated plus-maze test has been widely used for screening of anxiolytic drugs and for exploring neurobiological bases of anxiety. In this study, we validated a new automated system that enables to record exploratory behavior in the elevated plus-maze test. This system, called cyberplus, consisted of ten pairs of photoelectric cells strategically located in several parts of the apparatus, and seemed to be sensitive to the position of the animal's forepaws, so it would yield scores in anxiety measurements and locomotor activity similar to those obtained by following the traditional procedure, that is, by analyzing videotapes by experienced observers. In order to assess this hypothesis, we exposed rats to the elevated plus-maze test and compared the scores obtained by cyberplus with the values recorded by two independent observers, conducting a correlational study with both kinds of recording procedures. The results obtained suggest the utility of cyberplus as a behavioral recording automated system in the elevated plus-maze test, making data collection and data analysis easier in exploring pharmacological and neurobiological bases of anxiety.     

Effects of diazepam on the behaviour of weaned pigs in three putative models of anxiety

The present study examined the effects of diazepam (a widely used anxiolytic benzodiazepine) on the behavioural response of pigs to three novel experimental situations used to measure anxiety-related behaviour in rodents. Twelve weaned pigs (two pairs from each of the three litters) were tested in an elevated plus-maze at the age of 6 weeks, a light/dark test at the age of 7 weeks and an open-field test at the age of 8 weeks. Six of the pigs were pre-treated with diazepam (valium) and the other six with saline (control). In the elevated plus-maze, diazepam-treated pigs had a higher number of entries into open arms (P=0.04), spent more time on open arms (P=0.07), and had a higher number of total arm entries (P=0.05) than pigs from the control group. However, diazepam had no significant effects on behaviour in the light/dark test (i.e., latency to enter lit compartment, number of entries into lit compartment and the time spent in lit compartment) or the open-field test (i.e., number of lines crossed, number of entries into centre). In summary, the anxiolytic effects of diazepam on the pigs' behaviour were only demonstrated in the elevated plus-maze, where the time spent on open arms and the number of entries into open arms could be interpreted as measures of anxiety in pigs.     

Avoidance behaviour and anxiety in rats irradiated with a sublethal dose of gamma-rays

The aim of this study was to assess, whether a sublethal dose of gamma-rays will influence the avoidance behaviour and anxiety in rats and whether the response to radiation depends on time of day of its application. Adult male Wistar rats were tested in elevated plus-maze, in hot plate test and in the light/dark box in 4 regular intervals during a day. After two weeks the animals were irradiated with a whole-body dose 6 Gy of gamma-rays. One day after irradiation the animals were repeatedly tested in the same way, as before irradiation. In the plus-maze test an increased level of anxiety was established. The irradiation significantly decreased the locomotor activity of rats, but the extent of exploratory and comfortable behaviour were not altered. After irradiation, an elevated aversion to the thermal stimulus was observed in the hot plate test. The effects of radiation were more pronounced in the light period of the day, than in the dark one. No significant differences in aversion to light were detected after irradiation. The obtained results indicate, that sublethal doses of ionizing radiation can markedly influence the reactivity of animals to adverse stimuli, their motoric activity and emotional status, as well.     

Individual differences in the elevated plus-maze and the forced swim test

The elevated plus-maze is an apparatus composed of enclosed and open (elevated) arms and time spent in the open arms by a rat can be increased/decreased by anxiolytic/anxiogenic agents. In the forced swim test, floating behavior is used as an index of behavioral despair and can be decreased by antidepressant agents. As the comorbidity between anxiety and depression is a remarkable issue in human behavioral disorders, a possible relationship between the behaviors seen in the cited tests is of great relevance. In the present study, fifty-four male rats (Rattus norvegicus) were submitted to a plus-maze session and to a 2-day forced swim protocol. According to their time in the open arms, they were divided into three groups: Low Open, Medium Open and High Open. Some plus-maze measures were found to be coherent with time in the open arms and are suggested to also be reliable anxiety indexes. In the forced swim test, the Low Open group showed decreases in floating duration from forced swim Session 1 to Session 2, an alteration opposite to that observed in the other groups (particularly, the Medium Open group). The Low Open group also showed increases in floating latency, again in sharp contrast with the alteration found in the other groups. Accordingly, positive and negative correlation were found between time in the open arms and floating duration and latency, respectively. Results are compared to previous studies and mediation of the effect by reactivity to aversive stimulation or alterations induced by open arm exposure is discussed.     

Peculiarities of exploration behavior of socially deprived rats in stress situation

Male Wistar rats (n = 18) were studied at the age of 120 days after social deprivation in the period from the 22nd to 70th days of postnatal development. They displayed significant lower activity in the open field, elevated plus-maze, and Porsolt test than control animals (n = 19). Decreased exploratory activity was found to be related with higher level of anxiety. This was confirmed by their avoidance of open arms of the elevated plusmaze and reactions to approaching hand. These animals had problems in the maze arm choice. They showed low time variability of choice-related actions (waiting in the center of the open field or elevated plus-maze and change in the movement direction along an open-field wall, including the movement per se, and arm choice and changing, i.e., alternative choice). Shorter time of choice-related actions was observed in situations of unequal alternatives.     

Effect of phenobarbitone administration to pregnant rats on anxiety in offsprings

Adults Charles-Foster rats were prenatally treated to phenobarbitone (10 mg/kg, i.p.) from day 13 to 21 of gestation, this being the critical period of neural development. Pregnant control rats were similarly treated with equal volume of vehicle. Adult rat offsprings at 8-9 weeks of age were subjected to open-field exploratory behaviour, elevated plus-maze and elevated zero-maze tests. The rat offsprings displayed significantly increased ambulation and rearings in an open-field arena when compared to control offsprings whereas self-grooming and faecal droppings remain unchanged. On elevated plus-maze test these prenatally treated rat offsprings spent significantly less time on open arms and more time and more number of entries in enclosed arms as compared to controls. Prenatally exposed rats also showed significant less time on open arms, less number of head dips and stretched attend postures on elevated zero-maze test indicating increased anxiogenic behavioural pattern in these animals. The results suggest that prenatal exposure to phenobarbitone leaves a lasting effect on the anxiety state of the offsprings.     

Increased circulating cholecystokinin contributes to anorexia and anxiety behavior in mice overexpressing pancreatic polypeptide

We have previously reported that pancreatic polypeptide (PP) overexpressing mice display thin phenotype with delayed gastric emptying and decreased food intake. In the present study, we further examined if CCK contributes to anorexia and anxiety behavior in PP overexpressing mice. Plasma CCK levels in PP overexpressing mice and their littermates were determined by radioimmunoassay using antisera specific to sulfated CCK-8 and CCK-33. To elucidate the role of CCK in PP overexpressing mice, CCK-1 receptor antagonist (L-364,718) or saline was administered intraperitoneally and food intake was measured for 2 h. CCK-2 antagonist (L-365,260) or saline was injected intraperitoneally and the elevated plus-maze test was performed to assess anxiety. Plasma CCK levels were significantly increased in PP overexpressing mice. Administration of L-364,718 increased food intake in PP overexpressing mice compared to the saline-injected PP overexpressing group, while L-364,718 did not increase food intake in non-transgenic littermates. PP overexpressing mice exhibited anxiety in the plus-maze test. Administration of CCK-2 receptor antagonist (L-365,260) reversed the decreased percentage of entry into the open arms in PP overexpressing mice. These results indicated that elevated CCK may contribute to anorexic and anxious phenotype of PP overexpressing mice.     

Sex differences in behavioral and corticosterone responses to mild stressors in ICR mice are altered by ovariectomy in peripubertal period

Among rodents, females are generally considered to be highly responsive in terms of emotionality under stressful conditions, and have higher corticosterone levels and activity. In this study, we examined sex differences in mice by evaluating anxiety behaviors and corticosterone responses to mild stressors. In our first experiment, we analyzed the behavioral and corticosterone responses to the elevated plus-maze test and open-field test in male and female mice, and compared sex differences. Principal component analysis (PCA) was used to investigate the correlation of these responses between males and females. The corticosterone level was higher in females under both basal and stressed conditions. In the behavioral response, higher locomotor activity was seen in females in the elevated plus-maze test. PCA showed little association among anxiety behavior, locomotor activity, and corticosterone secretion. In our second experiment, we examined the activational effects of sex steroids on the corticosterone response to the elevated plus-maze test by gonadectomizing male and female mice and using testosterone or estrogen capsules as hormonal replacements. Sex differences at the basal corticosterone level were not altered by the hormonal milieu in adults, however the higher corticosterone level of females in response to stress was diminished by ovariectomy, although replacement with neither testosterone nor estrogen had any effect. These results suggest that the sex difference in novelty exposure observed in the form of a greater hypothalamic-pituitary-adrenal (HPA) axis response in female ICR mice is controlled by ovary-derived factors in adults.     

Anxiolytic-like effect of the selective neuropeptide Y Y2 receptor antagonist BIIE0246 in the elevated plus-maze

The involvement of Neuropeptide Y (NPY) in the pathophysiology of mood disorders has been suggested by clinical and preclinical evidence. NPY Y1 and Y2 receptors have been proposed to mediate the NPY modulation of stress responses and anxiety related behaviors. To further investigate the role of Y2 receptors in anxiety we studied the effect of BIIE0246, a selective Y2 receptor antagonist, in the elevated plus-maze test. Rats treated with 1.0 nmol BIIE0246 showed an increase in the time spent on the open arm of the maze. In addition, to study the effects of the Y2 antagonism on NPY protein level, NPY-like immunoreactivity was measured in different brain regions following treatment with BIIE0246, but no statistically significant effects were observed. These results suggest that BIIE0246 has an anxiolytic-like profile in the elevated plus-maze.     

Augmentation of the NO-cGMP cascade induces anxiogenic-like effect in mice.

Several studies have reported the anxiolytic-like effects of various nitric oxide synthase inhibitors in distinct animal models. However, in the context of anxiety, the possible involvement of cyclic GMP, believed to be one of the main targets of NO, remains obscure. Cyclic GMP is degraded by the specific phosphodiesterases in the brain. Therefore, we studied the effect of the selective phosphodiesterase type 5 inhibitor sildenafil in the mouse elevated plus-maze test of anxiety and in the open field test of locomotion. We found that sildenafil (0.05-10 mg/kg i.p.) alone did not affect the behavior of animals in the plus-maze or open field tests, but the anxiogenic beta-carboline DMCM given in a subconvulsive dose (2 mg/kg i.p.) decreased the time spent on open arms in the elevated plus-maze. Treatment with the NO precursor L-arginine (200 mg/kg i.p.) did not modify the behavior of animals in the plus-maze, however, when sildenafil (1 mg/kg i.p.) was administered in combination with L-arginine (200 mg/kg i.p.), both the time spent on the open arms and the percentage of open arm visits were significantly decreased. We conclude that augmentation of the NO-cGMP cascade induces anxiogenic-like effect in mice.     

Changes in anxiety level of Wistar rats under the influence of maximally-diluted solution of a plant alkaloid

A very dilute solution of Nux Vomica 200 plant alkaloid was applied onto the tongue of Wistar rats. The level of rats' anxiety was estimated by their behavior in elevated plus-maze. The dry application led to substantial changes in rats' behavior. In "passive" rats previously defined by the forced swimming tests changes in behavior were most prominent. The possible influence of the drug under study on the adaptive abilities of this group of animals is discussed.     

The effects of 5-HT1B characterizing agents in the mouse elevated plus-maze

Although the serotonergic system has been implicated in the modulation of anxiety states, the specific receptor subtypes that mediate these states require clarification. The effects of drugs that act preferentially at 5-HT1B receptors were evaluated on the behavior elicited in the elevated plus-maze, an animal model of anxiety. Variations in the intensity of light affected mouse behavior in the plus-maze; lower light intensity increased the entries to and time spent on the open arm in a manner similar to that seen with stress-attenuating circumstances. Opposite effects were observed in high light-intensity, similar to effects seen under elevated stress conditions. Chlordiazepoxide produced increased entries and time spent on the open arm, whereas pentylenetetrazol (PTZ) produced opposite effects. The preferential 5-HT1B agents TFMPP and mCPP exhibited a profile similar to PTZ. The effects of TFMPP in the plus-maze were reversed by chlordiazepoxide, but not by the benzodiazepine receptor antagonist flumazenil, which suggests that this effect is not directly mediated by benzodiazepine receptors. The decreased entries and time spent on the open arm of the maze following TFMPP or mCPP administration was possibly mediated by an antagonistic action at 5-HT1B receptors, since this effect was reversed by the selective 5-HT1B agonist CGS 12066B. The present study further demonstrates the utility of mouse behavior in the elevated plus-maze as a model for identifying anxio-modulatory substances.     

Long-term isolated housing causes anxiety in C57BL/6J female mice

Behaviour of female C57BL/6J strain mice was studied in the elevated plus-maze and Porsolt's tests after either long-lasting individual housing or keeping with daily shifting group-housed females (social instability). After 2-3 months, an increased level of anxiety in the individually housed females was revealed in the elevated plus-maze. However, in 3 months the least passive behaviour in the Porsolt's was in the individually housed females. No changes were found in behaviour of females individually housed at 3 weeks of age for 4 months. Also, females with preliminary social contacts with males and following individual housing for one month had not any abnormalities in the used behavioural tests. Social instability conditions did not significantly influence the females' plus-maze behaviour, but decreased the passive behaviour in the Porsolt's test.     

Postanesthetic Effects of Isoflurane on Behavioral Phenotypes of Adult Male C57BL/6J Mice

Isoflurane was previously the major clinical anesthetic agent but is now mainly used for veterinary anesthesia. Studies have reported widespread sites of action of isoflurane, suggesting a wide array of side effects besides sedation. In the present study, we phenotyped isoflurane-treated mice to investigate the postanesthetic behavioral effects of isoflurane. We applied comprehensive behavioral test batteries comprising sensory test battery, motor test battery, anxiety test battery, depression test battery, sociability test battery, attention test battery, and learning test battery, which were started 7 days after anesthesia with 1.8% isoflurane. In addition to the control group, we included a yoked control group that was exposed to the same stress of handling as the isoflurane-treated animals before being anesthetized. Our comprehensive behavioral test batteries revealed impaired latent inhibition in the isoflurane-treated group, but the concentration of residual isoflurane in the brain was presumably negligible. The yoked control group and isoflurane-treated group exhibited higher anxiety in the elevated plus-maze test and impaired learning function in the cued fear conditioning test. No influences were observed in sensory functions, motor functions, antidepressant behaviors, and social behaviors. A number of papers have reported an effect of isoflurane on animal behaviors, but no systematic investigation has been performed. To the best of our knowledge, this study is the first to systematically investigate the general health, neurological reflexes, sensory functions, motor functions, and higher behavioral functions of mice exposed to isoflurane as adults. Our results suggest that the postanesthetic effect of isoflurane causes attention deficit in mice. Therefore, isoflurane must be used with great care in the clinical setting and veterinary anesthesia.     

Effects of Milnacipran in Animal Models of Anxiety and Memory

Serotonin (5-HT) and noradrenaline (NA) are involved in both pathogenesis and recovery from depression and anxiety. We examined the effects of acute and chronic treatment with milnacipran, a serotonin/noradrenaline reuptake inhibitors (SNRIs) antidepressant, on anxiety and memory retention in rats. Male Wistar rats received acute or chronic administration of milnacipran (12.5, 25 or 50 mg/kg) or saline (control group). The animals were separately submitted to elevated plus-maze, inhibitory avoidance and open-field tasks 1 h after injection, in the acute group, or 23 h after last injection, in the chronic group. Our results showed an anxiolytic-like effect after chronic administration of milnacipran at doses of 25 and 50 mg/kg. The treatment does not interfere in memory retention and habituation to a novel environment at any doses studied. These findings support that milnacipran, an established SNRIs antidepressant, can also be useful in the treatment of anxiety disorders.     

Neurons in the amygdala with response-selectivity for anxiety in two ethologically based tests

The amygdala is a key area in the brain for detecting potential threats or dangers, and further mediating anxiety. However, the neuronal mechanisms of anxiety in the amygdala have not been well characterized. Here we report that in freely-behaving mice, a group of neurons in the basolateral amygdala (BLA) fires tonically under anxiety conditions in both open-field and elevated plus-maze tests. The firing patterns of these neurons displayed a characteristic slow onset and progressively increased firing rates. Specifically, these firing patterns were correlated to a gradual development of anxiety-like behaviors in the open-field test. Moreover, these neurons could be activated by any impoverished environment similar to an open-field; and introduction of both comfortable and uncomfortable stimuli temporarily suppressed the activity of these BLA neurons. Importantly, the excitability of these BLA neurons correlated well with levels of anxiety. These results demonstrate that this type of BLA neuron is likely to represent anxiety and/or emotional values of anxiety elicited by anxiogenic environmental stressors.     

Effects of prenatal disturbance in the brain testosterone metabolism on anxiety level and behavior of rats in a novel environment

The effects of maternal administration of the aromatase inhibitor 1,4,6-androstatrien-3,17-dione (ATD) during the last week of gestation on formation of behavior in a novel environment were studied in male and female offspring. The "open field" and the elevated plus-maze tests were used. The results showed that there were a significant elevation of the anxiety level and emotionality in ATD-treated 30-day-old female rats, whereas at the age of 90 days, the elevation of these behavioral parameters was observed both in males and females. There was no a sexual dimorphism in behavioral response to a novel environment such as locomotor activity, time of immobilization, total duration of grooming reaction, and anxiety level between adult control male and treated female rats. These data suggest that prenatal inhibition of the brain testosterone metabolism alters the formation of sexual dimorphism of the anxiety and behavioral response to a novel environment in adulthood.     

Role of sex steroids in development of anxiety in female mice

Level of sex steroid hormones being changed throughout an estrous cycle influences physiological and behavioral features of female subjects. To test how estrogen and progesterone affect the anxiety level in mice the ovariectomy (OVX) followed by hormone treatment was carried out. After 1 week of recovery period estradiol benzonate (20 micrograms, s/c) was injected once a day during 7 consequent days. By the 7th day in addition to EB injection progesterone (500 micrograms, s/c) was also injected. Four hours later the mice were tested in elevated plus-maze to measure the anxiety level. Control animals were treated with sesame oil only. Behavioral data obtained demonstrate that the hormonal treatment altered anxiety state in experimental animals. In plusmaze paradigm, it has been demonstrated that progesterone-treated mice revealed the lowest level of open arm activity. In contrast, these mice showed the highest grooming activity as compared to other experimental groups. Immunohistochemical data on progesterone receptor (PR), immunoreactivity in brain have shown that the manipulation with different hormonal treatments modified the number of PR-ir cells in many brain areas. Our data suggest that sex steroid hormones play an important role in induction of anxiety level, as measured by elevated plus-maze, and this action might be partially mediated through the classical steroid receptors.     

The effect of different stages of the sex cycle on rat behavior in a plus maze

Anxiety and motor activity of female white rats in the elevated plus-maze were studied at different stages of the reproduction cycle (estrus, diestrus, pregnancy and lactation). The level of anxiety was lower, and that of locomotor and exploratory activity was higher during estrus and lactation than during diestrus and pregnancy. Exposure to chronic pain of threshold intensity did not induce behavioral changes in pregnant rats. There was no difference between the control and experimental animals in the level of plasma corticosterone.     

Pharmacological Activity of Ruthenium Complexes trans-[RuCl2(L)4] (L = Nicotinic or i-Nicotinic acid) on Anxiety and Memory in Rats

Many biological properties have been attributed to ruthenium complex I (trans-[RuCl₂(nic)₄]) and ruthenium complex II (trans-[RuCl₂ (i-nic) ₄]) including nitric oxide synthase inhibition. In this study, we evaluated pharmacological effects of these complexes on anxiety and memory formation. Memory was evaluated with inhibitory avoidance and habituation to an open-field and anxiety was tested with elevated plus-maze. Adult male Wistar rats (250 to 350 g) received intraperitoneal injections of vehicle, ruthenium complex I (45.2, 90.4, or 180.7 μmol/kg), or ruthenium complex II (0.08, 4.5, or 13.6 μmol/kg) 30 min prior open-field training or elevated plus-maze test and 30 min or 0 h after training. No effects were observed in the anxiety parameters and habituation to an open-field. The ruthenium complexes impaired memory retention compared with vehicle group in the inhibitory avoidance, as when administrated 30 min prior as immediately after training. The memory impairment induced by ruthenium complexes may be due to their nitric oxide synthase inhibition capacity.