Restraint-induced hypoactivity in an elevated plus-maze

Rodents submitted to restraint stress show decreased activity in an elevated plus-maze (EPM) 24 h later. The objective of the present study was to determine if a certain amount of time is needed after stress for the development of these changes. We also wanted to verify if behavioral tolerance of repeated daily restraint would be detectable in this model. Male Wistar rats were restrained for 2 h and tested in the EPM 1, 2, 24 or 48 h later. Another group of animals was immobilized daily for 2 h for 7 days, being tested in the EPM 24 h after the last restraint period. Restraint induced a significant decrease in the percent of entries and time spent in the open arms, as well as a decrease in the number of enclosed arm entries. The significant effect in the number of entries and the percentage of time spent in the open arms disappeared when the data were submitted to analysis of covariance using the number of enclosed arm entries as a covariate. This suggests that the restraint-induced hypoactivity influences the measures of open arm exploration. The modifications of restraint-induced hypoactivity are evident 24 or 48 h, but not 1 or 2 h, after stress. In addition, rats stressed daily for seven days became tolerant to this effect.

Strain-dependent effects of diazepam and the 5-HT2B/2C receptor antagonist SB 206553 in spontaneously hypertensive and Lewis rats tested in the elevated plus-maze

The 5-HT2B/2C receptor antagonist SB 206553 exerts anxiolytic effects in rat models of anxiety. However, these effects have been reported for standard rat strains, thus raising the issue of SB 206553 effects in rat strains displaying different levels of anxiety. Herein, the effects of SB 206553 in a 5-min elevated plus-maze test of anxiety were compared to those of the reference anxiolytic, diazepam, in two rat strains respectively displaying high (Lewis rats) and low (spontaneously hypertensive rats, SHR) anxiety. Diazepam (0.37, 0.75, or 1.5 mg/kg; 30 min before testing) increased in a dose-dependent manner the behavioral measures in SHR, but not in Lewis rats. On the other hand, SB 206553 (1.25, 2.5, or 5 mg/kg; 30 min before testing) failed to alter the anxiety parameters in both strains, whereas it increased closed arm entries in Lewis rats, suggesting that it elicited hyperactivity in the latter strain. Accordingly, the hypolocomotor effect of the nonselective 5-HT2B/2C receptor agonist m-chlorophenylpiperazine (1.5 mg/kg ip 20 min before a 15-min exposure to an activity cage) was prevented by the 1.25 and 2.5 mg/kg doses of SB 206553 in Lewis rats and SHR, respectively. Compared with SHR, Lewis rats may display a lower response to benzodiazepine-mediated effects and a more efficient control of locomotor activity by 5-HT2B/2C receptors.

The interaction of housing condition and acute immobilization stress on the elevated plus-maze behaviors of protein-malnourished rats

Protein malnutrition induces structural, neurochemical and functional alterations in the central nervous system, leading to behavioral alterations. In the present study, we used the elevated plus-maze (EPM) as a measure of anxiety to evaluate the interaction between acute immobilization and housing conditions on the behavior of malnourished rats. Pups (6 males and 2 females) were fed by Wistar lactating dams receiving a 6% (undernourished) or 16% (well-nourished) protein diet. After weaning, the animals continued to receive the same diets ad libitum until 49 days of age when they started to receive a regular lab chow diet. From weaning to the end of the tests on day 70, the animals were housed under two different conditions, i.e., individual or in groups of three. On the 69th day, half of the animals were submitted to immobilization for 2 h, while the other half were undisturbed, and both groups were tested 24 h later for 5 min in the EPM. Independent of other factors, protein malnutrition increased, while immobilization and social isolation per se decreased, EPM exploration. Analysis of the interaction of diet vs immobilization vs housing conditions showed that the increased EPM exploration presented by the malnourished group was reversed by acute immobilization in animals reared in groups but not in animals reared individually. The interaction between immobilization and housing conditions suggests that living for a long time in social isolation is sufficiently stressful to reduce the responses to another anxiogenic procedure (immobilization), while living in groups prompts the animals to react to acute stress. Thus, it is suggested that housing condition can modulate the effects of an anxiogenic procedure on behavioral responses of malnourished rats in the EPM.

Changes in the biogenic amine content of the prefrontal cortex, amygdala, dorsal hippocampus, and nucleus accumbens of rats submitted to single and repeated sessions of the elevated plus-maze test

It has been demonstrated that exposure to a variety of stressful experiences enhances fearful reactions when behavior is tested in current animal models of anxiety. Until now, no study has examined the neurochemical changes during the test and retest sessions of rats submitted to the elevated plus maze (EPM). The present study uses a new approach (HPLC) by looking at the changes in dopamine and serotonin levels in the prefrontal cortex, amygdala, dorsal hippocampus, and nucleus accumbens in animals upon single or double exposure to the EPM (one-trial tolerance). The study involved two experiments: i) saline or midazolam (0.5 mg/kg) before the first trial, and ii) saline or midazolam before the second trial. For the biochemical analysis a control group injected with saline and not tested in the EPM was included. Stressful stimuli in the EPM were able to elicit one-trial tolerance to midazolam on re-exposure (61.01%). Significant decreases in serotonin contents occurred in the prefrontal cortex (38.74%), amygdala (78.96%), dorsal hippocampus (70.33%), and nucleus accumbens (73.58%) of the animals tested in the EPM (P < 0.05 in all cases in relation to controls not exposed to the EPM). A significant decrease in dopamine content was also observed in the amygdala (54.74%, P < 0.05). These changes were maintained across trials. There was no change in the turnover rates of these monoamines. We suggest that exposure to the EPM causes reduced monoaminergic neurotransmission activity in limbic structures, which appears to underlie the "one-trial tolerance" phenomenon.

Dose-response effects of systemic anandamide administration in mice sequentially submitted to the open field and elevated plus-maze tests

The endocannabinoid system is involved in the control of many physiological functions, including the control of emotional states. In rodents, previous exposure to an open field increases the anxiety-like behavior in the elevated plus-maze. Anxiolytic-like effects of pharmacological compounds that increase endocannabinoid levels have been well documented. However, these effects are more evident in animals with high anxiety levels. Several studies have described characteristic inverted U-shaped dose-response effects of drugs that modulate the endocannabinoid levels. However, there are no studies showing the effects of different doses of exogenous anandamide, an endocannabinoid, in animal models of anxiety. Thus, in the present study, we determined the dose-response effects of exogenous anandamide at doses of 0.01, 0.1, and 1.0 mg/kg in C57BL/6 mice (N = 10/group) sequentially submitted to the open field and elevated plus-maze. Anandamide was diluted in 0.9% saline, ethyl alcohol, Emulphor® (18:1:1) and administered ip (0.1 mL/10 g body weight); control animals received the same volume of anandamide vehicle. Anandamide at the dose of 0.1 mg/kg (but not of 0.01 or 1 mg/kg) increased (P < 0.05) the time spent and the distance covered in the central zone of the open field, as well as the exploration of the open arms of the elevated plus-maze. Thus, exogenous anandamide, like pharmacological compounds that increase endocannabinoid levels, promoted a characteristic inverted U-shaped dose-response effect in animal models of anxiety. Furthermore, anandamide (0.1 mg/kg) induced an anxiolytic-like effect in the elevated plus-maze (P < 0.05) after exposing the animals to the open field test.

L-histidine provokes a state-dependent memory retrieval deficit in mice re-exposed to the elevated plus-maze

The effects of L-histidine (LH) on anxiety and memory retrieval were investigated in adult male Swiss Albino mice (weight 30-35 g) using the elevated plus-maze. The test was performed on two consecutive days: trial 1 (T1) and trial 2 (T2). In T1, mice received an intraperitoneal injection of saline (SAL) or LH before the test and were then injected again and retested 24 h later. LH had no effect on anxiety at the dose of 200 mg/kg since there was no difference between the SAL-SAL and LH-LH groups at T1 regarding open-arm entries (OAE) and open-arm time (OAT) (mean ± SEM; OAE: 4.0 ± 0.71, 4.80 ± 1.05; OAT: 40.55 ± 9.90, 51.55 ± 12.10, respectively; P > 0.05, Kruskal-Wallis test), or at the dose of 500 mg/kg (OAE: 5.27 ± 0.73, 4.87 ± 0.66; OAT: 63.93 ± 11.72, 63.58 ± 10.22; P > 0.05, Fisher LSD test). At T2, LH-LH animals did not reduce open-arm activity (OAE and OAT) at the dose of 200 mg/kg (T1: 4.87 ± 0.66, T2: 5.47 ± 1.05; T1: 63.58 ± 10.22; T2: 49.01 ± 8.43 for OAE and OAT, respectively; P > 0.05, Wilcoxon test) or at the dose of 500 mg/kg (T1: 4.80 ± 1.60, T2: 4.70 ± 1.04; T1: 51.55 ± 12.10, T2: 43.88 ± 10.64 for OAE and OAT, respectively; P > 0.05, Fisher LSD test), showing an inability to evoke memory 24 h later. These data suggest that LH does not act on anxiety but does induce a state-dependent memory retrieval deficit in mice.

H1 but not H2 histamine antagonist receptors mediate anxiety-related behaviors and emotional memory deficit in mice subjected to elevated plus-maze testing

This study investigated the role of H1 and H2 receptors in anxiety and the retrieval of emotional memory using a Trial 1/Trial 2 (T1/T2) protocol in an elevated plus-maze (EPM). Tests were performed on 2 consecutive days, designated T1 and T2. Before T1, the mice received intraperitoneal injections of saline (SAL), 20 mg/kg zolantidine (ZOL, an H2receptor antagonist), or 8.0 or 16 mg/kg chlorpheniramine (CPA, an H1 receptor antagonist). After 40 min, they were subjected to the EPM test. In T2 (24 h later), each group was subdivided into two additional groups, and the animals from each group were re-injected with SAL or one of the drugs. In T1, the Student t-test showed no difference between the SAL and ZOL or 8 mg/kg CPA groups with respect to the percentages of open arm entries (%OAE) and open arm time (%OAT). However, administration of CPA at the highest dose of 16 mg/kg decreased %OAE and %OAT, but not locomotor activity, indicating anxiogenic-like behavior. Emotional memory, as revealed by a reduction in open arm exploration between the two trials, was observed in all experimental groups, indicating that ZOL and 8 mg/kg CPA did not affect emotional memory, whereas CPA at the highest dose affected acquisition and consolidation, but not retrieval of memory. Taken together, these results suggest that H1 receptor, but not H2, is implicated in anxiety-like behavior and in emotional memory acquisition and consolidation deficits in mice subjected to EPM testing.

Low-dose thioperamide injected into the cerebellar vermis of mice immediately after exposure to the elevated plus-maze impairs their avoidance behavior on re-exposure to the apparatus

The present study investigated the effect of thioperamide (THIO), an H3 histaminergic receptor antagonist, microinjected into the cerebellar vermis on emotional memory consolidation in male Swiss albino mice re-exposed to the elevated plus-maze (EPM). We implanted a guide cannula into the cerebellar vermis using stereotactic surgery. On the third day after surgery, we performed behavioral tests for two consecutive days. On the first day (exposure), the mice (n=10/group) were exposed to the EPM and received THIO (0.06, 0.3, or 1.5 ng/0.1 µL) immediately after the end of the session. Twenty-four hours later, the mice were re-exposed to the EPM under the same experimental conditions, but without drug injection. A reduction in the exploration of the open arms upon re-exposure to the EPM (percentage of number of entries and time spent in open arms) compared with the initial exposure was used as an indicator of learning and memory. One-way analysis of variance (ANOVA) followed by the Duncan post hoc test was used to analyze the data. Upon re-exposure, exploratory activity in the open arms was reduced in the control group, and with the two highest THIO doses: 0.3 and 1.5 ng/0.1 µL. No reduction was seen with the lowest THIO dose (0.06 ng/0.1 µL), indicating inhibition of the consolidation of emotional memory. None of the doses interfered with the animals' locomotor activity. We conclude that THIO at the lowest dose (0.06 ng/0.1 µL) microinjected into the cerebellum impaired emotional memory consolidation in mice.

GPR30 activation decreases anxiety in the open field test but not in the elevated plus maze test in female mice

The GPR30 is a novel estrogen receptor (ER) that is a candidate membrane ER based on its binding to 17beta estradiol and its rapid signaling properties such as activation of the extracellular-regulated kinase (ERK) pathway. Its distribution in the mouse limbic system predicts a role for this receptor in the estrogenic modulation of anxiety behaviors in the mouse. A previous study showed that chronic administration of a selective agonist to the GPR30 receptor, G-1, in the female rat can improve spatial memory, suggesting that GPR30 plays a role in hippocampal-dependent cognition. In this study, we investigated the effect of a similar chronic administration of G-1 on behaviors that denote anxiety in adult ovariectomized female mice, using the elevated plus maze (EPM) and the open field test as well as the activation of the ERK pathway in the hippocampus. Although estradiol benzoate had no effect on behaviors in the EPM or the open field, G-1 had an anxiolytic effect solely in the open field that was independent of ERK signaling in either the ventral or dorsal hippocampus. Such an anxiolytic effect may underlie the ability of G-1 to increase spatial memory, by acting on the hippocampus.

Longitudinal study of daily variation of rats' behavior in the elevated plus-maze

We conducted a longitudinal study about daily variation of Wistar male rats' behavior in the elevated plus-maze (EPM) evaluated in the 1st, 2nd, 3rd, 6th, 12th, and 18th months of life. Animals were submitted to the plus-maze in 12 sessions at 2-h intervals (n=72, 6 per time point). Spontaneous rest-activity rhythm of four animals was assessed by observation of 24-h videotape records. Time series were analyzed by Cosinor method. Behavioral rates on the six occasions and in light and dark phases were compared by means of two-way ANOVA with repeated measures. Exploratory behavior in EPM was smaller in the light phase and in older animals. Higher values of open and closed arms exploration were observed in the first and third months of the dark phase, and in the first month of the light phase. Adjustment to the 24-h period was significant at all stages for rest-activity data, number of entries in closed arms, and time on center, and for three to five stages for open-arm exploration. In general, 24 h variability was more pronounced in younger animals compared with older ones. The present study showed that: (1) a significant amount of total variability of the behavioral indexes analyzed could be attributed to 24 h variation, (2) light/dark phases differences in EPM exploration were present at all developmental stages, (3) older Wistar rats explored less the EPM and were less active in their home cage compared with younger ones, and (4) behavioral indexes (EPM) decrease was phase related and partially related to a reorganization of rest-activity rhythm. (C) 2003 Elsevier B.V. All rights reserved.

Social separation and diazepam withdrawal increase anxiety in the elevated plus-maze and serotonin turnover in the median raphe and hippocampus

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Anxiogenic-like effects of mCPP microinfusions into the amygdala (but not dorsal or ventral hippocampus) in mice exposed to elevated plus-maze

Serotonin (5-HT) can either increase or decrease anxiety-like behaviour in animals, actions that depend upon neuroanatomical site of action and 5-HT receptor subtype. Although systemic studies with 5-HT(2) receptor agonists and antagonists suggest a facilitatory role for this receptor subtype in anxiety, somewhat inconsistent results have been obtained when such compounds have been directly applied to limbic targets such as the hippocampus and amygdala. The present study investigated the effects of the 5-HT(2B/2C) receptor agonist mCPP bilaterally microinjected into the dorsal hippocampus (DH: 0, 0.3 1.0 or 3.0 nmol/0.2 mu l), the ventral hippocampus (VH: 0, 0.3, 1.0 or 3.0 nmol/0.2 mu l) or the amygdaloid complex (0, 0.15, 0.5, 1.0 or 3.0 nmol/0.1 mu l) in mice exposed to the elevated plus-maze (EPM). Test sessions were videotaped and subsequently scored for conventional indices of anxiety (percentage of open arm entries and percentage of open arm time) and locomotor activity (closed arm entries). Results showed that mCPP microinfusions into the DH or VH failed to affect any behavioural measure in the EPM. However, when injected into the amygdaloid complex, the dose of 1.0 nmol of this 5HT(2B/2C) receptor agonist increased behavioural indices of anxiety without significantly altering general activity levels. This anxiogenic-like effect of mCPP was selectively and completely blocked by local injection of a behaviourally-inactive dose of SDZ SER-082 (10 nmol/0.1 mu l), a preferential 5-HT(2C) receptor antagonist. These data suggest that 5HT(2C) receptors located within the amygdaloid complex (but not the dorsal or ventral hippocampus) play a facilitatory role in plus-maze anxiety in mice. (c) 2007 Elsevier B.V. All rights reserved.

Activation of 5-HT2C receptors in the dorsal periaqueductal gray increases antinociception in mice exposed to the elevated plus-maze

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Fos-like immunoreactivity in central nervous system of mice simultaneously exposed to the elevated plus-maze and nociception

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)