Behavioral effects of "vehicle" microinjected into the dorsal periaqueductal grey of rats tested in the elevated plus maze

To investigate the behavioral effects of different vehicles microinjected into the dorsal periaqueductal grey (DPAG) of male Wistar rats, weighing 200-250 g, tested in the elevated plus maze, animals were implanted with cannulas aimed at this structure. One week after surgery the animals received microinjections into the DPAG of 0.9% (w/v) saline, 10% (v/v) dimethyl sulfoxide (DMSO), 2% (v/v) Tween-80, 10% (v/v) propylene glycol, or synthetic cerebrospinal fluid (CSF). Ten min after the injection (0.5 µl) the animals (N = 8-13/group) were submitted to the elevated plus maze test. DMSO significantly increased the number of entries into both the open and enclosed arms when compared to 0.9% saline (2.7 ± 0.8 and 8.7 ± 1.3 vs 0.8 ± 0.3 and 5.1 ± 0.9, respectively, Duncan test, P<0.05), and tended to increase enclosed arm entries as compared to 2% Tween-80 (8.7 ± 1.3 vs 5.7 ± 0.9, Duncan test, P<0.10). In a second experiment no difference in plus maze exploration was found between 0.9% saline- or sham-injected animals (N = 11-13/group). These results indicate that intra-DPAG injection of some commonly used vehicles such as DMSO, saline or Tween-80 affects the exploratory activity of rats exposed to the elevated plus maze in statistically different manners

Influence of lactation on motor activity and elevated plus maze behavior

Lactating rats show less noise-induced freezing and fewer inhibitory responses on the 6th day post-delivery when submitted to water and food deprivation in a classical conflict paradigm. Lactating mice go more often to the illuminated chamber in a light-dark cage and stay longer in it than virgin females. The present study was designed to assess the influence of this physiological state, i.e. lactation, on the elevated plus maze (EPM) and open-field behavior in adult female rats. Total (TL) and central (CL) locomotion and rearing (RF) frequencies were measured in an open-field. Number of entries into the open and closed arms as well as the time spent in each of these arms were measured in the EPM. Percent time spent and number of entries into the open arms were calculated and compared. In the open-field, TL was significantly decreased (115 ± 10.6 vs 150 ± 11.6) while CL and RF did not differ from those presented by virgin rats. In the EPM, lactating rats displayed a significant reduction in percent time spent (10.9 ± 1.5 vs 17.4 ± 2.3) in the open arms as well as a tendency to a reduction in percent entries into the open arms (35.7 ± 4.7 vs 45.7 ± 4.3). These results show that the physiological state of lactation modulates the open-field and EPM behaviors in rats

Paradoxical increase of exploratory behavior in the elevated plus-maze by rats exposed to two kinds of aversive stimuli

Albino rats were submitted to a 24-h period of social isolation (individual housing) combined with 0, 1, 2 or 3 twenty-four-hour periods of exposure to different vivaria (novelty) and tested in the elevated plus-maze. Results, reported as mean ± SEM for N = 12, show that the time (in seconds) spent in the open arms by rats exposed to novelty for 0, 1, 2 and 3 days was 28.3 ± 4.4, 31.6 ± 3.2, 29.1 ± 3.5 and 25.0 ± 3.3, respectively, when grouped in the same vivarium; 29.6 ± 2.7, 7.6 ± 2.1, 9.6 ± 4.4 and 28.5 ± 3.7 when grouped in different vivaria; 2.9 ± 1.1, 1.8 ± 1.0, 2.7 ± 1.1 and 0 ± 0 when isolated in the same vivarium, and 2.6 ± 1.1, 31.5 ± 8.2, 24.8 ± 4.2 and 0 ± 0 when isolated in different vivaria. The number of entries into the open and closed arms followed a similar trend. This indicates that, separately, both exposure to novelty and isolation are aversive manipulations. Paradoxically, when novelty was combined with a concomitant 24-h period of social isolation prior to testing, the decrease in exploratory behavior caused by either of the two aversive manipulations alone was reverted. These results are indicative that less intense anxiety triggers mechanisms mediating less energetic behavior such as freezing, while higher levels trigger mechanisms mediating more vigorous action, such as flight/fight behavior, since the combination of two aversive situations resulted in more exploratory behavior than with either alone. They are also suggestive of habituation to the effects of novelty, since exposure to it for 3 days produced exploratory behavior similar to that of controls

Memory-impairing effects of local anesthetics in an elevated plus-maze test in mice

Post-training intracerebroventricular administration of procaine (20 µg/µl) and dimethocaine (10 or 20 µg/µl), local anesthetics of the ester class, prolonged the latency (s) in the retention test of male and female 3-month-old Swiss albino mice (25-35 g body weight; N = 140) in the elevated plus-maze (mean ± SEM for 10 male mice: control = 41.2 ± 8.1; procaine = 78.5 ± 10.3; 10 µg/µl dimethocaine = 58.7 ± 12.3; 20 µg/µl dimethocaine = 109.6 ± 5.73; for 10 female mice: control = 34.8 ± 5.8; procaine = 55.3 ± 13.4; 10 µg/µl dimethocaine = 59.9 ± 12.3 and 20 µg/µl dimethocaine = 61.3 ± 11.1). However, lidocaine (10 or 20 µg/µl), an amide class type of local anesthetic, failed to influence this parameter. Local anesthetics at the dose range used did not affect the motor coordination of mice exposed to the rota-rod test. These results suggest that procaine and dimethocaine impair some memory process(es) in the plus-maze test. These findings are interpreted in terms of non-anesthetic mechanisms of action of these drugs on memory impairment and also confirm the validity of the elevated plus-maze for the evaluation of drugs affecting learning and memory in mice

Anxiogenic-like effect of acute and chronic fluoxetine on rats tested on the elevated plus-maze

The selective serotonin reuptake inhibitor fluoxetine (FLX) is widely prescribed for depression and anxiety-related disorders. On the other hand, enhanced serotonergic transmission is known to be classically related to anxiety. In this study, the effects of acute (5.0 mg/kg) and chronic (5.0 mg/kg, 22 days) FLX were investigated in both food-deprived and non-deprived rats tested in the elevated plus-maze. Significant main effects of the three factors (drug, food condition and administration regimen) were observed, but no interaction between them. The administration of either acute or chronic FLX resulted in an anxiogenic effect, as detected by a significant reduction in the percentage of time spent in the open arms and in the percentage of open arm entries. Food deprivation yielded an anxiolytic-like profile, probably related to changes in locomotor activity. The administration regimen resulted in an anxiolytic profile in chronically treated rats, as would be expected after 22 days of regular handling. The anxiogenic action of acute FLX is consistent with both its neurochemical and clinical profile. The discrepancy between the anxiogenic profile of chronic FLX and its therapeutic uses is discussed in terms of possible differences between the type of anxiety that is measured in the plus-maze and the types of human anxiety that are alleviated by fluoxetine.

The relationship between anxiety and depression in animal models: a study using the forced swimming test and elevated plus-maze

The present study evaluated the correlation between the behavior of mice in the forced swimming test (FST) and in the elevated plus-maze (PM). The effect of the order of the experiments, i.e., the influence of the first test (FST or PM) on mouse behavior in the second test (PM or FST, respectively) was compared to handled animals (HAND). The execution of FST one week before the plus-maze (FST-PM, N = 10), in comparison to mice that were only handled (HAND-PM, N = 10) in week 1, decreased % open entries (HAND-PM: 33.6 ± 2.9; FST-PM: 20.0 ± 3.9; mean ± SEM; P<0.02) and % open time (HAND-PM: 18.9 ± 3.3; FST-PM: 9.0 ± 1.9; P<0.03), suggesting an anxiogenic effect. No significant effect was seen in the number of closed arm entries (FST-PM: 9.5 (7.0-11.0); HAND-PM: 10.0 (4.0-14.5), median (interquartile range); U = 46.5; P>0.10). A prior test in the plus-maze (PM-FST) did not change % immobility time in the FST when compared to the HAND-FST group (HAND-FST: 57.7 ± 3.9; PM-FST: 65.7 ± 3.2; mean ± SEM; P>0.10). Since these data suggest that there is an order effect, the correlation was evaluated separately with each test sequence: FST-PM (N = 20) and PM-FST (N = 18). There was no significant correlation between % immobility time in the FST and plus-maze indexes (% time and entries in open arms) in any test sequence (r: -0.07 to 0.18). These data suggest that mouse behavior in the elevated plus-maze is not related to behavior in the forced swimming test and that a forced swimming test before the plus-maze has an anxiogenic effect even after a one-week interval.

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.

Effects of emotional reactivity on inhibitory avoidance in the elevated T-maze

The possibility of the presence of inter-individual emotional differences and the memory performance of rats was examined in the elevated T-maze. Two kinds of aversively motivated behaviors, inhibitory avoidance and escape learning, were measured. Based on the number of trials to achieve a learning criterion, rats were divided into two subgroups with either low or high avoidance reactivity (LAR or HAR, respectively). Retention test avoidance latencies showed that HAR animals had better avoidance memory (Mann-Whitney rank sum test, P = 0.0035). No such differences were found for the escape component of this test. These data suggest that individual emotional differences affect inhibitory avoidance performance, which may help to explain the dispersion of the data observed in other studies using this paradigm.

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.

The inactivation of the basolateral nucleus of the rat amygdala has an anxiolytic effect in the elevated T-maze and light/dark transition tests

Pharmacological evidence indicates that the basolateral nucleus of the amygdala (BLA) is involved in the mediation of inhibitory avoidance but not of escape behavior in the elevated T-maze test. These defensive responses have been associated with generalized anxiety disorder (GAD) and panic disorder, respectively. In the present study, we determined whether the BLA plays a differential role in the control of inhibitory avoidance and escape responses in the elevated T-maze. Male Wistar rats (250-280 g, N = 9-10 in each treatment group) were pre-exposed to one of the open arms of the maze for 30 min and 24 h later tested in the model after inactivation of the BLA by a local injection of the GABA A receptor agonist muscimol (8 nmol in 0.2 µL). It has been shown that a prior forced exposure to one of the open arms of the maze, by shortening latencies to withdrawal from the open arm during the test, improves the escape task as a behavioral index of panic. The effects of muscimol in the elevated T-maze were compared to those caused by this GABA agonist in the avoidance reaction generated in the light/dark transition test. This defensive behavior has also been associated with GAD. In the elevated T-maze, intra-BLA injection of muscimol impaired inhibitory avoidance (control: 187.70 ± 14.90 s, muscimol: 37.10 ± 2.63 s), indicating an anxiolytic effect, without interfering with escape performance. The drug also showed an anxiolytic effect in the light/dark transition test as indicated by the increase in the time spent in the lighted compartment (control: 23.50 ± 2.45 s, muscimol: 47.30 ± 4.48 s). The present findings point to involvement of the BLA in the modulation of defensive responses that have been associated with GAD.

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.

Anxiolytic effects of repeated treatment with an essential oil from Lippia alba and (R)-(-)-carvone in the elevated T-maze

Lippia alba (Mill.) N.E. Brown (Verbenaceae) is widely used in different regions of Central and South America as a tranquilizer. The plant’s anxiolytic properties, however, merit investigation. The present study evaluated the effects of repeated daily (14 days) intraperitoneal (ip) treatment with an essential oil (EO) from a chemotype of L. alba (LA, chemotype II, 12.5 and 25 mg/kg; N = 6-8) and (R)-(-)-carvone (25 mg/kg; N = 8-12), the main constituent of this chemotype, on male Wistar rats (weighing 250 g at the beginning of the experiments) submitted to the elevated T-maze (ETM). The ETM allows the measurement of two defensive responses: inhibitory avoidance and one-way escape. In terms of psychopathology, these responses have been related to generalized anxiety and panic disorder, respectively. Treatment with the EO impaired ETM avoidance latencies, without altering escape, in a way similar to the reference drug diazepam (P < 0.05) (avoidance 2: control = 84.6 ± 35.2; EO 12.5 mg/kg = 11.8 ± 3.8; EO 25 mg/kg = 14.6 ± 2.7; diazepam = 7 ± 2.1). (R)-(-)-carvone also significantly altered this same response (P < 0.05; avoidance 1: control = 91.9 ± 31.5; carvone = 11.6 ± 1.8; diazepam = 8.1 ± 3.3). These results were not due to motor changes since no significant effects were detected in an open field. These observations suggest that LA exerts anxiolytic-like effects on a specific subset of defensive behaviors that have been implicated in generalized anxiety disorder, and suggest that carvone is one of the constituents of LA responsible for its action as a tranquilizer.