Five sessions of repeated EPM as a paradigm to cause prolonged anxiety
Using the EPM test we explore the possibility that mice (n = 22) tested every 30 min for five times in the EPM can undergo a form of prolonged anxiety, which is maintained either 24 h or 21 days later (Fig. 1 A, B1). One-way repeated measures analysis of variance (One Way RM ANOVA) in mice submitted to this paradigm, shows significant changes in the percentage of time spent in open arms when we compare the first two sessions of EPM with the others (Fig. 1 B1) (p < 0.001 vs. 1° session and p < 0.05 vs. 2° session). The reduction of the percentage of time spent in open arms is maintained either in 24 h (n = 22) or 21 days sessions (n = 10). Furthermore, also the second session of EPM displays a substantial decrement in the percentage of time spent in open arms if correlated with the first one (p < 0.05; 1° vs. 2° session, F (6, 114) = 31.82).
Exploration, locomotor activities and anxiety, represented as number of total and open arms entries, show a strong reduction in the five daily sessions. The 24 h later session displays diminished values of total and open entries, which are comparable with those in the fifth trial. After 3 weeks these parameters are still significantly decreased but proportional to the second session of EPM. From these data we can assert that the level of anxiety induced in mice with our paradigm, increases over each session, reaching a significant value already in the second one, and a partial recovery is present only in the last trial, when mice rediscover a greater locomotor activity and propensity for exploration (Fig. 1 B2) (p < 0.001 vs. 1°, 2°, 7° session; p < 0.05 vs. 3° session; F (6, 114) = 143.12, total entries; p < 0.001 vs. 1° session; p < 0.05 vs. 2° session; F (6, 114) = 42.13, open entries). The continuous avoidance of the open arms, along all sessions of the paradigm, clarifies the impossibility of mice to habituate themselves to repeated exposures.
A different group of ten mice were tested in the EPM for only one session and retested after 24 h. When we compare the two trials, a significant change of percentage of time spent in open arms is present within this group (Fig. 1 C1) (p < 0.05 vs. 1° session; F (1, 9) = 13.31). The session after 24 h, when compared with the same one of mice that have undergone five repeated EPMs, displays an indicative difference (control vs. 1 EPM = p < 0.05 in 6° session; F (1, 60) = 42.12). These results show that a single trial on the EPM is sufficient to affect the level of anxiety of a second trial 24 h later, and highlights, above all, that mice submitted to five repeated sessions of EPM, in a single day, have a drastic reduction of percentage of time spent in open arms after 24 h that is a greater compared to mice having a single experience in the EPM.
A single experience in EPM was not able to change completely the locomotor activity, in fact the entries in the total arms between the first and the session 24 later are comparable (Fig. 1 C2) (p > 0.05 vs. 1° session; F (1, 9) = 4.80, total entries). A single exposition to EPM, instead, is sufficient to reduce the number of entries in the open arms after 24 h (p < 0.05 vs. 1° session; F (1, 9) = 22.22, open entries). Both the number of entries in the total and open arms, compared with the parameters for the same session of mice tested five times, are significantly greater (control vs. 1 EPM = p < 0.001 in 6° session; F (1, 60) = 102.15, total entries; p < 0.05 in 6° session; F (1, 60) = 53.27, open entries). The results emphasize how repeated experiences in the EPM can strongly reduce the exploration of the arms and increase the anxiety-like behavior, while mice which have undergone a single session maintain a great propensity for exploration and a lower level of anxiety.
We performed the open field test on naïve mice and those which have previously undergone a five -time repeated EPM, to investigate more in-depth anxiety-related behaviors and to assess novel environment exploration or the general locomotor activity [18]. Software related to the open field test reported, for each mouse tested one time for 30 min, both the travel distance of the central and total zone and the spontaneous activities.
Comparing the travel distance, a measure of exploratory behavior, of the two groups during the first few minutes in the central zone, we were able to assess the exploration of a novel environment and to analyze the anxiety-like behavior [12]. Dividing the duration of the test in time bins of 5 min (Fig. 2 A1), we found that mice which had undergone repeated EPM travelled less distance compared to naïve ones (repeated EPM mice: 463.05 ± 90.08 cm vs. naïve: 675.28 ± 80.69 cm; p < 0.05; F (5, 180) = 5.34; 0-5 min) juxtaposed also by less time spent (Fig. 2 A5), in the central area during the first 5 min (repeated EPM mice: 5.66 ± 0.86 s vs. naïve: 9.00 ± 1.15 s; p < 0.05; F (5, 180) = 10.71; 0-5 min).
Extending the statistical comparison of the travel distance to all the 5-min intervals of the central or total zone, we examined the habituation to an increasingly familiar environment which shows a strong reduction along the duration of the test in the group of mice which previously endured up to five times EPM (Fig. 2 A1, B1). The decreased exploration and locomotor activity are verifiable from the paths in the maps, which are less dense (Fig. 2 E1, E2). We also scored spontaneous activities which provide measures of the level of interest in the novelty of the environment, and of general physical motor abilities. These different parameters include vertical (rearing), ambulatory (horizontal fast activity), stereotypic (when the animal is moving with the presence of repetitive, invariant behaviors such as grooming, rearing and head bobbing thus to break the same beam or set of beams), jumping (escape latency) counts and entries. The significant difference of behavioral changes obtained during the open field test consistently confirms that mice that have previously undergone repeated EPM manifest an increased anxiety which is heavily increased mostly in the last 15 min of the test, when the environment should become more familiar.
NMDA receptors in the paradigm of prolonged behavioral anxiety
In order to exclude an involvement of learning and memory, a process NMDA receptor-mediated [19] and to confirm the anxiolytic effect of the antagonist MK-801 [20], we have administered, to a group of 16 mice, a dose of 0.25 mg/kg, i.p., 30 min before the beginning of the repeated EPM test (Fig. 3 A1). Two way ANOVA + Tukey post hoc evidences significant differences in the time spent in the open arms of the first and the second session of mice treated with a single dose of MK-801 i.p. compared with the control group (control vs. +MK-801 = p < 0.001 vs. 1° and 2° session; F (5, 216) = 29.48). Significant changes are present in the percentage of time spent in open arms of the first and second sessions when compared to the others of the same group (p > 0.05, 1° vs. 2° session; p < 0.001, vs. 1° and 2° session; p < 0.05, 2° vs. 3° session; F (5, 75) = 18.58). The analysis of the number of entries in the total arms (Fig. 3 A2) clearly shows that MK-801, at this dosage, influences the locomotor activity inducing hyperactivity. From the plot, MK-801, in fact, seems also to cause an amnesic effect, affecting the spatial orientation and increasing significantly, when compared with the control group, the exploration of the arms in the EPM in all daily sessions and 24 h later (control vs. +MK-801 = p > 0.05, in 1° session; p < 0.001 in 2°, 3°, 4°, 6° session; p < 0.05 in 5° session; F (5, 174) = 29.49, total entries). MK-801 reduces its effect, but is still significant if compared with control mice, only in the fifth session of the total entries (control vs. +MK-801 = p < 0.05, 1° and 2° vs. 5° session; F (5, 40) = 4.81, total entries). Mice treated with MK-801 display a significant decrement in the number of entries in the open arms after the second session (p > 0.05, 1° vs. 2° session; p < 0.001, vs. 1° and 2° session; p < 0.05, 2° vs. 3° session; F (5, 75) = 20.57, open entries). Comparing the same parameter between MK-801-treated and control mice, the drug shows no more significant outcomes from the session subsequent to the third (control vs. +MK-801 = p < 0.001 in 1°, 2°, 3° session; F (5, 216) = 33.69, open entries).
During the behavioral experiments, due to their complex pharmacological profile, the NMDA receptor antagonists could produce several adverse motor effects: increased locomotor activity, turning behavior, head weaving, body rolling, and stereotyped motor patterns [21]. To avoid these outcomes, we have evaluated the importance of NR2B, which has been proved to be involved in anxiety, through the Tyr-1472 phosphorylation [22], in fear memory [23] but does not affect the spatial memory in the Morris water maze test [24]. We have injected nine mice with a dosage of ifenprodil of 10 mg/kg i.p. 30 min before the first session (Fig. 3 B1). At this concentration the NR2B antagonist does not induce any change in the time spent in the open arms, which seems to be similar to the control group (control vs. + ifenprodil 10 mg/kg = p > 0.05 in all sessions; F (5, 174) = 25.56), while the internal statistical analysis of the group presents important differences between the first and the second sessions when compared with the others (p < 0.001, 1° vs. 5°, 6° session; p < 0.05, 1° vs. 3°, 4° session; p < 0.05, 2° vs. 5° session; F (5, 40) = 7.94).
From the plot (Fig. 3 B2) we can appreciate how the ifenprodil, correlated with the control group, negatively affects the locomotor activity in the first session, while in the 24 h later session, it increases the total entries (control vs. + ifenprodil 10 mg/kg = p < 0.001, in 1° session; p > 0.05 in 2°, 3°, 4°, 5° session; p < 0.05 in 6° session; F (5, 174) = 104.02, total entries). The entries in the open arms, on the other hand, maintain values comparable to control group (control vs. + ifenprodil 10 mg/kg = p > 0.05 in all sessions; F (5, 174) = 45.88, open entries). The statistic inside the group of mice treated with ifenprodil displays a reduction, either in total or open arms entries, from the first to the fifth session (p < 0.001, all vs. 1° session; p < 0.001, 2° vs. 5°, 4° session; p < 0.05, 2° vs. 3° session; p < 0.05, 6° vs. 5° session; p < 0.05, 3° vs. 5° session; F (5, 40) = 35.61, total entries; p < 0.001, 1° vs. 5°, 4°, 3°, 6° session; p < 0.05, 1° vs. 2° session; p < 0.05, 2° vs. 5°, 4° session; F (5, 40) = 18.25, open entries).
A second group of ten mice were treated with a higher dosage of ifenprodil 20 mg/kg i.p. 30 min before the test (Fig. 3 C1). Time in the open arms is significantly increased in the second session (control vs. + ifenprodil 20 mg/kg = p < 0.05, in 2° session; p > 0.05 in 1°, 3°, 4°, 5°, 6° session; F (5, 210) = 44.00), after which the percentage of time spent in open arms become, for each session, comparable to the control group. The comparison of each session shows that the last four sessions are heavily diminished (p < 0.001, 1° vs. 5°, 4°, 3°, 6° session; p < 0.001, 2° vs. 5°, 4° session; p < 0.05, 2° vs. 3°, 6° session; F (5, 70) = 15.55). At this dosage ifenprodil (Fig. 3 C2) has no effect in the number of total entries along the five consecutive sessions, but greatly increases the same dimension in the 24 h later trial (control vs. + ifenprodil 20 mg/kg = p < 0.001 in 6° session; p > 0.05 in 1°, 2°, 3°, 4°, 5° session; F (5, 210) = 167.30, total entries). The treatment seems to ameliorate the anxiety increasing the entries in the open arms of the first trial, but without affecting remaining ones (control vs. + ifenprodil 20 mg/kg = p < 0.001 in 1° session; p > 0.05 in 2°, 3°, 4°, 5°, 6° session; F (5, 210) = 78.01, open entries). The internal comparison of the total entries inside the groups manifests a clear decrease along the sessions (blue bars) with a slight increase on the 24 h later trial, which is comparable to the third one (p < 0.001, 1° vs. all sessions; p < 0.001, 2° vs. 3°, 4°, 5° session; p < 0.05, 2° vs. 6° session; p < 0.001, 6° vs. 5°, 4° session; p < 0.05, 3° vs. 5° session, F (5, 70) = 86.61, total entries). The entries in the open arms present an indicative differences when correlated with the first and the second session (p < 0.001, 1° vs. all sessions; p < 0.001, 2° vs. 5° session; p < 0.05, 2° vs. 3°, 4° session; F (5, 70) = 33.83, open entries).
L-type voltage-gated calcium channels (L-VGCCs) in the repeated EPM paradigm
To speculate the influence of calcium homeostasis in anxiety-related behavior, we have injected an L-type VGCCs blocker, a class of receptors which are also recognized to contribute to LTP in several areas of the brain [25, 26], and which plays an important role in cued fear conditioning [27] with no effect on the acquisition of spatial, working and reference memory [28, 29].
An intraperitoneal administration of nimodipine (10 mg/kg) in a group of eight mice 30 min before the first session of repeated EPM is unable to reduce the anxiety behavior (Fig. 3 D1). Almost all sessions are comparable to the control mice’s one, but conversely to our expectations, in the second and third one, nimodipine seems to increase the anxious responses (control vs. + nimodipine = p < 0.001 in 2° session; p < 0.05 in 3° session; p > 0.05 in 1°, 4°, 5°, 6° session; F (5, 168) = 27.58). The internal statistic of the group treated with nimodipine shows a remarkable difference only between the first and the other sessions (p < 0.001, 1° vs. all sessions; F (5, 35) = 9.72). From Fig. 3 D2 it is possible to appreciate how, correlated to the control group, the nimodopine reduces the locomotor activity, the exploration and increase the anxiety-related behavior until the third trial, after which it returns to levels comparable to the control mice (control vs. + nimodipine = p < 0.001 in 1°, 2° session; p < 0.05 in 3° session; F (5, 168) = 61.21, total entries; p < 0.001 in 1°, 2° session; p < 0.05 in 3° session; F (5, 168) = 32.30, open entries). The statistical analysis of the mice treated with nimodipine has shown an indicative decrement of the values after the first session of both entries in the total or open arms (p < 0.001, 1° vs. all sessions; F (5, 35) = 12.03, total entries; p < 0.001, 1° vs. all sessions; F (5, 35) = 8.93, open entries).
Effects of opioid antagonist naloxone
It is known that opioid receptors are linked with anxiety by the use of genetic knockout mice [30, 31]. Activation of opioid receptors are related to ACs [32]. We have treated a group of 11 mice (Fig. 3 E1) with naloxone (5 mg/kg, i.p.), an opioid antagonist (μ,δ,k). Statistical analysis reveals a significant effect in the time spent in the open arms, compared to the control group, only in the first session with no effects on the others (control vs. + naloxone = p < 0.001 in 1° session; p > 0.05 in 2°, 3°, 4°, 5°, 6° session; F (5, 181) = 39.29). Statistical analysis of the group indicates that there is a strong decrease of the effect after the first trial (p < 0.001, 1° vs. all sessions; F (5, 45) = 18.14).
Total entries (Fig. 3 E2) do not point out any differences in the locomotor activity of the control group (control vs. + naloxone = p > 0.05, in all sessions; F (5, 181) = 123.67). A statistical reduction is evident in all the sessions when compared with the first and the second one (p < 0.001, 1° vs. all sessions; p < 0.001, 2° vs. 5°, 4° session; p < 0.05, 2° vs. 3° session; F (5, 45) = 53.31, total entries). The reduction of anxiety (Fig. 3 E2) in the first session is confirmed by an increase, compared to the control group, in the number of entries in the open arms (control vs. + naloxone = p < 0.001 in 1° session, p > 0.05 in 2°, 3°, 4°, 5°, 6° session; F (5, 181) = 82.81). The statistic inside the group notifies a decrease of number of entries if compared with the first and the second trials (p < 0.001, 1° vs. all sessions; p < 0.05, 2° vs. 5°, 4° session; F (5, 45) = 46.14, open entries).
Role of AC1 and AC8 in repeated EPM
While AC1 plays important role in pain-related LTP in the ACC in mice [33] confirmed by the analgesic effects of NB001 [12], an AC1inhibitor, and seems to reduce anxiety induced by irritable bowel syndrome (IBS) [3], studies in AC8 KO mice and single-nucleotide polymorphisms (SNPs) located in the AC8 (ADCY8) gene in humans, restrict its involvement to mental disorders as depression and anxiety [16, 34]. Using 29 AC1 KO mice (Fig. 4 A1), we investigate the role of AC1 in this anxiety test. Mice undergoing repeated EPM exhibit, if compared to control group, an increment in the percentage of time spent in open arms only the first session, with no relevant differences in the other trials (control vs. AC1KO = p < 0.05 in 1° session; p > 0.05 in 2°, 3°, 4°, 5°, 6° session; F (5, 290) = 26.92). The first two sessions of the AC1 KO group present important changes compared with the others (p < 0.001, 1° vs. all sessions; p < 0.05, 2° vs. 5°, 6° session; F (5, 136) = 33.38).
Total entries in the arms and in the open arms display no differences when compared to the control group (Fig. 4 A2), with values going down until the fifth one (control vs. AC1KO = p > 0.05 in all sessions; F (5, 290) = 112.37, total entries; p > 0.05 in all sessions; F (5, 290) = 59.16, open entries). In the 24 h later session both the parameters are comparable with those in the fourth one (p < 0.001, 1° vs. all sessions; p < 0.001, 2° vs. all sessions; p < 0.001, 3° vs. 5° session; p < 0.05, 6° vs. 5° session; F (5, 136) = 96.02, total entries; p < 0.001, 1° vs. all sessions; p < 0.001, 2° vs. 5° session; p < 0.05, 2° vs. 6°, 4° session; F (5, 136) = 47.15, open entries).
Performing the repeated EPM test on a group of twenty-eight AC8 KO mice (Fig. 4 B1), we examine the role of AC8. Surprisingly the percentage of time spent in open arms is maintained constant along all the sessions until 24 h later, when correlated with control group (control vs. AC8KO = p < 0.001 in 3°, 4°, 5° session; p < 0.05 in 1°, 2°, 6° session; F (5, 288) = 4.14). The statistic inside the group of the time in the open arms does not show any change along the entire test (p > 0.05 in all sessions; F (5, 135) = 2.15). After the first session, either the total entries or the entries in the open arms (Fig. 4 B2) show values remarkably higher than the control ones (control vs. AC8KO = p > 0.05 in 1° session; p < 0.05 in 2° session; p < 0.001 in 3°, 4°, 5°, 6° session; F (5, 246) = 92.01, total entries; p > 0.05 in 1° session; p < 0.05 in 2°, 5° session; p < 0.001 in 3°, 4°, 6° session; F (5, 288) = 16.00, open entries). The statistical analysis inside the group highlights that the number of total entries appears to decrease after the first two sessions (p < 0.001, 1° vs. all sessions; p < 0.001, 2° vs. 5° session; p < 0.05, 2° vs. 4°, 6°, 3° session; F (5, 100) = 32.24, total entries) while, for the number of entries in the open arms, after the first one (p < 0.001, 1° vs. 5° session; p < 0.05, 1° vs. 4°, 6°, 3° session; F (5, 135) = 6.87, open entries).