Ethopharmacological analysis of the open elevated plus-maze in mice

Behavioural Brain Research 246 (2013) 76–85

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Ethopharmacological analysis of the open elevated plus-maze in mice Tatiani Sorregotti a,b , Joyce Mendes-Gomes a , Javier Leonardo Rico a,c , Robert John Rodgers d , Ricardo Luiz Nunes-de-Souza a,b,e,∗ a

Laboratory of Pharmacology, School of Pharmaceutical Sciences, University of Estadual Paulista, UNESP, Araraquara, SP 14801-902, Brazil Joint Graduate Program in Physiological Sciences, UFSCar/UNESP, Rod. Washington Luís, Km 235, São Carlos, SP 13565-905, Brazil Laboratory of Animal Behavior, Fundación Universitaria Konrad Lorenz, Bogotá, Colombia d Behavioural Neuroscience Laboratory, Institute of Psychological Sciences, University of Leeds, Leeds, England, United Kingdom e Institute for Neuroscience and Behavior-IneC, USP, Ribeirão Preto, SP 14040-901, Brazil b c

h i g h l i g h t s     

Behavioural profile of mice exposed to the open elevated plus-maze (oEPM). Analysis revealed depth exploration, cautious exploration of arms and risk assessment. Acute fluoxetine or imipramine had no effects on defensive behaviour. Alprazolam, diazepam and chronic fluoxetine attenuated defensiveness. oEPM induces defensive behaviours that are sensitive to panicolytic-like drugs.

a r t i c l e

i n f o

Article history: Received 18 December 2012 Received in revised form 20 February 2013 Accepted 24 February 2013 Available online 5 March 2013 Keywords: Open elevated plus-maze Factor analysis Defensive behaviour Anxiolytics and anxiogenics Antidepressants Mice

a b s t r a c t Exposure of rodents to an open elevated plus-maze (oEPM) elicits antinociception and increases plasma corticosterone levels. However, no studies have yet assessed the defensive behaviour repertoire of animals in this modified test. In Experiment 1, factor analysis was employed to characterise the behavioural profile of mice exposed to the oEPM. Experiments 2 and 3 assessed the effects of acute alprazolam (0.5–1.5 mg/kg; diazepam 0.5–1.5 mg/kg), pentylenetetrazole (10.0–30.0 mg/kg), yohimbine (2.0–6.0 mg/kg), mCPP (0.3–3.0 mg/kg), and acute and chronic fluoxetine (10.0–30.0 mg/kg) and imipramine (1.0–15.0 mg/kg) on behaviours identified in Experiment 1. The factor analyses revealed that behaviour in the oEPM can largely (77% total variance) be accounted for in terms of 3 factors: factor 1 (‘depth exploration’; e.g. head-dipping on the arms), factor 2 (‘cautious exploration of arms’; e.g. flatback approach), and factor 3 (‘risk assessment’; stretched attend postures – SAP). Experiments 2 and 3 showed that, over the dose range used, alprazolam selectively attenuated all measures of defensiveness. Similar, though more modest, effects were seen with diazepam. Confirming the intensity of the emotional response to the oEPM (nociceptive, endocrine and behavioural), relatively few significant behavioural changes were seen in response to the anxiogenic compounds tested. Although acute fluoxetine or imipramine treatment failed to modify behaviour in the oEPM, chronic fluoxetine (but not chronic imipramine) attenuated total flat back approach and increased head dipping outside the central square. Together, the results indicate that the oEPM induces behavioural defensive responses that are sensitive to alprazolam and chronic fluoxetine. © 2013 Elsevier B.V. All rights reserved.

1. Introduction Animal tests for anxiety are used as tools to study the neural bases of emotion and to identify potential anxiolytic drugs [1]. In

∗ Corresponding author at: Laboratory of Pharmacology, School of Pharmaceutical Sciences, University Estadual Paulista, UNESP, Araraquara, SP 14801-902, Brazil. Tel.: +55 16 3301 6983; fax: +55 16 3301 6980. E-mail address: [email protected] (R.L. Nunes-de-Souza). 0166-4328/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.bbr.2013.02.035

this context, popular tests of anxiety-like behaviour in rodents, such as the plus-maze [2], zero-maze [3] and T-maze [4], are based on spatial preference i.e. preference for walled areas over open areas [e.g. 5]. The elevated plus maze (EPM) or elevated X-maze, a test based on the natural aversion of rodents to open spaces [2,5–8], has been widely used for almost three decades. The elevated apparatus is in the shape of a plus sign, with two open and two enclosed arms, each with an open roof [2,7]. Anxiety-like behaviour is traditionally evaluated by the percentage of entries and time spent on the open arms [9], while the absolute number of closed arm entries

T. Sorregotti et al. / Behavioural Brain Research 246 (2013) 76–85

is considered a measure of motor activity [10]. The EPM was originally developed by Handley and Mithani (1984) [2] and then more thoroughly validated for rats and mice by Pellow et al. (1985) [7] and Lister (1987) [6], respectively. Since its inception, the standard elevated plus-maze test (sEPM) in particular has been the most widely-used preclinical paradigm both for studying mechanisms of anxiety and screening for novel anxiolytic agents [reviews: 1, 11]. However, it is clear [e.g. 12] that the sEPM is relatively insensitive to certain classes of anxiolytic (e.g. buspirone, SSRIs), resulting in attempts to improve the sensitivity spectrum through the use of prior stress protocols (e.g. electric shock, social isolation, social defeat) [13–16]. Another way to approach this problem is to exploit the fact that the open arms represent the principal source of aversive stimulation in the sEPM [5,7,17,18]. Indeed, in the escape component of the elevated T-maze protocol, animals placed directly on an open arm learn very quickly to flee to an area of relative safety (i.e. a closed arm), a behaviour pattern that is particularly sensitive to panicolytic as opposed to anxiolytic agents [e.g. 19, 20]. The test is not only used to evaluate the potential of anxiolytic and anxiogenic drugs [2,7,11] but also permits research on the influence of aversion on nociception [21–23]. In one of the earliest studies of this type, Rodgers and Lee (1990) [24] demonstrated that exposure of mice to the EPM not only elicits behavioural defensive responses but also induces antinociception assessed with the tail-flick test. For a number of years, our laboratory has been studying stress-induced antinociception in mice using a modified version of the sEPM comprising four open arms, the so-called open or oEPM [e.g. 18, 25, 26, 27, 28, 29, 30]. Animals exposed to this apparatus show a very much more intense and longer-lasting antinociceptive response than that seen in response to the sEPM, a reaction that is accompanied by high plasma corticosterone levels [27], thereby confirming the aversive nature of this modified test environment. Moreover, oEPM-antinociception is prevented with systemic injection of low doses of the serotonin 5-HT1A receptor agonist, 8-OH-DPAT [17], as well as to intra-amygdala injection of midazolam [17,18]. Interestingly, infusions of midazolam into this forebrain limbic structure also attenuate the behavioural indices of anxiety in mice exposed to the EPM [17], suggesting that amygdaloid benzodiazepine/GABAA receptors play a role in both oEPM-induced antinociception and anxiety. Although the sEPM has been extensively validated as an anxiety test for rats [2,7] and mice [6,8], the oEPM has thus far only been used to investigate the mechanisms underlying anxiety/fear/stressinduced antinociception. As such, the full repertoire of defensive responses displayed in the oEPM remains to be characterised, both behaviourally and pharmacologically. In the current study, we employed factor analysis in order to profile the behaviour of mice in the oEPM. Given that strongly correlated measures should respond similarly to pharmacological interventions, two further experiments examined the behavioural effects of acute treatment with diazepam, alprazolam, pentylenetetrazole, yohimbine and mCPP (1-(3-chlorophenyl) piperazine), as well as the effects of acute and chronic treatment with fluoxetine and imipramine.

2. Materials and methods 2.1. Subjects Subjects were adult male Swiss mice (São Paulo State University, UNESP, SP, Brazil) weighing 25–35 g at testing. They were housed in groups of 5 per cage (33 × 15 × 13 cm) and maintained under a normal 12 h light cycle (lights on at 7 a.m.) in a temperature (23 ± 1 ◦ C) – controlled environment. Food and water were freely available except during the brief test periods. All mice were experimentally naive, and experimental sessions were carried out during the light phase of the cycle (09.00–15.00 h). All experimental protocols carried out were approved by the Sao Paulo State University Research Ethics Committee (CEP/FCF/CAr protocol 26/2010).

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2.2. Drugs Diazepam and alprazolam (Formil Química Ltda., Brazil) were suspended in physiological (0.9%) saline containing 2% Tween 80, while pentylenetetrazole (PTZ; Sigma-Aldrich, USA), fluoxetine hydrochloride (Tocris, USA), imipramine hydrochloride (Sigma–Aldrich, USA), yohimbine (Sigma–Aldrich, USA) and mCPP ((1-(3-chlorophenyl) piperazine; Tocris, USA) were dissolved in physiological saline. The doses used were based on previous studies [4,28,31–36]. All drugs were injected intraperitoneally (i.p.) in a volume of 10 ml/kg. 2.3. Apparatus The oEPM is similar to the sEPM described by Lister (1987) [6], except that it has four open arms (30 × 5 × 0.25 cm) raised 38.5 cm above floor level on a wooden pedastel. Each arm of the oEPM was divided into three sections (10 × 5 cm), which were designated as proximal, medial and distal areas relative to the central square (5 × 5 cm). 2.4. General procedure All tests were conducted during the light phase of the light/dark cycle, under the illumination of a 100-W light bulb (50 lux on the floor of the apparatus). All mice were individually placed on the central square always facing the same arm and allowed to explore the oEPM freely for 5 min. Between each test, the apparatus was cleaned with ethanol 20% and dried with paper towels. All sessions were recorded by a vertically mounted camera linked to a monitor and DVD recorder. Animals were tested in an order counterbalanced for treatment condition. Test DVDs were scored by a highly trained observer (intrarater reliability ≥0.90) using the software “X-plo-rat 2005”, developed by Dr. Morato’s group at Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto, USP (the software can be freely downloaded at http://scotty.ffclrp.usp.br/X-Plo-Rat.html). In accordance with detailed profiling in the sEPM [e.g. 8, 10, 37], the behavioural parameters comprised both spatiotemporal and ethological measures. Spatiotemporal measures were the frequency of entries and time spent on the central platform and in each section of the arms, while ethological measures comprised the frequency of stretched attend postures (SAP; exploratory posture in which the body is stretched forward but the animal’s hind paws remain in position, followed by retraction to original position), the duration of flat back approach (FBA; time spent slowly moving forward with the body stretched), and the frequency of head dipping (HD; an exploratory behaviour in which the animal scans over the sides of the maze towards the floor) [37–40]. 2.4.1. Experiment 1: factor analysis To understand the inter-relationships between behaviours elicited by exposure to the oEPM, data from 100 naïve mice were subjected to three successive factor analyses. Initially, separate analyses were performed on the spatiotemporal measures (analysis 1) and on the ethological measures (analysis 2), with a view both to understanding behavioural structure and reducing redundancy in the total number of variables scored. The outcomes of these two analyses were then combined in a third factor analysis which yielded the 3-factor behavioural structure used in Experiments 2 and 3. 2.4.2. Experiment 2: acute effects of diazepam, alprazolam, pentylenetetrazole, yohimbine, mCPP, fluoxetine and imipramine This experiment investigated the effects of acute treatment with two benzodiazepines (diazepam and alprazolam), three anxiogenic compounds (PTZ, yohimbine and mCPP), and fluoxetine and imipramine. Following three days acclimatization to local laboratory conditions, mice were randomly allocated to treatment condition (n = 6–11): saline or saline/Tween vehicle as appropriate, diazepam (0.5, 1.0 and 1.5 mg/kg), alprazolam (0.5, 1.0 and 1.5 mg/kg), PTZ (10.0; 20.0 and 30.0 mg/kg), yohimbine (2.0, 4.0 and 6.0 mg/kg), mCPP (0.3, 1.0 and 3.0 mg/kg), fluoxetine (10.0, 20.0 and 30.0 mg/kg) or imipramine (1.0, 5.0 and 15.0 mg/kg). Treatments were administered i.p. 30 min before testing and behaviour scored as described in General Procedure (see Section 2.4). 2.4.3. Experiment 3: chronic effects of imipramine and fluoxetine The third experiment assessed the behavioural effects of chronic treatment with fluoxetine and imipramine. Following three days of acclimatization to local laboratory conditions, mice were randomly allocated (n = 5–8) to treatment condition which comprised daily i.p. injections for 15 consecutive days with saline, fluoxetine (10.0, 20.0, 30.0 mg/kg), or imipramine (1.0, 5.0, 15.0 mg/kg). On the 15th day, treatments were administered 30 min before testing, and behaviour scored as described in General Procedure (see Section 2.4). 2.4.4. Statistics In Experiment 1, the factor analyses were performed by principal component solution with orthogonal rotation (varimax) of the factor matrix. This method ensures that the extracted factors are independent of one other and should, therefore, reflect distinct biological phenomena. The Kaiser test (eigenvalues ≥ 1) was used to confirm the factors extracted, and factor loadings less than 0.5 were discarded.

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Table 1 Ethological description of behaviour displayed by mice (N = 100) in a 5-min open elevated plus-maze session. Parameter

Mean ± SEM

Time spent (s) Central square Proximal areas Medial areas Distal areas

121.68 82.21 44.38 51.85

± ± ± ±

6.47 3.06 2.70 3.93

11.94 18.16 12.68 5.93 48.71

± ± ± ± ±

0.50 0.89 0.82 0.41 2.55

3.05 1.33 0.70 0.52 5.59

± ± ± ± ±

0.43 0.27 0.25 0.19 0.48

± ± ± ± ±

0.15 0.42 0.55 0.17 0.17

Frequency of entries Central square Proximal areas Medial areas Distal areas Total Grooming (s) Central square Proximal areas Medial areas Distal areas Total Flat back (s) Central square Proximal areas Medial areas Distal areas Total

1.43 5.45 7.10 1.93 15.91

Table 2 Orthogonal factor loadings for spatiotemporal measures in mice tested in the open EPM. Behavioural parameter

Factors 1

Time spent in the central square (s) Time spent in the proximal areas (s) Time spent in the medial areas (s) Time spent in the distal areas (s) Entries in the central square Entries in the proximal areas Entries in the medial areas Entries in the distal areas % of variance

−0.75 0.98 0.66 0.76 0.84 0.92 0.96 0.96 65.05

13.58 10.53 4.28 8.39 38.78

± ± ± ± ±

0.75 0.59 0.41 0.79 1.66

Stretched Attend Postures (Freq.) Central square Proximal areas Medial areas Distal areas Total

18.96 9.50 4.95 12.29 45.70

± ± ± ± ±

1.06 0.53 0.38 0.90 1.41

This dataset was used in the three factor analyses reported in Tables 2–4.

For Experiments 2 and 3, all datasets were initially submitted to Levene’s test for homogeneity of variance. Where Levene’s test yielded significance, results were transformed to the log, square root or cube root, and then confirmed for homogeneity of variance before one-way analysis of variance (ANOVA). Significant ANOVA outcomes were analyzed by Duncan’s test. Where heterogeneity of variance remained following all transforms, data were analysed by nonparametric KruskalWallis tests followed, where significant, by pairwise Mann-Whitney tests. A P-value of 0.05 or less was required for significance.

3. Results

14.47

Factor loadings greater than 0.5 are shown. Minus signs indicate the direction of the particular loading. Criteria: eigenvalue ≥ 1.

Table 3 Orthogonal factor loadings for ethological measures in mice tested in the open EPM. Behavioural parameter

Factors 2

1

Head dipping (Freq.) Central square Proximal areas Medial areas Distal areas Total

2

Flat back in the central square (s) Flat back in the proximal areas (s) Flat back in arm-ends (s) SAP in the central square SAP in the proximal areas SAP in the arm-ends Head dipping in the central square Head dipping in the proximal areas Head dipping in the arm-ends % of variance

3

4

0.90 0.95 0.93 0.72 0.96 0.59 0.93

39.89

0.89 0.84 21.01

13.57

11.98

Factor loadings greater than 0.5 are shown. Minus signs indicate the direction of the particular loading. Criteria: eigenvalue ≥ 1.

were found to load highly and positively on factor 1, whereas time spent in the central square loaded highly but negatively on the same factor, it was concluded that the second factor analysis should focus on specific behaviours displayed on the central square, proximal areas and “arm ends” (i.e. medial + distal areas). Factor analysis 2 (Table 3), which focused on several defensive exploratory behaviours (FBA; SAP; HD) displayed in different areas of the apparatus (central square, proximal areas and arm ends), yielded four factors accounting for 86.4% of the total variance. Importantly, no co-loadings were observed for any measure. As the duration of FBA back in all three areas of the apparatus loaded heavily on factor 1, it was concluded that a single (total) FBA score could be used in future analyses. Furthermore, since HD frequency in both the proximal areas and arm ends loaded heavily on factor 2 whereas HD on the central square loaded heavily on factor 3, it was concluded that HD seen on the arms and on the central square

3.1. Experiment 1: factor analysis The results of Experiment 1 are summarized in Tables 1–4. Table 1 outlines the behavioural profile of the animals exposed to the oEPM. The animals spent more time in the center and in the proximal zone of the apparatus (≈68%), where they mainly displayed defensive behaviours such as stretched attend postures (62.27%) and head dipping (62.17%). Table 2, which shows the results of a factor analysis applied purely to the spatiotemporal measures (entries and time spent in the central square, proximal, medial and distal areas), yielded two factors accounting for 79.5% of the total variance. With the sole exception of time spent in the proximal areas, which loaded highly on factor 2, all behavioural measures loaded on factor 1. As the number of entries into each of the four areas (i.e. centre, proximal, medial and distal) loaded highly and positively on factor 1, it was concluded that a single (total) entries measure could be used in future analyses. Since times spent in the medial and distal areas

Table 4 Orthogonal factor loadings for spatiotemporal and ethological measures in mice tested in the open EPM. Behavioural parameter

Factors 1

Time spent in the central square (s) Time spent in the proximal areas (s) Time spent in the arm-ends (s) Total of entries Total flat back (s) SAP in the central square SAP in the proximal areas SAP in the arm-ends Head dipping in the central square Head dipping in the arms % of variance

−0.81

2

3 0.89

0.79 0.56

0.61 0.84

−0.84 0.72 0.82 −0.57 0.75 48.41

16.08

12.55

Factor loadings greater than 0.5 are shown. Minus signs indicate the direction of the particular loading. Criteria: eigenvalue ≥ 1.

T. Sorregotti et al. / Behavioural Brain Research 246 (2013) 76–85

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Table 5 Effect of anxiolytic and anxiogenic compounds on behaviour of Swiss male mice exposed to the open EPM. Drug (mg/kg) PTZ 0 10 20 30 Yohimbine 0 2 4 6 mCPP 0 0.3 1.0 3.0 Fluoxetine acute 0 10 20 30 Imipramine acute 0 1 5 15 Imipramine chronic 0 1 5 15

SAP central square

SAP proximal areas

SAP arm-ends

Head dipping in the arms

15.33 12.36 16.73 15.22

± ± ± ±

2.65 2.34 2.52 3.05

12.89 9.91 15.36 8.56

± ± ± ±

1.90 1.37 2.00 1.56

36.00 39.82 40.55 24.44

± ± ± ±

2.95 4.00 3.63 3.35*

24.22 27.64 8.18 6.33

± ± ± ±

6.09 8.58 1.80* 1.00*

17.00 11.75 12.43 7.00

± ± ± ±

2.37 2.30 2.26 1.79*

9.25 11.38 12.00 8.25

± ± ± ±

1.47 0.98 2.40 1.41

40.38 47.75 40.86 32.50

± ± ± ±

6.27 2.24 5.28 5.70

21.75 13.25 7.14 10.13

± ± ± ±

26.50 26.00 34.50 32.29

± ± ± ±

2.86 4.49 5.40 3.80

15.50 17.25 17.50 19.71

± ± ± ±

1.77 1.88 1.27 1.78

27.33 35.13 28.13 30.00

± ± ± ±

3.92 2.79 4.67 4.61

18.33 20.50 16.38 25.43

9.17 18.86 15.00 14.71

± ± ± ±

3.05 4.19 3.15 4.93

7.83 10.86 11.57 6.86

± ± ± ±

0.54 2.48 2.22 0.96

37.67 37.43 35.71 32.57

± ± ± ±

5.33 6.86 4.77 2.92

12.43 13.78 16.14 18.13

± ± ± ±

2.88 1.64 2.40 3.88

7.43 9.11 11.00 13.38

± ± ± ±

1.80 1.25 2.48 2.09

25.57 33.11 39.86 33.00

± ± ± ±

20.71 20.86 13.80 18.86

± ± ± ±

3.15 1.34 3.80 2.82

9.00 14.43 11.20 16.00

± ± ± ±

1.40 1.46 1.93 2.65

36.00 29.43 42.00 20.71

± ± ± ±

Total flat back (s)

n

7.15 5.45 9.84 5.05

± ± ± ±

1.83 1.57 1.37 1.80

4.42 3.82 1.84 1.83

11.98 6.22 2.46 1.88

± ± ± ±

2.32 1.89 0.85* 0.82*

8 8 7 8

± ± ± ±

2.85 5.21 2.88 4.53

9.29 23.71 14.14 12.68

± ± ± ±

1.94 1.45* 1.87 1.93

6 8 8 7

13.83 17.43 11.29 3.86

± ± ± ±

3.17 9.37 4.26 1.35

3.15 10.38 13.42 19.58

± ± ± ±

1.61 3.85 3.84 5.64

6 7 7 7

4.81 4.15 2.90 3.82

32.14 21.33 12.86 21.88

± ± ± ±

9.05 6.07 2.72 6.53

7.78 9.90 11.85 11.08

± ± ± ±

2.86 3.83 2.75 1.92

7 9 7 8

2.45 7.18 6.07 3.31

22.43 13.71 30.80 24.57

± ± ± ±

4.14 3.77 6.64 3.41

13.05 5.84 8.65 7.29

± ± ± ±

2.92 1.30 2.50 1.39

7 7 5 7

9 11 11 9

Data are presented as mean values (±S.E.M). * P < 0.05 vs. control group.

Table 6 Effect of anxiolytic and anxiogenic compounds on behaviour of Swiss male mice exposed to the open EPM. Drug (mg/kg) PTZ 0 10 20 30 Yohimbine 0 2 4 6 mCPP 0 0.3 1.0 3.0 Fluoxetine acute 0 10 20 30 Imipramine acute 0 1 5 15 Imipramine chronic 0 1 5 15

Time spent central square (s)

Time spent proximal areas (s)

Time spent arm-ends (s)

Total of entries

n

76.41 ± 7.85 60.04 ± 6.00 78.56 ± 12.38 101.25 ± 19.95

78.22 ± 2.66 81.22 ± 4.88 78.63 ± 4.82 81.47 ± 11.28

145.37 ± 8.84 158.75 ± 9.36 142.81 ± 9.66 117.28 ± 21.63

93.11 ± 12.16 101.64 ± 8.82 78.64 ± 8.61 56.33 ± 7.73*

9 11 11 9

60.87 50.31 56.03 53.03

± ± ± ±

10.84 4.85 5.74 5.70

68.32 83.26 83.07 78.55

± ± ± ±

3.43 3.54 6.80 3.14

170.8 166.43 160.90 168.42

± ± ± ±

12.63 5.72 10.33 7.37

93.38 106.88 103.29 101.88

± ± ± ±

9.98 8.30 12.35 13.79

8 8 7 8

115.47 89.85 139.09 110.42

± ± ± ±

6.85 9.43 17.83 11.32

80.72 91.82 70.73 73.95

± ± ± ±

8.29 5.67 7.38 4.72

103.81 118.33 89.67 115.72

± ± ± ±

12.16 9.52 15.19 13.85

53.50 58.25 48.63 69.57

± ± ± ±

8.65 6.00 8.26 11.50

6 8 8 7

73.46 73.95 70.45 73.63

± ± ± ±

21.33 7.61 8.49 13.11

72.14 79.77 79.75 62.87

± ± ± ±

4.78 4.60 7.94 4.68

154.41 146.28 149.81 163.50

± ± ± ±

17.00 10.61 14.41 13.90

114.00 103.43 86.57 83.86

± ± ± ±

17.79 12.38 10.41 9.09

6 7 7 7

64.28 81.52 71.27 91.56

± ± ± ±

8.85 12.46 7.51 10.67

68.41 83.37 73.34 76.20

± ± ± ±

6.97 5.96 6.02 4.57

167.32 135.11 155.39 132.23

± ± ± ±

12.92 15.44 6.56 10.34

71.86 94.89 80.43 81.00

± ± ± ±

5.09 11.99 6.57 7.66

7 9 7 8

88.82 110.99 71.05 96.19

± ± ± ±

11.57 18.40 15.62 13.99

68.69 88.43 82.21 93.04

± ± ± ±

6.87 9.12 13.13 9.24

142.49 100.57 146.74 110.77

± ± ± ±

8.73 19.82 16.09 14.96

71.43 68.14 84.00 46.43

± ± ± ±

5.25 16.46 13.43 9.85

7 7 5 7

Data are presented as mean values (± S.E.M). * P < 0.05 vs. control group.

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represented two independent measures. Finally, as SAP in different areas of the apparatus loaded on 3 separate factors (factor 1: arm ends; factor 3: proximal areas; factor 4: central square), it appeared that SAP could not be reduced to a single score. Factor analysis 3 (Table 4), performed on the combined spatiotemporal and ethological measures distilled from the first two analyses, produced 3 factors accounting for 77.04% of the total variance. Factor 1, showed high positive loadings for time spent and HD frequency in the arm ends and a high negative loading for time spent and SAP on the central square, and seems to best reflect time-consuming depth exploration towards the distal end of the arms. Factor 2 was associated with high and positive loadings for total FBA and SAP at the arm ends, suggesting a relationship to defensive/cautious exploration of the arms. Unsurprisingly, in view of the reliance of factor 1 and factor 2 measures on movement to and from the central square, total entries co-loaded modestly on both factors. Lastly, factor 3 presented high positive loadings for time spent and SAP in the proximal areas coupled with a modest negative loading for HD on the central square, possibly suggesting risk assessment (information gathering; approach/avoid conflict) at the junction between the start point (central square) and the more distal reaches of the arms.

3.2. Experiment 2: acute effects of diazepam, alprazolam, PTZ, yohimbine, mCPP, fluoxetine and imipramine 3.2.1. Two indices of anxiety Results are summarised in Figs. 1 and 2 and Tables 5 and 6. Alprazolam (Fig. 1A–C): Measures with positive loadings on factor 1 (see Table 4), were significantly increased by alprazolam: HD on the arms [F(3,35) = 6.46, P < 0.05; Fig. 1C] and the time spent in the arm ends [F(3,35) = 12.00, P < 0.05; Fig. 1A]. Duncan’s tests showed that HD on the arms was increased by 0.5 and 1.0 mg/kg alprazolam, while time spent in the arm ends was increased by all doses of this agent. In contrast, measures with negative loadings on factor 1 were significantly decreased by all doses of alprazolam (Fig. 1A and B): time spent in central square [F(3,35) = 5.89, P < 0.05; Fig. 1A] and SAP in the central square [F(3,35) = 8.32, P < 0.05; Fig. 1B]. Alprazolam (all doses) also reduced measures with positive loadings on factor 2: total FBA [F(3,35) = 19.10, P < 0.05; Fig. 1C] and frequency of SAP in the arm ends [F(3,35) = 6.47, P < 0.05; Fig. 1B]. Measures with positive loadings on factor 3 were also differentially affected by alprazolam, with all doses reducing time spent [F (3,35) = 9.68, P < 0.05; Fig. 1A] and SAP [F(3,35) = 10.76, P < 0.05; Fig. 1B] in the proximal areas of the apparatus. Non-parametric analysis showed that the mice treated with 0.5 mg/kg alprazolam

Fig. 1. Effects of alprazolam (0–1.5 mg/kg, n = 9–10) on (A) time spent in the central square, proximal areas and end of the arms, (B) SAP frequency in the central square, proximal areas and end of the arms, and (C) total entries, frequency of head dipping in the arms, and total duration of flat back approach. Data are presented as mean ± S.E.M. *P < 0.05 vs. control group.

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Fig. 2. Effects of diazepam (0–1.5 mg/kg, n = 8) on (A) time spent in the central square, proximal areas and end of the arms, (B) SAP frequency in the central square, proximal areas and end of the arms, and (C) total entries, frequency of head dipping in the arms and total duration of flat back approach. Data are presented as mean ± S.E.M. *P < 0.05 vs. control group.

increased total entries [H(3,39) = 10.47, P < 0.05; Fig. 1 C]. However, subsequent analyses of covariance confirmed that the former behavioural effects of alprazolam were independent of its effects on total entries. Diazepam (Fig. 2A–C): Although diazepam had comparatively fewer effects than alprazolam, it had an overlapping profile with significant reductions in key measures on each factor: factor 1 (SAP frequency in the central square [F(3,28) = 4.25, P < 0.05; Fig. 2B]; factor 2 (duration of FBA [F(3,28) = 8.05, P < 0.05; Fig. 2C] and factor 3 (SAP in proximal areas [F(3,28) = 4.32, P < 0.05; Fig. 2B]). Although no other behavioural measures showed a significant drug effect [F(3,28) ≤ 1.85, P > 0.05], alprazolam-like trends were observed for time spent on the central square (decreased), time spent in arm ends, total entries and HD in the arms (all increased). PTZ, yohimbine and mCPP: Somewhat surprisingly, most behavioural measures were unaffected by the anxiogenic compounds pentylenetetrazole (PTZ; 0–30 mg/kg) [F(3,36) ≤ 1.61, P > 0.05], yohimbine (0–6 mg/kg) [F(3,27) ≤ 2.72, P > 0.05] and mCPP (0–3.0 mg/kg) [F(3,25) ≤ 2.76, P > 0.05]. However, as summarised in Tables 5 and 6, the two higher doses of the GABAA receptor-related drug PTZ decreased HD in the arms [F(3,36) = 4.06, P < 0.05], while 30.0 mg/kg additionally reduced SAP in the arm ends [F(3,36) = 4.09, P < 0.05] and total entries [F(3,36) = 4.30, P < 0.05]. Nevertheless, subsequent analyses of covariance indicated that the reduction in arm entries seen with the higher dose of PTZ may have been

responsible for the reduction in HD [F(3,35) = 1.49, P < 0.05] and SAP measures [F(3,35) = 2.49, P = 0.08]. Treatment with the two higher doses of yohimbine decreased total FBA [F(3,27) = 7.11, P < 0.05] while the 6.0 mg/kg decreased SAP frequency in the central square [F(3,27) = 3.60, P < 0.05]. Post hoc analysis also revealed that the lower dose of mCPP (0.3 mg/kg) significantly increased total FBA [F(3,25) = 12.00, P < 0.05]. Fluoxetine and imipramine: As shown in Tables 5 and 6, no behavioural effects were seen following acute treatment with fluoxetine (0–30.0 mg/kg; [F (3.23) ≤ 2.61, P > 0.05]) or imipramine (0–15.0 mg/kg; [F (3.27) ≤ 1.79, P > 0.05]). 3.3. Experiment 3: chronic effects of imipramine and fluoxetine Results are summarised in Fig. 3 and Tables 5 and 6. As shown in Tables 5 and 6, chronic treatment with imipramine (0–15 mg/kg) failed to reliably alter any behaviours displayed measure in mice exposed to the oEPM [F(3,22) ≤ 2.34, P > 0.05]. However, as shown in Fig. 3C, chronic treatment with fluoxetine (0–30 mg/kg; Fig. 3) produced a clear dose-dependent reduction in total FBA [F(3,23) = 4.97, P < 0.05; significant at all doses], coupled with an increase in HD in the arms [F(3,23) = 5.38, P < 0.05; Fig. 3C] which was significant at the intermediate dose of 20.0 mg/kg. No other behavioural measures were significantly influenced by this compound [F (3.23) ≤ 2.81, P > 0.05].

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Fig. 3. Effects of chronic treatment with fluoxetine (0–30.0 mg/kg, n = 5–8) on (A) time spent in the central square, proximal areas and end of the arms, (B) SAP frequency in the central square, proximal areas and end of the arms, and (C) total entries, frequency of head dipping in the arms and total duration of flat back approach. Data are presented as mean ± S.E.M. *P < 0.05 vs. control group.

4. Discussion The current research was designed to comprehensively describe the range of defensive behaviours displayed by naïve mice when allowed to freely explore the oEPM (Experiment 1) and to assess the sensitivity of such behaviours to a range of drugs that have been shown to attenuate (alprazolam, diazepam, fluoxetine and imipramine) or exacerbate (PTZ, yohimbine and mCPP) defensive behaviour (Experiments 2 and 3). Although factor analysis has been extensively used to understand the structure of rat and mouse behaviour in the sEPM [e.g. 6, 8, 10], it has not as yet been applied to behaviours observed in the oEPM. Therefore, in Experiment 1, the oEPM behavioural records for 100 naïve mice were subjected to a series of three factor analyses, the first two of which were used to understand the relationships between the various spatiotemporal and ethological measures recorded. Factor analysis 1 (2-factor structure) validated the future use of a single total entries score, together with separate scores for the amounts of time spent on the central square, proximal areas of the arms, and ‘medial plus distal’ areas of the arms. The second factor analysis (4-factor structure) exploited this understanding to focus on specific defensive exploratory behaviours (i.e. FBA, SAP and HD) as expressed in or on particular spatial zones of the apparatus. The outcome of that analysis validated the future use of a single (total) measure of FBA, together with separate measures for HD on the central square and on the arms, and separate

measures for SAP on the central square, proximal areas and the arm ends. The refined spatiotemporal and ethological measures were together entered into a third factor analysis which produced a 3factor structure acounting for 77% of the total variance (see Table 4). Unlike factor analyses performed on data generated in the sEPM, which identified separate factors related to open arm avoidance, general locomotor activity and rearing [6,8,10,41], all 3 factors identified in the oEPM were unsurprisingly (given the ‘no hiding place’ structure of the maze) related to different components of the rodent defensive repertoire [42]. Thus, variables loading on factor 1 indicated a relationship to depth exploration (HD) from the open arms, variables loading on factor 2 a relationship to cautious/defensive exploration of the arms (FBA), and variables on factor 3 a relationship to risk assessment (SAP) in the proximal areas. Most parsimoniously, following movement from the central square starting area, mice spent time in the proximal areas of the open arms from which they gathered (via SAP) information relevant to approach/avoid decision-making [8,10,43]. When adopted, an approach strategy was cautious in the sense of flat-back, as opposed to normal curved back, locomotion [44]. Interestingly, the variable ‘total entries’ co-loaded on factors 1 and 2. However, even more so than is the case in the sEPM [e.g. 10, 41] or even the more recently-developed rat exposure test [36], it would be most unwise to consider total entries a measure of general locomotor activity untainted by an emotional component.

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The above interpretation of oEPM behaviour was reinforced by the results of our pharmacological investigations. Thus, the results of Experiment 2 show that, over the entire dose range tested, acute treatment with the triazolobenzodiazepine alprazolam decreased the total duration of FBA as well as the frequency of SAP in all areas of the maze. When displayed in tests of antipredator behaviour [e.g. 45] and in the standard elevated plus-maze (sEPM), these behaviours have been linked to risk assessment and, as such, their reduction would be entirely consistent with an attenuation of anxiety [46]. Consistent with observations in the sEPM [e.g. 10], alprazolam reduced time spent on the central platform and proximal areas of the arms while increasing both time spent and head-dipping at the arm-ends. The stimulation of exploratory head-dipping in elevated mazes is a characteristic effect of benzodiazepines [e.g. 3, 47, 48] and, along with the observed increase in total arm entries (0.5 mg/kg), confirms that the effects of alprazolam in the oEPM were not secondary to general behavioural suppression. Interestingly, while alprazolam affected virtually all behavioural measures in the oEPM, diazepam attenuated only total FBA and SAP frequency in the central square (1.5 mg/kg) and proximal areas (1.0 and 1.5 mg/kg). It is unlikely that this weaker profile is due to the dose range of diazepam used since higher doses of this compound would most likely have induced muscle relaxation/sedation [e.g. 49, 50]. Overall, these results suggest that the behavioural profile displayed by mice in the oEPM is more sensitive to the panicolytic agent alprazolam than to the anxiolytic agent diazepam. Somewhat surprisingly, acute treatment with the anxiogenic compounds PTZ, yohimbine and mCPP [e.g. 28, 4, 51, 52, 53, 54] failed to produce major effects on the behaviour of mice exposed to the oEPM (Tables 5 and 6). Nevertheless, some changes consistent with an anxiogenic-like action were observed: thus, the frequency of HD in the arm-ends was significantly reduced by 20–30 mg/kg PTZ while the higher of these doses also produced non-significant changes in time spent on the central square (reduced) as well as time spent and SAP (increased) on the arm-ends [10,28,47]. However, it is pertinent to note that the highest dose of PTZ also produced a significant decrease in total entries [see also 10, 31, 53, 55], an effect that may reflect a sedative or even pro-convulsant action of the compound [28]. Thus, the behavioural changes seen with PTZ most likely reflect a combination of sedative and anxiogenic-like responses. Previous research has shown that the ␣2-adrenoceptor antagonist yohimbine induces anxiogenic-like effects in animals exposed to various tests of anxiety [53,56–59] and no effect [4,36]. Although yohimbine was also active in the oEPM (reductions in SAP in the central square and total FBA), these effects would be more consistent with an anxiolytic (as opposed to an anxiogenic)-like action. In this context, it is pertinent to note that others have also reported anti-anxiety effects with this compound. For example, Cole et al. [60] showed that yohimbine produced anxiolytic-like effects in mice exposed to the sEPM. This drug also produced anti-anxiety effects in animals exposed to the punished drinking test (Vogel test) [56] and to shock-induced ultrasonic vocalization [61]. Similar to PTZ and yohimbine, mCPP also produced weak effects on defensive behaviour in mice exposed to the oEPM. Only the lower dose used (0.3 mg/kg) increased total FBA, suggesting a mild anxiogenic-like effect. This finding is consistent with reports that mCPP increases acoustic startle in mice [32], facilitates inhibitory avoidance acquisition in mice exposed to the elevated T-maze [4], and dose-dependently induces anxiogenic-like behavioural changes in mice exposed to the sEPM [62]. Taken together, present results failed to demonstrate a robust enhancement of mouse defensiveness in the oEPM following acute treatment with three anxiogenic compounds differing in their mode of action (PTZGABAA receptors; yohimbine-␣2 -adrenoceptors; mCPP-5-HT2C

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receptors). As the limited responses to these agents cannot readily be attributed to inadequate dose ranges, it seems likely that they are due to the highly aversive nature of the oEPM and, hence, a ceiling effect. The present study also investigated the effects of acute treatment with fluoxetine, a selective serotonin re-uptake inhibitor, and imipramine, a dual serotonin and noradrenaline re-uptake inhibitor. In contrast to effects seen with the benzodiazepines, acute treatment with the fluoxetine and imipramine did not result in any behavioural changes in mice exposed to the oEPM. The effects of these compounds have been shown to vary from anxiolytic [63], through no effect [4,6,7,33,46,64,65], to anxiogenic [65–67]. In accord with previous negative findings, acute treatment with neither fluoxetine nor imipramine exerted any behavioural effects in the oEPM. However, antidepressants are clinically effective only after a period of chronic treatment [68]. In the current study, however, daily treatment with imipramine (0–15.0 mg/kg) for 15 days completely failed to alter the behaviour of mice in the oEPM. Although chronic imipramine has been found to attenuate escape responses in the mouse defence test battery (MDTB) [69], and impair the acquisition of inhibitory avoidance and prolonge escape latency from the open arms in the elevated T-maze in rat [70], such treatment has been found to be ineffective in the elevated T-maze in mice [4,33; but see 70] and rat exposure [36] tests. In contrast to acute treatment (which may enhance defensive responding), chronic treatment with SSRIs such as fluoxetine attenuates flight, defensive attack and risk assessment in tests of antipredator behaviour [69]. Furthermore, unlike chronic imipramine, chronic fluoxetine treatment reduces the defensive behavior of mice exposed to the elevated T-maze [33] and rat exposure test [36]. Consistent with these findings, which may result from the down-regulation of postsynaptic 5-HT receptors in the limbic forebrain [71], our results show that chronic fluoxetine decreased the total duration of FBA and, at a dose of 20.0 mg/kg, also increased frequency of head dipping on the arms of the oEPM. These effects, while modest in comparison, overlap with those of the benzodiazepines and given the lack of effect on total entries, suggest a rather selective effect of this SSRI on defensive behaviour. When the results of drug treatment are considered in the light of the initial factor analyses (Table 4), it would indeed seem that all three factors reflect (albeit somewhat different) aversive responses to the oEPM. This inference is based largely on the results obtained with alprazolam which significantly influenced measures from all three factors; e.g. increasing HD (factor 1), reducing FBA (factor 2) and reducing SAP (factor 3). Furthermore, as would be predicted, alprazolam increased measures with positive loadings of factor 1 while reducing measures with negative loadings on the same factor. Diazepam and chronic (but not acute) fluoxetine had similar, though more modest, effects on oEPM behaviour while the anxiogenic compound PTZ produced opposite (i.e. inhibitory) effects on at least some parameters e.g. head-dipping on the arms and total entries. In conclusion, therefore, present results indicate that the mouse oEPM induces behavioural defensive responses that are sensitive to anxiolytic-like compounds, and especially the triazolobenzodiazepine alprazolam. Since potent benzodiazepines, such as alprazolam and clonazepam, are renowned for their efficacy in the treatment panic disorder [72–74], further oEPM studies with other panicolytic agents would appear warranted.

Acknowledgements We thank Dr. André Ramos (Federal University of Santa Catarina) for his suggestion on factorial analysis and Elisabete Z.P. Lepera and Rosana F.P. Silva for their technical assistance. This study was supported by FAPESP, CNPq and PADC/FCF-UNESP. T. Sorregotti

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and J. Mendes-Gomes were supported by FAPESP fellowship (Proc. 2010/13441-7 and 05/01988-3) and R.L. Nunes-de-Souza received a CNPq research fellowship (Proc. 303580/2009-7).

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