Behavioural Brain Research 203 (2009) 35–42 Contents lists available at ScienceDirect Behavioural Brain Research journal homepage: www.elsevier.com/locate/bbr Research report Variations in illumination, closed wall transparency and/or extramaze space influence both baseline anxiety and response to diazepam in the rat elevated plus-maze Nicolas Violle, Frédérique Balandras, Yves Le Roux, Didier Desor, Henri Schroeder ∗ URAFPA, INRA UC340, Nancy-Université, Vandœuvre-lès-Nancy, France article info Article history: Received 18 December 2008 Received in revised form 10 April 2009 Accepted 13 April 2009 Available online 21 April 2009 Keywords: Elevated plus-maze Rat Baseline anxiety level Illumination Closed wall type Extramaze space size Diazepam Test sensitivity abstract Numerous methodological-related variables have been demonstrated to influence the baseline anxiety level of rodents exposed to the elevated plus-maze (EPM), raising questions about the sensitivity of this test for the detection of the effects of anxiolytic drugs. Thus, the present study was designed (1) to assess the combined effects of illumination (40-lx red or white light), closed wall type (walls made of translucent or opaque material) and extramaze space size (small or spacious experimental room) on rat behaviour, and (2) to investigate the effects of such parameters on the relevance of the maze for detecting the effects of diazepam orally administrated at the anxiolytic dose of 3mg/kg. Results indicate that illumination and closed wall type are two main independent parameters that are able to modify the open arm avoidance. Moreover, the closed wall type interacts with the extramaze space size since the reduction of the open arm exploration induced by opaque closed walls is two-fold stronger in the spacious experimental room than in the small one. Finally, the diazepam anxiolytic activity is significantly detected in our laboratory in specific EPM conditions (maze with opaque walls, use of a red light, maze located in a spacious experimental room). In conclusion, the present study demonstrates that an inappropriate baseline anxiety level due to the methodological use of the EPM can dramatically reduce the sensitivity of the maze for the detection of benzodiazepine-related compounds. This study also provides new insights into the perception of the EPM open space in rats. © 2009 Elsevier B.V. All rights reserved. 1. Introduction The elevated plus-maze (EPM) is one of the most popular paradigm for the study of anxiety-related behaviour in rodents [9,33]. Since its first introduction by Handley and Mithani [25], this test has been used in numerous studies including the assessment of anxiety-modulating properties of pharmacological substances [10,23,39,48,49], the understanding of neurobiological or geneti- cal substrates of anxiety [3,17], and the investigation of the effects of various environmental parameters (e.g. pain, exposure to toxic agents, environmental enrichment, earlier weaning,...) on anxiety- related behaviours [4,22,27,34,43,45]. The EPM testing consists in measuring the behaviour of a naive rat that can freely explore a maze elevated from the ground to heights of 50–70 cm [35,49] and composed of two opposed open arms (i.e. without walls) and two opposed closed arms (i.e. with ∗ Corresponding author at: URAFPA, INRA UC340, Faculté des Sciences et Tech- niques, B.P. 239, 54506 Vandoeuvre-lès-Nancy Cedex, France. Tel.: +33 383 68 48 94; fax: +33 383 68 48 88. E-mail address: henri.schroeder@scbiol.uhp-nancy.fr (H. Schroeder). walls) arranged in such a way as to form a cross. Typically, naive rats enter less frequently and spend less time in the open arms than in the closed arms. A rat treated with an anxiolytic drug increases ratios of both open/closed arm entries and open/closed arm times, whereas an anxiogenic drug produces an opposite effect [35]. More- over, the measurement of additional ethological-derived items such as risk assessment activities or vertical exploration has been shown to increase the efficacy and the sensitivity of the EPM [9,20,40,44]. In order to improve the quality of the behavioural measure- ments, especially when ethological-derived items are observed, some laboratories modified the original EPM apparatus and/or the procedure validated by Pellow et al. [35]. Two alterations are usually applied. First, EPM testing is often performed during the light phase although rats are nocturnal rodents. To overcome this problem, some laboratories maintain the animals under a reversed light/dark cycle and expose the rats to the maze during the dark phase of the cycle [5,31]. In this case, a red dim light is often used because it has been demonstrated that wavelengths over than 600 nm are poorly visible for rats [18,28,29]. Second, some laboratories use a modified version of the EPM with closed arms made of translucent Plexiglas walls in order to facilitate the observation of ethological items such as closed arm rears and open arm head dips [1,2]. Paradoxically, 0166-4328/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.bbr.2009.04.015