Previous maze experience required to increase open arms avoidance in rats submitted to the elevated plus-maze model of anxiety

Abstract

Studies have shown an increased open arm avoidance in rats re-exposed to the elevated plus-maze (EPM), which suggests a qualitative shift in emotional states from an unconditioned (Trial 1) to a learned (Trial 2) form of fear response, but a precise source of aversion has not been determined. Using rats submitted to the EPM or various EPM-derived configurations, this study was designed to investigate what previous maze experiences in Trial 1 are required to increase avoidance of open arms in EPM Trial 2. Results obtained from rats submitted to the EPM or EPM-derived configurations confirmed the increased open arms avoidance in Trial 2. Rats confined to either open or enclosed arms failed to show the increased avoidance of open arms in Trial 2. The results are discussed in terms of the minimum prerequisite in Trial 1 to elicit an avoidance learning response to open arms in Trial 2, and also the implications of an acquired fear response in rats for the study of the biological basis of anxiety.

Introduction

A series of experiments conducted in 1955 studied the relationship between fear and exploratory behavior in an elevated Y-maze composed of a varying numbers of open and enclosed arms [23]. Based on the fact that rats would explore the enclosed arms with a higher frequency than the open arms, one of the Montgomery’s main conclusions was that in this model, novel stimulation elicited both the exploratory and the fear drive, thus generating approach-avoidance conflict behavior. Thirty years later, Handley and Mithani [16], in an attempt to study the involvement of the noradrenergic systems in anxiety, developed a symmetrically constructed elevated X-maze consisting of two open and two enclosed arms, based on earlier observations by Montgomery [23]. Their assumptions were that the response by rats to this type of maze would be sensitive to the anxiolytic and anxiogenic drugs in rats, and therefore the effects of anxioselective drugs should be assessed in terms of preference for the open arms as simple ratios (open arm/total entries percentage). These predictions were subsequently confirmed (see [29] for a review) and validated for both rats [26] and mice [21]. The elevated plus-maze test (EPM) is the most popular of all currently available animal models of anxiety, based on the study of spontaneous behavior [4], [8]. The consistency and relevance of this model can be assessed in 900 published papers over the past 15 years [8], [16], [17], [19], [21], [26], [29], [39].

Rats are normally cautious when exploring open spaces and in the EPM, after an initial overall exploration, they avoid the open arms starting around the third minute of trial 1 [18], and instead remain in the two enclosed arms of the maze [2], [20], [26]. Although rats forced to stay in the open arms of the EPM show fear-like reactions such as freezing, defecation, and increased production of plasma corticosterone [26], the precise source of aversion has not been determined [17]. Treit et al. [39] showed that in the EPM, the lack of thigmotaxis in the open arms might be the main avoidance-promoting feature, rather than height or novelty.

As pointed out by Rodgers and Cole [29], there are two major groups of variables influencing behavior and/or drug response in the EPM: (1) organismic variables such as, species, genetic strain [27], [28], gender [19], [28], [37] and age [22]; and (2) procedural variables such as housing [22], prior handling [1], [9], prior stress [38], [41], and lighting levels [15], [24], which have all been reported to have significant effects on basal anxiety.

Although early results regarding the influence of previous maze experience in EPM performance showed that repeated testing did not modify baseline open arm entries or time [3], [11], [21], [26], nowadays there is an agreement that the retest of rats and mice in the EPM increases the open arm avoidance, therefore suggesting an anxiogenic tendency [31], [34], [39]. File et al. [11] described a phenomenon named “one trial tolerance” due to the lack of anxiolytic effect by benzodiazepines in the EPM for rats submitted to a single prior exposure to the apparatus. The main line of reasoning to explain the retest effect includes the fact that a 300 s prior experience in the EPM is able to release endogenous inverse agonists that bind to and alter the state of benzodiazepine receptors in brain areas, further inducing desensitization of benzodiazepine receptors [13]. This pharmacological evidence would reflect an enhancement in memory processes, therefore supporting the notion that a prior undrugged EPM experience induces a qualitative shift in anxiety/fear reaction from an unconditioned to an acquired phobic response [18], [33], [39].

An unanswered question related to test–retest or Trial 1–Trial 2 data in the EPM refers to the key feature in the avoidance learning process. The purpose of this study was to investigate what previous maze experiences in Trial 1 were required to alter baseline EPM results in Trial 2, confirming the hypothesis of a qualitative shift in anxiety/fear response.