Integrating the open field, elevated plus maze and light/dark box to assess different types of emotional behaviors in one single trial

Abstract

Current anxiety tests do not provide, individually, a pure and complete picture of an animal's emotional profile. Therefore, many authors test their experimental hypotheses using a series of anxiety-related tests, which are thought to reflect different facets of emotionality. The objective of this study was to investigate the potential usefulness of integrating three widely used behavioral tests, the open field (OF), elevated plus maze (EPM), and light/dark box (LDB), to assess a wider range of emotional and non-emotional behaviors within one single trial. A protocol was developed where rats could freely explore an OF that was physically connected to an EPM and a LDB during 15 min. Classical anxiety- and locomotion-related behaviors from each test were measured. Lewis and spontaneously hypertensive rats (SHR) inbred strains, known to present genetic differences in each of the individual tests, differed for all anxiety-related behaviors from the combined apparatus. Factor analyses revealed that similar anxiety- and locomotion-related factors were produced by the three tests applied either separately or in combination. Under both conditions, each test produced its own anxiety-related factor. Two benzodiazepines, chlordiazepoxide (at 5 and 10 mg/kg) and midazolam (at 0.75 mg/kg), facilitated the approach towards the EPM open arms, whereas pentylenetetrazole (10 mg/kg) specifically inhibited exploration of the three aversive areas (OF center, EPM open arms, LDB light compartment). Together, these results suggest that the new integrated apparatus may contribute to the study of anxiety, by providing a rapid, comprehensive and reliable method of assessing emotionality-related behaviors and its underlying components.

Introduction

Since the creation of the open field test, conceived in 1934 to provide objective measures of emotionality in rats [18], dozens of different tests aiming to assess anxiety-related behaviors in laboratory rodents have appeared in the literature, for reviews see Refs. [1], [13], [17], [23], [27], [37]. From the field of experimental psychology, the interest on such tests spread rapidly among neuroscientists, pharmacologists and, more recently, molecular geneticists. Today, these tests are part of the screening routine of many laboratories, not only of those working on drug development but also the ones dedicated to the behavioral and neurological study of mutant mice and rats [11], [17], [40].

Despite their wide diffusion, most users agree that none of the existing anxiety tests provides a pure, undisputed measure of emotional reactivity and that each individual test assesses only a fraction of an animal's emotional profile [6], [13], [27]. In order to minimize the consequences of such limitations, many authors submit their experimental subjects to a battery of different tests which, together, are expected to provide a more complete and reliable picture of an animal's emotional reactivity [11], [27]. Nevertheless, several concerns arise from the use of test batteries, such as the long-lasting influences of previous test experiences, which were found to be dependent on the animal's genotype [41], and the need for allowing inter-test intervals (of a few days or weeks) in order to minimize the effects of test history [24]. In addition to that, ideally, a test battery should not be excessively complex or time-consuming in order to allow its use in high-throughput behavioral phenotyping studies.

Another issue to be considered when using different tests is how to interpret treatment effects that are seen in one test but not in another. Data from correlational studies, in spite of being somewhat contradictory [28], [29], often indicate that there is little correlation among anxiety-related behaviors measured in different tests [16], [29], [38]. Such a lack of correlation is normally interpreted as a sign of difference in the psychobiological meanings of the various tests [16], [27], [38]. However, because an animal cannot be tested at the same time in two different tests and as most tests only assess temporary emotional states [23], one cannot be sure of how much of the observed inter-test inconsistency is due to real construct differences among tests and how much of it is caused by temporal changes in the emotional state of an animal that is submitted to different tests at different times [19].

In view of all the aforementioned matters, several authors have urged the behavioral neuroscience community to improve the current methods of behavioral testing. Such an improvement could be attained either by conceiving new tests and paradigms [15], [21] or by refining the behavioral analysis [22], [33] and/or the current design of existing tests [13], [14].

In the present study, we hypothesize that a more comprehensive, reliable and rapid assessment of the emotional profile of laboratory rodents could be obtained by physically combining different existing tests that are based on the free exploration of aversive environments. To this end, we propose a combination of the three most widely used tests of anxiety, namely the open field (OF), the elevated plus maze (EPM) and the light/dark box (LDB). With this integrated test, the experimenter should be able to assess different aspects of emotionality simultaneously, thus avoiding undesirable effects of previous test experiences, inter-test intervals and handling between tests. Two pilot studies were devised to determine: whether or not laboratory rats would indeed explore all areas of this integrated test; what would be the best starting place in the apparatus; and what would be the best duration of a test session. Once a general testing protocol was defined, another experiment was performed to verify whether the new test, provisionally called the “triple test”, would be capable of detecting previously known genetic differences in anxiety-related behaviors. Subsequently, factor analyses were performed on behavioral data obtained from the OF, EPM and LDB, used either separately or in combination, in order to elucidate the different behavioral dimensions assessed under each test condition. Finally, pharmacological experiments were carried out to investigate the effects of three anxiety-modulating drugs on the new combined apparatus.