Longitudinal study of daily variation of rats’ behavior in the elevated plus-maze Miriam Mendonc ¸a Morato Andrade*, Marta Frenesda Tome ´, Eneida Silveira Santiago, Ana Lu ´cia-Santos, Telma Gonc ¸alves Carneiro Spera de Andrade Department of Biological Sciences, FCL, Unesp/Assis, Av. Dom Antonio, 2100, Assis, SP 19806-173, Brazil Received 5 December 2001; received in revised form 3 September 2002; accepted 3 October 2002 Abstract We conducted a longitudinal study about daily variation of Wistar male rats’ behavior in the elevated plus-maze (EPM) evaluated in the 1st, 2nd, 3rd, 6th, 12th, and 18th months of life. Animals were submitted to the plus-maze in 12 sessions at 2-h intervals (n = 72, 6 per time point). Spontaneous rest – activity rhythm of four animals was assessed by observation of 24-h videotape records. Time series were analyzed by Cosinor method. Behavioral rates on the six occasions and in light and dark phases were compared by means of two-way ANOVA with repeated measures. Exploratory behavior in EPM was smaller in the light phase and in older animals. Higher values of open and closed arms exploration were observed in the first and third months of the dark phase, and in the first month of the light phase. Adjustment to the 24-h period was significant at all stages for rest – activity data, number of entries in closed arms, and time on center, and for three to five stages for open-arm exploration. In general, 24 h variability was more pronounced in younger animals compared with older ones. The present study showed that: (1) a significant amount of total variability of the behavioral indexes analyzed could be attributed to 24 h variation, (2) light/dark phases differences in EPM exploration were present at all developmental stages, (3) older Wistar rats explored less the EPM and were less active in their home cage compared with younger ones, and (4) behavioral indexes (EPM) decrease was phase related and partially related to a reorganization of rest – activity rhythm. D 2003 Elsevier Science Inc. All rights reserved. Keywords: Circadian rhythm; Elevated plus-maze; Development; Aging; Anxiolysis; Motor activity 1. Introduction The elevated plus-maze (EPM) has been widely used as a tool in the investigation of the psychological and neuro- chemical basis of anxiety and for screening new drugs. The EPM apparatus is based on rodents’ innate fear of open spaces. The EPM does not involve training or use of noxious stimuli and the test is simple and quick to perform. Animals are allowed to explore two open arms perpendic- ular to two closed arms, elevated from the floor. They usually spend more time in the closed arms than in the open arms. The EPM has been repeatedly validated for use with rats and mice, and more recently, for others species such as guinea pigs, voles, and hamsters [22,36,46]. The test is sensitive to both anxiolytic and anxiogenic manipulations. Despite the EPM advantages, there are considerable inter- laboratory differences that contribute to discrepancies among results, including a wide range of experimental animals used (age, gender, strain), maze construction, and procedures adopted (handling, prior exposure to other tests, illumination, among others) [21,23,37]. Little attention has been directed to age-dependent changes in daily variation of rats’ behavior in the EPM. Generally, there is a description in different investigations of animals’ weight (a characteristic that may change according to the number of animals housed in the cage) but not of animals’ age. The EPM apparatus has been used more frequently with adult rats. Imhof et al. [25], one of the few examples of works comparing the behavior in the EPM of animals with different age groups (45, 60, 90, 120, and 150 days old), submitted rats to the apparatus only during 0031-9384/03/$ – see front matter D 2003 Elsevier Science Inc. All rights reserved. PII:S0031-9384(02)00941-1 * Corresponding author. Tel.: +55-18-33025848; fax: +55-18- 33025849. E-mail address: mandrade@assis.unesp.br (M.M.M. Andrade). Physiology & Behavior 78 (2003) 125 – 133