Elsevier

Physiology & Behavior

Volume 78, Issue 1, January 2003, Pages 125-133
Physiology & Behavior

Longitudinal study of daily variation of rats' behavior in the elevated plus-maze

https://doi.org/10.1016/S0031-9384(02)00941-1Get rights and content

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.

Introduction

The elevated plus-maze (EPM) has been widely used as a tool in the investigation of the psychological and neurochemical 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 perpendicular 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 interlaboratory 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 the light phase and observed age-related changes in anxiety indexes.

Recently, some articles have been published analyzing time-of-day effect on animals' behavior in the EPM. Jones and King [26] compared the behavior of Sprague–Dawley rats submitted to a series of tests (among them the EPM) in only two different time points, and could not identify significant differences in the pattern of response of animals tested during the light phase compared with animals tested during the dark phase. On the other hand, studies conducted by Yannielli et al. [46], [47] have shown 24 h variations of hamsters' behavior in the EPM. The percentage of time on open arms and percentage of entries in open arms (behavioral indexes of anxiety) as well as the number of entries in both arms (locomotor activity index) reached a peak value during the dark phase. This pattern remained with a period of approximately 24 h in constant darkness, suggesting endogenous mechanisms related to the temporal variation. In a following study, Golombek et al. [19] observed changes of daily variation of behavioral indexes in EPM and of rest–activity rhythm in hamsters with age.

Biological rhythms undergo changes in the course of life span (for review, see [1], [7]). Studies of age-dependent changes in rhythmic parameters have shown similar trends among different rhythmic functions although not always simultaneously [44]. During early developmental stages, the synchronization of circadian rhythms with environment temporal cues takes place, the amplitude of the oscillation increases, the phase shifts, and ultradian periodicity weakens while circadian periodicity consolidates [8], [31], [43]. Furthermore, changes in rhythmic parameters seem to occur in opposite directions comparing early life to old age [27], [45].

As the knowledge about biological rhythmicity grew over the last decades, investigators usually choose one time point to perform behavioral tests, but usually do not ask themselves if their findings (obtained at that time point) could be generalized to others. For instance, there is evidence that the anxiolytic activity of a small dose of diazepam assessed in the EPM is observed only at some time points but not at others [46]. Considering the importance of biological rhythmicity underlying the expression of physiological and behavioral processes, it is necessary to verify the temporal pattern (for instance, using more time points) of animal's performance in the EPM. In addition, as temporal internal organization changes with age, findings are not obligatorily comparable among age groups.

The present work aims to provide a temporal framework (concerning 24 h variations and age-related changes) for studies that use the EPM. We conducted a longitudinal study about daily variations of Wistar male rats' behavior in the EPM evaluated in the 1st, 2nd, 3rd, 6th, 12th, and 18th months of life. Spontaneous rest–activity rhythm was evaluated at each occasion in order to measure whether changes in daily variation of rats' behavior in the EPM were related to age-dependent changes of the circadian system.

Section snippets

Subjects

A group of 72 male Wistar rats, born and reared in our laboratory, were housed in air-conditioned rooms (22±2 °C) on a 12:12 h light/dark cycle with approximately 270 lx (cool white fluorescent tubes, light phase) and 3 lx (lamp bulbs with red photograph filter, dark phase) at the center of the keeping room. Half of the animals were housed in one keeping room (lights on at 8 h, room LD) and half in another keeping room (lights on at 20 h, room DL). Food and water were provided ad libitum. In

Rats' behavior in the EPM

Behavioral indexes were significantly affected by age group and light/dark phases, and except for time spent on center, there were significant age and phase interactions (Table 1). Light phase values were smaller than dark phase ones. Post hoc comparisons showed reduced values of all behavioral indexes in the last months. Significant higher values of exploration of the open arms and more entries in closed arms were observed in the first and third months in the dark phase, and in the first month

Discussion

The results evinced a daily temporal pattern of rat's behavior in the EPM. Animals showed higher motor activity in the EPM, exploited more the open arms and also spent more time at the center of the apparatus during the dark phase. The higher rate of motor activity during the dark phase in the EPM may have propitiated the occurrence of higher exploration of open arms. However, increased motor activity during the dark phase was not the only factor affecting open-arm exploration, since the

Acknowledgements

This research was supported by grants from FAPESP to Miriam Mendonça Morato Andrade (98/00971-4), Ana Lúcia-Santos (99/06396-4), Eneida Silveira Santiago (98/00972-0) and a grant from CNPq/PIBIC to Marta Fresneda Tomé.

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