Longitudinal study of daily variation of rats' behavior in the elevated plus-maze
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.
References (47)
- et al.
Effect of electrolytic and neurotoxic lesions of the median raphe nucleus on anxiety and stress
Pharmacol. Biochem. Behav.
(2001) - et al.
Previous maze experience required to increase open arms avoidance in rats submitted to the elevated plus-maze model of anxiety
Behav. Brain Res.
(2000) - et al.
Behavioral profile of rats submitted to session 1–session 2 in the elevated plus-maze during diurnal/nocturnal phases and under different illumination conditions
Behav. Brain Res.
(2002) - et al.
Evolution of rat motor activity circadian rhythm under three different light patterns
Physiol. Behav.
(1991) - et al.
Ethopharmacological analysis of rat behaviour on the elevated plus-maze
Pharmacol. Biochem. Behav.
(1994) - et al.
Exposure to a novel stimulus reduces anxiety level in adult and aging rats
Physiol. Behav.
(2001) - et al.
Individual housing from rearing modifies the performance of young rats on the elevated plus-maze apparatus
Physiol. Behav.
(1996) - et al.
The influence of open arm ledges and maze experience in the elevated plus-maze
Pharmacol. Biochem. Behav.
(1996) - et al.
Anxiolytic effects in the plus-maze of 5-HT1A-receptor ligands in dorsal raphé and ventral hippocampus
Pharmacol. Biochem. Behav.
(1996) - et al.
Aging attenuates diurnal variation in hamster locomotion, anxiolysis and GABA turnover
Neurosci. Lett.
(1997)
Multiple serotonin mechanisms in animal models of anxiety: environmental, emotional and cognitive factors
Behav. Brain Res.
A review of the validity and variability of the elevated plus-maze as an animal model of anxiety
Pharmacol. Biochem. Behav.
Isolation during the play period in infancy decreases adult social interactions in rats
Behav. Brain Res.
Influence of gender and age on performance of rats in the elevated plus maze apparatus
Behav. Brain Res.
Influence of circadian phase and test illumination on pre-clinical models of anxiety
Physiol. Behav.
Body temperature and locomotor activity as marker rhythms of aging of the circadian system in rodents
Exp. Gerontol.
Role of resocialization and 5-HT1A receptor activation on the anxiogenic effects induced by isolation in the elevated plus-maze test
Physiol. Behav.
Validation of open–closed arm entries in an elevated plus-maze as a measure of anxiety in the rat
J. Neurosci. Methods
Effects of BOC-CCK-4 and L 365,260 on cortical 5-HT release in guinea-pigs on exposure to the elevated plus-maze
Neuropharmacology
Anxiogenic stimuli in the elevated plus-maze
Pharmacol. Biochem. Behav.
Locomotor response to an open field during C57BL/6J active and inactive phases: differences dependent on conditions of illumination
Physiol. Behav.
Social behavior and social motivation in adolescent rats: role of housing conditions and partner's activity
Physiol. Behav.
Daily activity and body temperature rhythms do not change simultaneously with age in laboratory mice
Physiol. Behav.
Cited by (55)
Three methods of behavioural testing to measure anxiety – A review
2024, Behavioural ProcessesAnxiety-related activity of ventral hippocampal interneurons
2022, Progress in NeurobiologyCitation Excerpt :When rats or mice are placed on the EPM and left to explore freely, the time spent exploring vs. avoiding open arms can be used as a measure of anxiety and the effects of anxiolytic drugs (Carobrez and Bertoglio, 2005; Cryan and Holmes, 2005; Walf and Frye, 2007). Repeated exposure of rodents to the EPM does not typically lead to substantial changes in anxiety behaviour or preference for the type of arms explored (Andrade et al., 2003; Schrader et al., 2018; Tucker and McCabe, 2017). In our study, we aimed to compare two groups of rats with the same genetic background but with distinct EPM exploration behaviours of the open arms, implying different anxiety levels of these two groups of rats (Hollis et al., 2015; Zalachoras et al., 2022).
Age-dependent changes in the medial prefrontal cortex and medial amygdala structure, and elevated plus-maze performance in the healthy male Wistar rats
2020, IBRO ReportsCitation Excerpt :However, Shoji and Miyakawa that have applied EPM on 2 to 25-month C57BL/6 J mice, did not report any significant impacts of aging on the percentages of the entries and time spent on open arms and anxiety (Shoji and Miyakawa, 2019). It should emphasize that various methodological differences include age, strain, or sex and number of the samples, and housing or testing conditions might lead to obtaining inconsistent findings in the EPM (Andrade et al., 2003; Nagy and Glaser, 1970). Although aging leads to the reduction of mPFC volume, it increased this parameter in the amygdala (MeA).
Malva sylvestris extract alleviates the astrogliosis and inflammatory stress in LPS-induced depression mice
2019, Journal of NeuroimmunologyA window into the brain: Tools to assess pre-clinical efficacy of biomaterials-based therapies on central nervous system disorders
2019, Advanced Drug Delivery Reviews