Effects of pretest manipulation on elevated plus-maze behavior in adolescent and adult male and female Sprague–Dawley rats

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Abstract

The elevated plus-maze (EPM) is vulnerable to variations in pretest circumstances when testing adult rodents. Because of an increasing interest in adolescence, the present experiments examined the impact of pretest manipulations on anxiety levels in the EPM among adolescent and adult Sprague–Dawley rats of both sexes. In Exp. 1, animals removed from their home cage and immediately placed on the EPM were compared to rats tested following 30 min of social isolation, or following 30-min exposure to a novel context. These pretest manipulations only modestly decreased anxiety levels at both ages. In Exp. 2, more varied pretest conditions were examined: testing directly from the home cage; testing following 30 min of social isolation in a novel environment; or a large saline injection and rehousing 18 h prior to a 30-min period of social isolation in a novelty situation before testing. In adults, anxiety levels decreased linearly as pretest perturbation increased, whereas adolescents showed comparable levels of anxiety with both the moderate and large perturbations. As a result, observed age differences in anxiety differed as a function of pretest circumstances. Therefore, caution is urged when using the EPM for across-age comparisons of anxiolytic and anxiogenic effects of pharmacological or other manipulations.

Introduction

The elevated plus-maze (EPM) is a widely used model for the study of anxiety-like behavior in rodents. This plus-shaped apparatus is elevated from the floor and consists of two arms with walls and two arms that are open platforms. Since the maze presents a conflict of motivation to explore the novel yet risky open arms against the motivation to remain safe in the enclosed arms, the proportion of time an animal spends in the open versus closed arms is believed to provide an index of that animal's anxiety level (for reviews see Carobrez and Bertoglio, 2005, Wall and Messier, 2001).

Substantial prior research has focused on validating the EPM as a behavioral assay of anxiety in adult rodents. Pharmacological studies have documented that anxiolytic drugs (e.g. diazepam) effectively increase the proportion of time animals spend in the open relative to the closed arms, reflecting decreased anxiety in these animals (Pellow et al., 1985, Wilson et al., 2004). In contrast, anxiogenic drugs (e.g. pentylenetetrazol) have been shown to have the opposite effect, with animals increasing the amount of time spent in the closed arms and avoiding the open arms to a greater extent (Wada and Fukuda, 1991, Wallis and Lal, 1998). Moreover, drugs that should not impact anxiety levels, such as haloperidol, have been shown to decrease overall locomotor activity on the maze while not affecting percentage of open arm activity (Pellow et al., 1985). File and colleagues further validated the EPM in terms of the hormonal responses to fearful and anxiety-provoking situations. They found that adult animals had much greater corticosterone (CORT) response following confinement to open arms than to closed arms in the EPM (Pellow et al., 1985), with other researchers also demonstrating similar increases in CORT levels following open arm confinement (Degroot et al., 2004, McCormick et al., 2008). These increases in CORT have been taken as evidence of increased anxiety.

Within the field of psychology, there has been increasing emphasis on adolescence as a time of notable increases in expression of mental health disorders including schizophrenia, substance abuse and depression (see Kessler et al., 2005 for review). Ontogenetic alterations are also seen in the expression of specific anxiety disorders and in the efficacy of their treatment with different classes of anxiolytic drugs (Foa et al., 2005). Characterized by the transition from dependence to independence, this ontogenetic phase is associated with numerous conserved neural, behavioral and hormonal features that are evident not only in human adolescents, but in organisms undergoing this transition in other species as well (Spear, 2000). These across-species commonalities in basic neurochemical and hormonal characteristics of adolescence provide support for the judicious use of animal models of adolescence, particularly under circumstances where such research with human youths would be difficult or unethical. In the rat, adolescence has been conservatively defined as postnatal days (P) 28 to 42 (Spear, 2000), although the exact onset and end of adolescence is a gradual one. During this period, adolescent rats (like their human counterparts) show an increased prevalence of behaviors such as risk-taking, novelty seeking and peer-directed social behaviors (for reviews see Adriani et al., 2004, Spear, 2000). The expression of these behaviors, combined with the challenging transitions that adolescents face (e.g. emigration from the family unit and establishment of mature relationships with peers), has lead to the hypothesis that adolescence may be a relatively stressful phase characterized by heightened anxiety and greater stress reactivity than seen among younger or more mature individuals (e.g. Spear, 2000, Walker et al., 2001).

To answer the question as to whether adolescents are more anxious than adults, and to explore the potential efficacy of novel anxiolytics among adolescents, researchers have primarily turned to the EPM and other well-established anxiety models developed and validated for use in adult rodents. Using these models, findings regarding ontogenetic changes in basal anxiety levels have been quite inconsistent, with adolescents reported to exhibit more (Hascoet et al., 1999, Slawecki, 2005), less (Imhof et al., 1993) or similar levels of anxiety as adults (Slawecki and Roth, 2004, Varlinskaya and Spear, 2002, Varlinskaya and Spear, 2008). While some of these inconsistencies may be related to differences across anxiety models (e.g. the light-dark box versus the EPM), a comparison of the results obtained within studies using the EPM alone have still yielded notable inconsistencies. Several EPM studies have reported that younger animals are more anxious than older animals (e.g. Doremus et al., 2003), whereas others have reported the opposite (e.g. Andrade et al., 2003, Imhof et al., 1993), or no age differences (e.g. Hefner and Holmes, 2007, Walker et al., 2004). Even within our own laboratory, comparison of control non-drug-exposed animals across different studies sometimes have yielded disparate conclusions regarding age-related differences in anxiety-like behavior (Doremus-Fitzwater and Spear, 2007, Doremus-Fitzwater et al., 2006, Doremus et al., 2003).

If tests such as the EPM are to be used to probe anxiety-related behaviors during adolescence, they should be validated for use with adolescents. As part of this process, factor analyses of several EPM data sets collected within our laboratory revealed primary underlying components of EPM behavior that were similar in both adolescents and adults, with anxiety-like measures consistently loading on the first factor and activity on the second (Doremus et al., 2006). While these results support the conclusion that similar constructs are being measured in adolescents and adults in this test, other issues also need to be addressed. Of particular importance, there is substantial evidence that basal anxiety levels in adults are sensitive to manipulations occurring prior to EPM testing (for reviews see Carobrez and Bertoglio, 2005, Hogg, 1996, Rodgers and Dalvi, 1997). To give but a few examples, studies have shown that pretest perturbations such as handling (Schmitt and Hiemke, 1998), transportation on a cart (Morato and Brandao, 1996) and housing condition (Haller and Halasz, 1999, Schmitt and Hiemke, 1998) each alter EPM anxiety levels among adults. Furthermore, results from our lab have suggested that the susceptibility of anxiety levels to be affected by pretest manipulations may vary with age, with, for instance a 5-min holeboard exposure prior to the EPM test differentially impacting anxiety levels in adolescents compared to adults (Doremus-Fitzwater and Spear, 2007). Effects of ontogenetic differences in response to pretest manipulations have not been systematically investigated, however, despite potential implications of age differences in pretest lability for conclusions drawn in across-age studies of anxiety-related indices in the EPM. Therefore, the purpose of the present study was to systematically explore the influence of pretest perturbation on anxiety-like behavior indexed in the EPM in both adolescent and adult rats.

Section snippets

General methods

At all times, animals used in the current experiments were maintained and treated in accordance with the guidelines for animal care established by the National Institutes of Health (Institute of Laboratory Animal Resources, Commission on Life Sciences, 1996), using protocols approved by the Binghamton University Institutional Animal Care and Use Committee (IACUC). In order to reduce the impact of litter effects, no more than one male and one female animal from a given litter was placed into any

Discussion

When the effects of mild pretest manipulations immediately prior to EPM testing were assessed in Exp. 1, both novelty exposure and, to a lesser extent, social isolation resulted in a modest, but significant reduction in anxiety-like behavior regardless of age or sex. When EPM data collected across a variety of experiments were examined, however, it seemed that more extreme levels of pretest perturbation resulted in more dramatic alterations in anxiety levels relative to animals tested directly

Acknowledgements

Supported by grants R01 DA019071, R37 AA12525 and R01 AA01735501 to Linda Spear and R01 AA12453 to Elena Varlinskaya.

References (59)

  • FernandesC. et al.

    The influence of open arm ledges and maze experience in the elevated plus-maze

    Pharmacol Biochem Behav

    (1996)
  • FryeC.A. et al.

    Estrous cycle and sex differences in performance on anxiety tasks coincide with increases in hippocampal progesterone and 3alpha,5alpha-THP

    Pharmacol Biochem Behav

    (2000)
  • HallerJ. et al.

    Acute effects of glucocorticoids: behavioral and pharmacological perspectives

    Neurosci Biobehav Rev

    (1998)
  • HascoetM. et al.

    Influence of age on behavioural response in the light/dark paradigm

    Physiol Behav

    (1999)
  • HefnerK. et al.

    Ontogeny of fear-, anxiety- and depression-related behavior across adolescence in C57BL/6J mice

    Behav Brain Res

    (2007)
  • HoggS.

    A review of the validity and variability of the elevated plus-maze as an animal model of anxiety

    Pharmacol Biochem Behav

    (1996)
  • HolsonR.R. et al.

    Principles and pitfalls in the analysis of prenatal treatment effects in multiparous species

    Neurotoxicol Teratol

    (1992)
  • ImhofJ.T. et al.

    Influence of gender and age on performance of rats in the elevated plus maze apparatus

    Behav Brain Res

    (1993)
  • JohnstonA.L. et al.

    Sex differences in animal tests of anxiety

    Physiol Behav

    (1991)
  • LalH. et al.

    Anxiogenic behavior in rats during acute and protracted ethanol withdrawal: reversal by buspirone

    Alcohol

    (1991)
  • LucionA.B. et al.

    Influence of early postnatal gonadal hormones on anxiety in adult male rats

    Physiol Behav

    (1996)
  • MarcondesF.K. et al.

    Estrous cycle influences the response of female rats in the elevated plus-maze test

    Physiol Behav

    (2001)
  • McCormickC.M. et al.

    Effects of chronic social stress in adolescence on anxiety and neuroendocrine response to mild stress in male and female rats

    Behav Brain Res

    (2008)
  • MoraS. et al.

    Effects of the estrous cycle and ovarian hormones on behavioral indices of anxiety in female rats

    Psychoneuroendocrinology

    (1996)
  • PalanzaP.

    Animal models of anxiety and depression: how are females different

    Neurosci Biobehav Rev

    (2001)
  • PellowS. et al.

    Validation of open:closed arm entries in an elevated plus-maze as a measure of anxiety in the rat

    J Neurosci Methods

    (1985)
  • RodgersR.J. et al.

    Anxiety, defence and the elevated plus-maze

    Neurosci Biobehav Rev

    (1997)
  • SchmittU. et al.

    Strain differences in open-field and elevated plus-maze behavior of rats without and with pretest handling

    Pharmacol Biochem Behav

    (1998)
  • SpearL.P.

    The adolescent brain and age-related behavioral manifestations

    Neurosci Biobehav Rev

    (2000)
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