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Testing Animal Anxiety in Rats: Effects of Open Arm Ledges and Closed Arm Wall Transparency in Elevated Plus Maze Test

The elevated plus maze test is a behavioral test for assessing animal anxiety in rodents. Although this test is widely applied in the field of behavioral ...

Meiji University

The elevated plus maze (EPM) is an established method for testing animal anxiety. However, EPM apparatuses and their features can differ among laboratories, most notably in the presence/absence of ledges on the open arm and/or the transparency/opaqueness of walls on the closed arm. The combined effects of these variable arm features on EPM behavior are not yet fully understood. In the present study, we prepared four types of EPM apparatus - open arms with (0.5 cm) or without (0 cm) ledges×closed arms with transparent or opaque walls - and compared the maze-exploration behavior of male Sprague-Dawley rats. We found that the presence of open arm ledges significantly increased the incidence of open arm exploration. Furthermore, time spent in the distal segment of the open arm was shortest in the apparatus that had open arms with no ledges and opaque closed arms (No-Ledges/Opaque), and was longest in the apparatus that had open arms with ledges and transparent closed arms (Ledges/Transparent). Additionally, the No-Ledges/Opaque apparatus could detect the effect of 0.5mg/kg diazepam, an anxiolytic drug, whereas the Ledges/Transparent apparatus could not. These results indicate that arm structure (features of both open and closed arms) significantly influences maze-exploratory behavior in rats, and that No-Ledges/Opaque apparatuses have higher detection sensitivity for anxiolytic effects of diazepam than that of Ledges/Transparent apparatuses.

Higher detection sensitivity of anxiolytic effects of diazepam by ledge-free open arm with opaque walled closed arm elevated plus maze in male rats.

Although perinatal exposure of female rats to estrogenic compounds produces irreversible changes in brain function, it is still unclear how the amount and timing of exposure to those substances affect learning function, or if exposure alters estrogen receptor α (ERα) expression in the hippocampus and cortex. In adult female rats, we investigated the effects of neonatal exposure to a model estrogenic compound, ethinyl estradiol (EE), on passive avoidance learning and ERα expression. Female Wistar-Imamichi rats were subcutaneously injected with oil, 0.02 mg/kg EE, 2 mg/kg EE, or 20 mg/kg 17β-estradiol within 24 h after birth. All females were tested for passive avoidance learning at the age of 6 weeks. Neonatal 0.02 mg/kg EE administration significantly disrupted passive avoidance compared with oil treatment in gonadally intact females. In a second experiment, another set of experimental females, treated as described above, was ovariectomized under pentobarbital anesthesia at 10 weeks of age. At 15-17 weeks of age, half of each group received a subcutaneous injection of 5 μg estradiol benzoate a day before the passive avoidance learning test. Passive avoidance learning behavior was impaired by the 0.02 mg/kg EE dose, but notably only in the estradiol benzoate-injected group. At 17-19 weeks of age, hippocampal and cortical samples were collected from rats with or without the 5 μg estradiol benzoate injection, and western blots used to determine ERα expression. A significant decrease in ERα expression was observed in the hippocampus of the estradiol-injected, neonatal EE-treated females. The results demonstrated that exposure to EE immediately after birth decreased learning ability in adult female rats, and that this may be at least partly mediated by the decreased expression of ERα in the hippocampus. 

A Single Neonatal Injection of Ethinyl Estradiol Impairs Passive Avoidance Learning and Reduces Expression of Estrogen Receptor α in the Hippocampus and Cortex of Adult Female Rats.

Correct perinatal oestrogen levels are critical for sexual differentiation. For example, perinatal exposure to oestrogen causes masculinization and defeminization of the brain in female rats and also induces delayed effects after maturation characterized by early onset of abnormal oestrus cycling. However, the mechanisms underlying the above effects of oestrogen remain to be fully determined. 17α-ethinyloestradiol (EE), a common synthetic oestrogen widely used in oral contraceptives, binds specifically to oestrogen receptors. In this study, we demonstrated the effects of a single neonatal injection of high- or low-dose EE on reproductive behaviours. Female rats within 24 h after birth were subcutaneously injected with sesame oil, EE (0.02, 2 mg kg ) and 17β-oestradiol (E ) (20 mg kg ). Between 11 and 15 weeks of age, sexual behaviour was tested twice in a paced mating situation. Latency to enter, lordosis and soliciting behaviour were recorded. Both high-dose EE- and E -treated females showed a significantly lower lordosis quotient, decreased soliciting behaviours, increased rejection and fighting numbers. Accessibility to males was also delayed by neonatal E exposure, although it was shortened by high-dose EE exposure. In contrast, low-dose EE-treated females did not exhibit impaired sexual behaviour. These results suggest that single neonatal exposure to a high dose of EE or E disturbs the normal development of the female brain, resulting in impaired sexual behaviours in a female-paced mating situation. Besides, the differences noted between high-dose EE- and E -treated females might be caused by different affinities of the oestrogen receptors, metabolic rates or mechanisms of action. Copyright © 2017 John Wiley & Sons, Ltd.

Effects of neonatal 17α-ethinyloestradiol exposure on female-paced mating behaviour in the rat.

Exposure to endocrine-disrupting chemicals may adversely affect animals, particularly during development. Tris(1,3-dichloroisopropyl) phosphate (TDCIPP) is an organophosphate with anti-androgen function in vitro that is present in indoor dust at relatively high concentrations. In male rats, androgens are necessary for the development of reproductive organs, as well as the endocrine and central nervous systems. However, we currently do not know the exact effects of TDCIPP exposure through suckling on subsequent reproductive behavior in males. Here, we show that TDCIPP exposure (25-250 mg kg via oral administration over 28 consecutive days post-birth) suppressed male sexual behavior and reduced testes size. These changes were dose-dependent and appeared first in adults rather than in juveniles. These results demonstrate that TDCIPP exposure led to normal body growth and appearance in juveniles, but disrupted the endocrine system and physiology in adults. Therefore, assays should be performed using adult animals to ensure accuracy, and to confirm the influence of chemical substances given during early mammalian life.

Early-life exposure to Tris(1,3-dichloroisopropyl) phosphate induces dose-dependent suppression of sexual behavior in male rats.

Two inbred strains of Sprague-Dawley rats, known as the Hatano high- and low-avoidance animals (HAA and LAA respectively), have been selectively bred for high versus low rates of avoidance responses in a shuttle-box avoidance task. To investigate differences in the sexual behavior of Hatano rats, male HAA, LAA and SD rats were tested from 12 to 15 weeks of age. LAA rats exhibited more rapid and frequent sexual behavior than HAA or SD rats, and such differences increased with repeated sexual experience. Plasma testosterone levels tended to be lower in LAA rats than in HAA or SD rats, suggesting that active sexual behavior in LAA rats is not related to these levels. Strain differences in mating behavior between HAA and LAA rats may be caused by emotional responses to novelty.

Maiko Kawaguchi

Laboratory of Animal Behavior and Environmental Science,
School of Agriculture

Meiji University

Testing Animal Anxiety in Rats: Effects of Open Arm Ledges and Closed Arm Wall Transparency in Elevated Plus Maze Test

Maiko Kawaguchi

Laboratory of Animal Behavior and Environmental Science,School of Agriculture

Meiji University

Testing Animal Anxiety in Rats: Effects of Open Arm Ledges and Closed Arm Wall Transparency in Elevated Plus Maze Test

Laboratory of Animal Behavior and Environmental Science,
School of Agriculture