Absence of repeated-trial tolerance to the anxiolytic-like effects of chlordiazepoxide in the rat triple test

The triple test, recently developed to assess anxiety-related behaviors in rodents, combines three widely used behavioral tests: the open field (OF), elevated plus maze (EPM) and light/dark box (LDB). The EPM and LDB, individually, are normally sensitive to the anxiolytic effects of benzodiazepines only in the first trial, due to the phenomenon of one-trial tolerance, which limits their use in longitudinal studies. The main objective of the present investigation was to verify whether the anxiolytic-like effects of chlordiazepoxide (CDZ), previously observed in naive animals submitted to the triple test, would persist after repeated testing. To this end, three experiments were carried out where male Wistar rats received CDZ (10 mg/kg) 30 min before the triple test for 2, 3 or 20 consecutive days. Except for the first day of drug treatment following a previous test experience in an undrugged state, CDZ had enduring anxiolytic-like effects under all schedules, promoting an increase in the exploration of the EPM open arms (and in some cases of the white compartment of the LDB), without affecting the number of closed-arm entries. The finding that rats did not develop tolerance to CDZ even with chronic treatment and repeated exposures to the triple test suggests that this new device is a promising tool to be used in longitudinal studies involving pharmacological manipulations of anxiety-related behaviors.

Research Highlights
► The new triple test is sensitive to the anxiolytic effects of chlordiazepoxide.
► The triple test provides anxiety measures from different devices simultaneously.
► The triple test does not present one-trial tolerance.
► The triple test can be useful for longitudinal studies using chronic drug treatment.