Anxiety- and stress-related disorders are commonly treated using combinations of centrally acting drugs. However, the behavioral outcomes of interactions between GABAergic and glutamatergic systems remain incompletely characterized. The present study investigated the effects of flumazenil, ketamine and midazolam, and their combinations, on anxiety-like behavior in adult male rats using aversive 22-kHz ultrasonic vocalization (AUV) and the elevated plus maze (EPM). Systemic administration of flumazenil (0.1-10 mg/kg, i.p.) produced a significant, dose-dependent reduction of AUV in Sprague Dawley rats, indicating intrinsic anxiolytic-like effects. Sub-anesthetic ketamine (2.5 mg/kg, i.p.) reduced AUV when administered alone in Wistar rats; however, co-administration with flumazenil was associated with dose-related changes in this effect, with higher flumazenil doses producing significant increases in AUV related to ketamine alone. Midazolam (0.25 mg/kg, i.p.) markedly reduced AUV in Wistar rats, whereas its combination with ketamine partially attenuated this reduction. In the EPM, midazolam alone increased open-arm exploration in Wistar rats, while co-administration with ketamine increased closed-arm time and reduced locomotor activity. Together, these findings highlight the behavioral consequences of interactions between ketamine and midazolam and suggest flumazenil as a modulator of acute ketamine-induced effects on anxiety-related behavior, although this interaction requires further investigation.

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