Pharmacological modulation of anxiety-like behavior by flumazenil, midazolam, and ketamine in rats: Evidence from aversive ultrasonic vocalizations and elevated plus maze.
Marcela Unterspann, Chrysostomos Charalambous, Martin Votava, Ladislav Hess, Jiri Sliva, Marek Lapka
Pharmacology, biochemistry, and behavior May 25, 2026 DOI: 10.1016/j.pbb.2026.174216 (opens in new tab)
Study at a glance
AI-extracted from the abstract| Characteristics | Preclinical experimental study Peer reviewed |
|---|---|
| Population | Adult male Sprague Dawley and Wistar rats |
| Interventions | Flumazenil Ketamine Midazolam |
| Dose | Flumazenil 0.1-10 mg/kg, i.p.; ketamine 2.5 mg/kg, i.p.; midazolam 0.25 mg/kg, i.p. |
| Measures | Aversive 22-kHz ultrasonic vocalization (AUV), Elevated Plus Maze (EPM) |
| Topics | Anxiety Ketamine Esketamine |
| Keywords | Animals Rats, sprague-dawley Rats, wistar Flumazenil Midazolam Anti-anxiety agents Vocalization, animal Maze learning Dose-response relationship, drug Male Gaba-a receptors Ultrasonic vocalization |
| Key findings | Flumazenil reduced aversive ultrasonic vocalizations dose-dependently, indicating an anxiolytic-like effect in rats, while sub-anesthetic ketamine also reduced these vocalizations. Higher flumazenil doses increased vocalizations when combined with ketamine, and midazolam's reduction of vocalizations was partially attenuated by ketamine. The authors suggest flumazenil may modulate acute ketamine effects on anxiety-related behavior, but note this interaction needs further investigation. |
Abstract
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.
Comparable studies
Other preclinical and animal studies on ketamine for anxiety, most cited first.
| Study | Year | Design | Participants |
|---|---|---|---|
| Psychedelics, but Not Ketamine, Produce Persistent Antidepressant-like Effects in a Rodent Experimental System for the Study of Depression Rats | 2020 | Preclinical study | |
| Psilocin and ketamine microdosing: effects of subchronic intermittent microdoses in the elevated plus-maze in male Wistar rats Wistar rats | 2018 | Experimental study | n = 40 |
| Repeated Dosing of Ketamine in the Forced Swim Test: Are Multiple Shots Better Than One? Rodents | 2021 | Review | |
| Psychedelics: A review of their effects on recalled aversive memories and fear/anxiety expression in rodents Rodents | 2024 | Review | |
| Sexually Dimorphic Behavioral Profile in a Transgenic Model Enabling Targeted Recombination in Active Neurons in Response to Ketamine and (2R,6R)-Hydroxynorketamine Administration Arc-CreERT2 × CAG-Sun1/sfGFP mice | 2020 | Experimental study |