AbstractThe benzodiazepine midazolam is widely used in critical care medicine. Midazolam has a clinically active metabolite, 1-hydroxymidazolam. The contribution of 1-hydroxymidazolam to the effects of midazolam is controversial. The aim of the current study was to compare the actions of midazolam and 1-hydroxymidazolam on network activity of cortical neurons. Midazolam depressed neuronal activity at a low concentration of 5 nM. When midazolam concentration was increased, it depressed neuronal discharge rates in a biphasic manner. In comparison, 1-hydroxymidazolam did not depress the cortical network activity at low nanomolar concentrations. Higher concentrations of 1-hydroxymidazolam consistently inhibited neuronal activity. Moreover, midazolam shortened cortical up states at low, but not at high concentrations, while the opposite effect was observed with 1-hydroxymidazolam. The network depressant action of midazolam at low concentrations was absent in slices from GABA_A receptor α₁(H101R)mutant mice. The α₁(H101R)mutation renders α₁-subunit containing GABA_A receptors insensitive towards benzodiazepines. This GABA_A receptor subtype is thought to mediate sedation. As midazolam is more potent than its metabolite 1-hydroxymidazolam, the major clinical effects are thus likely caused by midazolam itself. However, 1-hydroxymidazolam could add to the effects of midazolam, especially after the application of high doses of midazolam, and in case of impaired drug metabolism.