Role of neurosteroids in the anticonvulsant activity of midazolam

Abstract

Background and purpose: Midazolam is a short-acting benzodiazepine that is widely used as an i.v. sedative and anticonvulsant. Besides interacting with the benzodiazepine site associated with GABA(A) receptors, some benzodiazepines act as agonists of translocator protein (18 kDa) (TSPO) to enhance the synthesis of steroids, including neurosteroids with positive modulatory actions on GABA(A) receptors. We sought to determine if neurosteroidogenesis induced by midazolam contributes to its anticonvulsant action.

Experimental approach: Mice were pretreated with neurosteroid synthesis inhibitors and potentiators followed by midazolam or clonazepam, a weak TSPO ligand. Anticonvulsant activity was assessed with the i.v. pentylenetetrazol (PTZ) threshold test.

Key results: Midazolam (500-5000 µg·kg(-1) , i.p.) caused a dose-dependent increase in seizure threshold. Pretreatment with the neurosteroid synthesis inhibitors finasteride, a 5α-reductase inhibitor, and a functional TSPO antagonist PK 11195, reduced the anticonvulsant action of midazolam. The anticonvulsant action of midazolam was enhanced by the neurosteroidogenic drug metyrapone, an 11β-hydroxylase inhibitor. In contrast, the anticonvulsant action of clonazepam (100 µg·kg(-1) ) was reduced by finasteride but not by PK 11195, indicating a possible contribution of neurosteroids unrelated to TSPO.

Conclusion and implications: Enhanced endogenous neurosteroid synthesis, possibly mediated by an interaction with TSPO, contributed to the anticonvulsant action of midazolam. Enhanced neurosteroidogenesis may also be a factor in the actions of other benzodiazepines, even those that only weakly interact with TSPO.

Figure 1

Drug treatment protocols. In some experiments, clonazepam (25 or100 µg·kg⁻¹, i.p.) was used instead of midazolam.

Figure 2

Dose–response relationship for midazolam protection against tonic extension in response to i.v. PTZ infusion in mice. Midazolam was administered i.p. 15 min before the beginning of the PTZ infusion. Data points indicate mean ± SEM of threshold values from six to eight mice normalized with respect to the mean threshold value in the vehicle-treated control group, which was 51.5 ± 4.0 mg·kg⁻¹. Dashed lines indicate the limits of the SEM for the vehicle group. The mean threshold values for all groups other than the 100 µg·kg⁻¹ group are significantly different from the value in the vehicle group. *P < 0.001; one-way anova followed by Tukey's test.

Figure 3

Finasteride pretreatment reduces the seizure threshold elevation induced by midazolam. Finasteride (100 mg·kg⁻¹, i.p.) or vehicle was administered 5 min before the treatment with midazolam (500 µg·kg⁻¹, i.p.) or vehicle; 15 min after the second pretreatment, all animals were infused with PTZ. Bars indicate mean ± SEM of fractional threshold change values for tonic extension from 6–14 mice normalized with respect to the mean threshold value in the vehicle only control group, which was 59.2 ± 1.8 mg·kg⁻¹. In the absence of finasteride, midazolam caused a 2.3-fold increase in threshold (P < 0.001). Finasteride did not reduce the threshold significantly in the absence of midazolam (NS) but did reduce the threshold with midazolam pretreatment. *P < 0.001; one-way anova followed by Tukey's test.

Figure 4

Metyrapone elevates seizure threshold in the absence and presence of midazolam. Metyrapone (100 mg·kg⁻¹, i.p.) or vehicle was administered 15 min before treatment with midazolam or vehicle; 15 min after the second pretreatment, all animals were infused with PTZ. Bars indicate mean ± SEM of fractional change values in tonic extension threshold from six to nine mice normalized with respect to the mean threshold value in the vehicle only control group, which was 51.0 ± 4.4 mg·kg⁻¹. *P < 0.001, significantly different from vehicle only control group; •P < 0.001, significantly different from midazolam only group; one-way anova followed by Tukey's test.

Figure 5

PK 11195 pretreatment reduces the seizure threshold elevation induced by midazolam. PK11195 (15 mg·kg⁻¹, i.p.) was administered 30 min before the treatment with midazolam (500 µg·kg⁻¹, i.p.) or vehicle; 15 min after the second pretreatment, all animals were infused with PTZ. Bars indicate mean ± SEM of fractional threshold change values for tonic extension from 6–12 mice normalized with respect to the mean threshold value in the vehicle only control group (same as Figure 4). *P < 0.001, significantly different from vehicle only control group; •P < 0.001, significantly different from midazolam only group; one-way anova followed by Tukey's test.

Figure 6

Finasteride (upper panel) but not PK 11195 (lower panel) pretreatment reduces the seizure threshold elevation induced by clonazepam. Finasteride (100 mg·kg⁻¹, i.p.) or vehicle was administered 5 min before the treatment with clonazepam (100 µg·kg⁻¹, i.p.) or vehicle; 15 min after the second pretreatment, all animals were infused with PTZ. In the absence of finasteride, clonazepam caused a 2.8-fold increase in threshold (P < 0.001). Finasteride did not reduce the threshold significantly in the absence of midazolam (NS) but did reduce the threshold with clonazepam pretreatment (P < 0.001). PK11195 (15 mg·kg⁻¹, i.p.) was administered 30 min before the treatment with clonazepam or vehicle; 15 min after the second pretreatment, all animals were infused with PTZ. Bars indicate mean ± SEM of fractional threshold change values for tonic extension from 6–13 mice normalized with respect to the mean threshold value in the vehicle only control group, which was 46.9 ± 3.6 mg·kg⁻¹ in the experiment with finasteride and 51.9 ± 2.7 mg·kg⁻¹ in the experiment with PK 11195. *P < 0.001, significantly different from vehicle only control group; •P < 0.001, significantly different from clonazepam only group; one-way anova followed by Tukey's test.