Diazepam causes sedative rather than anxiolytic effects in C57BL/6J mice

Abstract

Diazepam has been broadly accepted as an anxiolytic drug and is often used as a positive control in behavioral experiments with mice. However, as opposed to this general assumption, the effect of diazepam on mouse behavior can be considered rather controversial from an evidence point of view. Here we revisit this issue by studying the effect of diazepam on a benchmark task in the preclinical anxiety literature: the elevated plus maze. We evaluated the minute-by-minute time-course of the diazepam effect along the 10 min of the task at three different doses (0.5, 1 and 2 mg/kg i.p. 30 min before the task) in female and male C57BL/6J mice. Furthermore, we contrasted the effects of diazepam with those of a selective serotoninergic reuptake inhibitor (paroxetine, 10 mg/kg i.p. 1 h before the task). Diazepam had no anxiolytic effect at any of the tested doses, and, at the highest dose, it impaired locomotor activity, likely due to sedation. Noteworthy, our results held true when examining male and female mice separately, when only examining the first 5 min of the task, and when animals were subjected to one hour of restrain-induced stress prior to diazepam treatment. In contrast, paroxetine significantly reduced anxiety-like behavior without inducing sedative effects. Our results therefore suggest that preclinical studies for screening new anxiolytic drugs should be cautious with diazepam use as a potential positive control.

Figure 1

Lack of anxiolytic effect of diazepam in the elevated plus maze. (A) (Left) Cumulative number of entries in the open arms along the 10-min session. (Middle and right) Mean (± SD) number of open arm entries after 5 (middle) and 10 min (right). White circles show data for individual animals. Animals were injected i.p. with vehicle or three doses of diazepam (0.5, 1.0 and 2.0 mg/kg) thirty minutes prior to behavior testing, as labeled. (B–D) Panels show the same as in (A), but for the percentage of the time spent in the open arms (B), and the total time animals spent moving (C) or in stretch-attend posture (D). No anxiolytic effect of diazepam is found in any of the doses, as inferred by no increase in the metrics shown in (A) and (B) (on the contrary, the highest diazepam dose decreased open arm exploration). Notice further that diazepam induces both a reduction in locomotor activity (C) and risk assessment behaviors (D). **p < 0.001, ***p < 0.0001, one-way ANOVA followed by Tukey’s post hoc test.

Figure 2

Lack of anxiolytic effect of diazepam in restraint-stressed animals. (A) (Left) Cumulative number of entries in the open arms of the maze along the 10-min session. (Middle and right) Mean (± SD) number of open arm entries after 5 (middle) and 10 min (right). White circles show data for individual animals. Animals were subjected to 1-h restraint-stress and then injected i.p. with vehicle or three doses of diazepam (0.5, 1.0 and 2.0 mg/kg) thirty minutes prior to behavior testing, as labeled. (B–D) Panels show the same as in (A), but for the percentage of the time spent in the open arms (B), and the total time animals spent moving (C) or in stretch-attend posture (D). Diazepam exhibited no statistically significant anxiolytic effect in this protocol, nor influenced locomotor activity. As in non-restrained animals (Fig. 1D), diazepam dose-dependently reduced the number of risk assessment behavior shown as reduced time in stretch-attend posture. *p < 0.01, **p < 0.001, ***p < 0.0001, one-way ANOVA followed by Tukey’s post hoc test. In A and B, outliers above the y-axis limit are not shown.

Figure 3

Anxiolytic effects of paroxetine in the elevated plus maze. (A) (Left) Cumulative number of entries in the open arms of the maze along the 10-min session. (Middle and right) Mean (± SD) number of open arm entries after 5 (middle) and 10 min (right). White circles show data for individual animals. Animals were treated with vehicle or paroxetine (10 mg/kg) 1 h prior to behavioral testing. (B–D) Panels show the same as in (A), but for the percentage of the time spent in the open arms (B), and the total time animals spent moving (C) or in stretch-attend posture (D). Paroxetine-treated animals exhibited a pronounced anxiolytic-like behavioral profile along with no change in locomotor activity and an increase in risk assessment behavior shown as increased time in stretch-attend posture. **p < 0.001, ***p < 0.0001, two-sample t-test.

Figure 4

Diazepam effects in the open field. (A) (Left) Cumulative distance moved in the open field. (Middle and right) Mean (± SD) distance after 5 (middle) and 10 min (right). White circles show data for individual animals. Animals were injected i.p. with vehicle or three doses of diazepam (0.5, 1.0 and 2.0 mg/kg) thirty minutes prior to behavior testing, as labeled. (B–D) As before, but for the time spent moving (B), number of entries in the center zone (C) and time spent there (D). Diazepam reduced locomotor activity and the number of entries in the center zone. *p < 0.01, ***p < 0.0001, one-way ANOVA followed by Tukey’s post hoc test.

Figure 5

Paroxetine effects in the open field. (A) (Left) Cumulative distance moved in the open field. (Middle and right) Mean (± SD) distance after 5 (middle) and 10 min (right). White circles show data for individual animals. Animals were treated with vehicle or paroxetine (10 mg/kg) 1 h prior to behavioral testing. (B–D) As before, but for the time spent moving (B), number of entries in the center zone (C) and time spent there (D). Paroxetine increased locomotor activity and did not majorly influence the exploration of the center zone. ^#p < 0.05, *p < 0.01, two-sample t-test.