Fig. 1: Chronic MDZ exposures at early development altered the overall physical and brain size attributes.

A Schematic experiment timeline showing the dose-escalation regimen of midazolam treatment starting on postnatal (P) day 3 with 1 mg/kg to 10 mg/kg on P21, similar to the previous pilot study [16, 17]. Animals were introduced to behavioral assays on P28, P45, and P60. B The growth curve demonstrates that MDZ-exposed animals (orange) gained weight slower than control saline animals (grey) during the exposure period (P3 to P21). Data represented as Mean ± SD. C, D Body weight recorded on early development (C, P7, P14, P21, n = 117–142/group/timepoint), and later development (D, P28, P45, P60, n = 38–78/group/timepoint) significantly reduced body weight in MDZ-exposed animals at P14 to P21. Two-way ANOVA with Sidak correction for multiple comparisons. There was no significant difference between the control and MDZ-exposed animals at later stages, as determined by Two-Way ANOVA with Sidak correction for multiple comparisons. E Overall weight gained a percentage of the post-treatment period (P28 to P60) with a significant difference as determined by Student t-test with Welch’s correction (**p <0.01, n = 39–41/group). F, G MDZ-exposed animals display significantly lower brain weight (F) but higher brain:body weight ratio at P21 (G). In addition, brain:body weight ratio of MDZ animals also displays a higher ratio than saline at P21 (****p < 0.001, n = 8–30/group/time point). Two-Way ANOVA. All data were represented as mean ± SEM, except (B). Data points for all panels and details on sample size and statistical analyses are provided in Source Data and Statistical Supplemental data files.