Beyond control: experimental control protocol slightly affects prepubescent mice behavior in a sex-specific manner

Study design and experimental tools are crucial for good quality science, and an important part of it is the choice of control groups to best test the hypothesis. Two of the most common control groups in research are needle prickling without substance injection (Sham) and/or vehicle injection (Saline). Very few studies investigated the behavioral effects of a single intraperitoneal (i.p) Sham and/or single i.p. Saline administration in mice, mostly using adult males. In this study, we investigated if a single i.p. Sham and/or i.p. Saline administration affects female and male prepubertal (4-weeks-old) mice behavior. After Sham or Saline injection, we examined exploratory/motor behavior (open field test - OFT), anxiety-like behavior (elevated plus-maze - EPM), and behavioral despair/depressive-like behavior (forced swimming test - FST). We observed that both Sham prepubertal females and males showed minor behavioral alterations in OFT and EPM, and Saline males showed behavioral alterations in OFT and FST. On the other hand, prepubertal Saline females showed an increase in exploratory behavior, risk assessment/anxiety-like behavior, and behavioral despair/depressive-like behavior. Thus, we observed that the experimental procedures used as control have mild (but not insignificant) consequences on prepubescent mice behavior. However, the consequences of control protocols in prepubescent females are stronger than in males. To the best of our knowledge, this is the first study investigating the consequences of Sham and Saline protocols in prepubertal mice. This study highlights how important is to include Naive animals (Negative control) together with Sham and/or Vehicle for a better and more honest interpretation of the data.