The Importance of Sex and Estrus as Biological Variables in Neuroscience Research

Females have been historically underrepresented in preclinical literature and sex differences remain underexplored in neuroscience, pharmacology, and behavioral research, particularly in rodent models (Beery and Zucker, 2011; Will et al., 2017; Mamlouk et al., 2020). A major reason for the omission of females is a concern over the natural fluctuations in gonadal hormone levels across their reproductive (estrus) cycle, which can affect behavior and neuroanatomy. This review presents an argument for examining sex and estrus stage as biological variables and discusses ways in which neurophysiological and behavioral data may (or may not) be impacted by females’ estrus stage. Evaluation of existing estrus staging protocols, approaches to minimize potential variables, and ways to optimize estrus reliability and replicability in undergraduate research experiences are presented as well. Finally, we included a small pilot study during laboratory members’ training to assess the inter- and intra-rater reliability of estrus identifications and further refine our protocol. We discuss the importance of training and education in undergraduate research laboratories that use preclinical models, as well as potential implications related to equity and the reproducibility crisis.

Abbreviations: DA – dopamine, CPP – conditioned place preference, FSH – follicle-stimulating hormone, GnRH – gonadotropin-releasing hormone, LH – luteinizing hormone, MOR – mu-opioid receptor, Nac – nucleus accumbens, VTA – ventral tegmental area

Keywords: female, sex difference, estrus cycle, mice, behavior, protocol, undergraduate research

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