Direct and Indirect Measurements of Sex Hormones in Rodents During Fear Conditioning

Antonio Florido, Laura Perez-Caballero, Eric Raúl Velasco, Patricia Molina, Ignacio Marin-Blasco, Raül Andero

Research output: Contribution to journalArticleResearchpeer-review


Fear conditioning (FC) is a widely accepted tool for the assessment of learning and memory processes in rodents related to normal and dysregulated acquired fear. The study of sex differences in fear learning and memory is vast and currently increasing. Sex hormones have proven to be crucial for fear memory formation in males and females, and several methods have been developed to assess this hormonal state in rats and mice. Herein, we explain a routine FC and extinction protocol, together with the evaluation of sex hormonal state in male and female rodents. We explain three protocols for the evaluation of this hormonal state directly from blood samples extracted during the procedure or indirectly through histological verification of the estrous cycle for females or behavioral assessment of social hierarchies in males. Although females have typically been considered to present great variability in sex hormones, it is highlighted that sex hormone assessment in males is as variable as in females and equally important for fear memory formation. The readout of these protocols has had a great impact on different fields of fear learning and memory study and appears essential when studying FC. The proven interaction with drugs involved in the modulation of these processes makes sex hormone assessment during FC a valuable tool for the development of effective treatments for fear-related disorders in men and women.

Original languageEnglish
Pages (from-to)e102
JournalCurrent Protocols
Issue number5
Publication statusPublished - 1 May 2021


  • blood extraction
  • confrontation tube test
  • fear conditioning
  • sex hormones
  • vaginal smear cytology


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