Developmental Biology

Ex vivo culture assays of biopsy specimens are advantageous for the experimental evaluation of human circadian characteristics. We developed a simple and non-invasive experimental evaluation method for monitoring the expression of circadian clock genes in an ex vivo culture assay using human hair follicles. This method imposes little burden on subjects. This assay is useful for validating correlations between circadian characteristics in hair follicles and intrinsic characteristics observed in physiological and behavioral studies. While they should be further validated, this ex vivo method constitutes a useful tool for estimating in vivo circadian characteristics.

C. elegans sleep during development is regulated by genes and cellular mechanisms that are conserved across the animal kingdom (Singh et al., 2014; Trojanowski and Raizen, 2016). C. elegans developmental sleep is usually assessed during the transition to adulthood, a 2.6 h time interval called lethargus (Raizen et al., 2008; Singh et al., 2011). During lethargus, animals cycle between periods of immobility (sleep bouts) and periods of active locomotion (motion bouts). Sleep bouts resemble sleep in other species based on behavioral criteria, including cessation of feeding and locomotion, increased arousal threshold for response to sensory stimulation, rapid reversibility, and homeostatic response to sleep loss. Several assays have been developed to study sleep in C. elegans (Belfer et al., 2013; Bringmann, 2011; Nelson et al., 2013; Raizen et al., 2008). Here, we contribute a detailed protocol for assessment of C. elegans sleep during lethargus, which has been used successfully by many research groups, incorporating simple microfluidic chambers, a low cost camera with lighting system, and computational analysis based on image subtraction. We note that this system could be easily adapted to assess sleep in any small animal.