Intermittent Short Sleep Protocol

Mice were randomized to receive 1, 2, or 4 weeks of intermittent short sleep (ISS1, ISS2, or ISS4) or rested conditions. Subsets of ISS4 mice and their rested controls were further randomized to 4-week unperturbed recovery prior to study. The rested control (Rest) group for all comparisons to ISS4 and ISS4+Rec was age-matched to ISS4+Rec mice (16 weeks of age for sleep, molecular, immunohistochemistry studies). A previously validated enriched, novel environment paradigm was used to promote spontaneous exploratory wakefulness.^13–15 On days 1–3 of each week, mice were moved with littermates to an enriched environment (600 × 400 × 600 cm with various climbing toys, water, food, and cage bedding) for 8 h from 09:00 to 17:00 and were then returned to their home cages with littermates and left unperturbed until the next ISS day, as summarized in the schedule in Figure 1. Ambient light (timing and intensity) and temperature were held constant across the novel environments and home cages. Across trials, mice were continuously observed, and when mice were still, new climbing toys, and bedding/nesting materials were introduced. This particular sleep loss paradigm does not influence plasma corticosterone levels.⁸ On days 4–7 of each week, mice were left undisturbed in their home cages with their littermates. Rest mice were placed into a similar enriched environment for one hour (17:00 to 18:00) on ISS days and then returned to their home cages. The recovery subsets of mice (ISS4+Rec and Rest) received 4 weeks of undisturbed rest in their home cages, with normal lighting, food, and water.

Intermittent Short Sleep (ISS) Study Paradigm. Adult male mice were randomized to the two groups, ISS (top) and Rested controls (Rest) below. Black and white bars highlight lights-on and lights-off times respectively. For both ISS and Rest mice, red bars highlight times of exploratory wakefulness exposures for ISS mice, from Zeitgeber hours 4–12 (10:00–18:00) and for Rest mice Zeitgeber hour 11–12 (17:00–18:00). At all other times mice are returned to home cages and left undisturbed. Brackets show times of a continuous recovery opportunity for 4 days. This pattern was repeated 3 times and mice were then randomized to recovery or no recovery.