Background

The functions of sleep and its long-term impact on behavior and physiology have been largely understudied. Studies focusing on the acute effect of sleep deprivation (SD) in juvenile mice, rats, and cats revealed that sleep affects synaptic structure and plasticity during adolescent development (Frank et al., 2001; Dumoulin Bridi et al., 2015; Shaffery et al., 2006; Shaffery et al., 2002; Maret et al., 2011; Yang et al., 2014; W. Li et al., 2017; de Vivo et al., 2017). However, whether these changes result in long-lasting behavioral changes remains unknown. To evaluate the impact of long-term sleep perturbation, especially during development, chronic sleep disruption is required, and so is longitudinal sleep monitoring using electroencephalogram (EEG) along the manipulation process.

Automated SD in rodents often uses physical stimulations or setups that keep the animal in constant motion, such as using continuously moving treadmills or rotating wheels (Colavito et al., 2013). In an alternating platform setup, the animal was forced to move between two small platforms that continuously and alternatively move above and below water (Pierard et al., 2007). In a setup of spinning disk above a water tank, the disk rotated whenever sleep onset was detected, and the animal would need to stay awake and mobile in order not to fall into the water (they woke up in the case they did fall) (Rechtschaffen et al., 1983). Another method is the so-called “grid over water,” where placing the animal on a grid floor suspended over the water surface significantly reduced NREM and REM sleep and increased sleep latency, but the reduction of sleep was only in half (Shinomiya et al., 2003). REM-selective deprivation can be achieved by placing the animal on a small platform surrounded by and slightly above the water surface (Morden et al., 1967). The animal was allowed to have NREM sleep by crouching on the platform; however, upon transition to a REM episode, the loss of muscle tone caused the animal to touch or fall into the water and thus aroused the animal (Morden et al., 1967). These automated SD paradigms all produce stress, anxiety, and/or excessive physical stimuli, making them unsuitable for adolescent mice, which likely have heightened vulnerability to stressors (Romeo, 2013). In comparison, more mild methods such as the “gentle touch” protocol requires constant monitoring of the mouse behavior or EEG by an experimenter, as whenever the mouse exhibits signs of sleep (e.g., staying still for longer than 5 s, or low-frequency EEG), the experimenter directly intervenes by touching the animal using a soft tool (e.g., a brush), keeping the animal awake (Eban-Rothschild et al., 2016; W. Li et al., 2017; Yang et al., 2014). However, this method is labor-intensive, and may not be easily replicated between experimenters; thus, it is not ideal if high throughput or chronic intervention is desired. Another “gentle” protocol requires exposing the animal to novel objects, a different environment, and/or nesting materials, which leads to exploratory or nest-building behaviors, respectively, and helps maintain wakefulness. This method does not cause significant stress (Kopp et al., 2006), but the animal tends to become habituated quickly, and the forced wakefulness usually does not last longer than 2 h. In addition, pre-exposure to the enrichment materials may be a confounder, if a behavior task such as the novel object recognition test is performed subsequently.

Here, we report an alternative protocol for automated SD in mice, which is effective for 4 h or longer and robust across many days, does not cause significant stress even in adolescent animals, and can be performed in high throughput (up to four mice at a time for each chamber) without human intervention. The protocol is ideal for chronic sleep disruptions over days, in both developing and adult mice. In combination with a closed loop algorithm, it is also possible to achieve REM-selective automated deprivation.