To describe and characterize a previously undescribed type of sleep in C. elegans, where deprivation would be feasible without disrupting vital functions of the animal, I started by recording undisturbed adult animals on food for hours, aiming at detecting possible bouts of spontaneous behavioral quiescence at this stage. Doing this also required a tool for automatic and precise detection of immobility, which would speed up the process compared to manual scoring and yield more accurate measurements than a simple analysis of centroid speed, as it would properly separate complete immobility from small nose movements. The method was developed at the beginning of the project, too. The results of the analysis showed for the first time that behavioral immobility is present also in the adult stage, and not only during development or following stress, as previously reported, though it is relatively short in duration in the first case. Though it is difficult to set a duration threshold to distinguish simple pauses throughout locomotion, e.g. during foraging or before reorientations, from real sleep bouts, an investigation around sleep-defining criteria did offer a partial answer. Sleep-defining criteria are those criteria that allow labelling different behavioral states across different animal species as sleep, and they include: immobility (often combined with species-specific postures), reversibility, increased arousal thresholds, and -most often- rebound after deprivation. I tested these criteria for what we tentatively called “adult sleep”, and I found that it is satisfying the main behavioral criteria for sleep definition. So, it is evident that the immobility highlighted in adulthood is at least partially composed by true sleep periods, and that the label of “adult sleep” is appropriate.
The next step was to measure adult sleep in various available mutants which have previously shown to be defective in developmentally timed or stress-induced sleep, so to assess whether the same genes are also able to modulate adult sleep. The results were mixed. First, very importantly, mutants which are defective for stress-induced sleep did not show reduced adult sleep compared to wild type worms, nor a change in the duration of the immobility periods. This is crucial as it shows that the quiescence analyzed it is not caused by stress the animals might have been exposed to during the experiments. Second, some other mutants showed a specific modulation in the duration of the immobility periods. This seem to link a specific molecular pathway, previously implicated in the regulation of developmentally timed sleep, in adult sleep too. Also, it hinted at the involvement of a specific interneuron, RIS, previously known as the main regulator of developmentally timed sleep, in the regulation of this type of sleep too.
Therefore, I took advantage of an imaging line previously developed in the lab, expressing both the calcium indicator GCaMP and the red fluorescent protein wCherry specifically in RIS. Given the presence of two fluorescent signals, one stable and one that is modulated by neural activity, this line allows single-neuron calcium imaging in freely moving animals (where the stable signal, i.e. the cherry fluorescence, serves for movement artifacts correction). I imaged the activity of RIS throughout different ages - in pre-lethargic L4 larval stage, during L4 lethargus (i.e. the last instance of developmentally-timed sleep in the worm’s life cycle), adulthood – with or without sensory stimulation which would cause periods of sleep deprivation and subsequent rebounds. To analyze such data, I also completed a new analyzer tool to extract signal throughout subsequent frames and from different cellular compartments (as the calcium indicator was expressed in the cytoplasm of the neuron).
