The initial phase of the project involved designing new experimental paradigms in which mechanosensory entrainment - or circadian modulations of mechanosensory behaviour - could be tested – with one of the tested hypotheses being that mechanical vibrations do not necessarily entrain the clock (external ‘vibration entrainment’) but possibly the flies’ vibration-induced activity feeds back into the clock (internal ‘activity entrainment’). Vibrated flies were constantly monitored by video tracking and high-resolution activity monitoring. Mechanical stimulus paradigms were designed to minimise input from external stimuli and maximise stimulus-induced activity in order to test our hypothesis. Interestingly, preliminary results suggest that not all mechanically-evoked activity rhythms are associated with an entrainment of the flies’ clock, posing the question if activity alone is sufficient to set the clock.
The key molecular players that help maintain circadian rhythmicity are well documented, therefore we tested for their expression in tissues important for mechanosensation – one major model organ of mechanosensation in Drosophila is Johnston’s organ (JO), a multi-cellular chordotonal organ (ChO). Chordotonal neurons are stretch-receptors, which serve both as exteroceptors (sensing external vibrations) and as proprioceptors (sensing the animals’ own movements); most notably, however, ChOs have been implicated in both mechanosensory and thermosensory entrainment of the fly’s circadian clock. We dissected JO tissue at different time points across a day and quantified expression of core clock genes. We compared clock gene expression levels between time points in JO and a control tissue, the fly’s head (as the site of the ‘central clock’). Rhythmic oscillations of core clock genes were found in JO; oscillations in JO were in phase with oscillations in the fly’s head.
One significant question in circadian biology is how multiple entrainment stimuli are being processed when presented in conflict. When light and temperature oscillate in phase we observed seemingly ‘normal’ activity patterns; when light and temperature were opposing each other, with an ‘unnatural’ 180 phase difference in between the two of them, light dominated the entrainment, that means the temperature stimulus was basically neglected. However, when the phase of these two conditions was offset by a smaller margin, of only a few hours, then temperature became a much more prominent entrainment stimulus, at intermediate phase differences of 5-7 hours, finally, behavioural coherence, and circadian entrainment, broke down: a unique, aberrant locomotor activity pattern was observed and molecular oscillations of core clock collapsed (Harper et al. 2016).
This is clear evidence that these sensory inputs are weighted and integrated to elicit the appropriate behavioural (and molecular) responses. Overall it alludes to a complexity that may not be fully untangled by conventional experiments.
