Imaging Neurons In Free-moving Drosophila Larvae Using Bilateral Illumination Light-sheet Microscope

Previous studies on Drosophila neurons and neural circuits have been conducted in immobilized or dissected larvae.In order to know how neurons behave during behavior,it is necessary to monitor the activity of neurons in free-moving larva.Here,we set up a bilateral illumination light sheet microscope system based on Gaussian laser beam,with low photo-damage and high temporal resolution of as high as 50 Hz.In addition,we also designed a microfluidic chip of certain specifications to limit the movement of larvae along z axis.Using a 30 μm thick light sheet,we successfully recorded the calcium imaging signals of neurons in ventral nerve cord during larval movement.We explored the activity pattern of PMSIs neurons in forward peristalsis in first-instar larvae and simultaneously recorded the activities of PMSIs and the contraction of body joints.It was found that when larvae move forward,PMSIs are excited sequentially from the posterior to the anterior,which is consistent with the order of body segments contraction.Interestingly,when the peristaltic wave moves to A1 segment,the wave-like activation of PMSIs is seen in reverse order from the anterior to the posterior,which has never been reported in previous studies.The establishment of the bilateral illumination light sheet microscope system lay a foundation for exploring the in-vivo functional neuron imaging of free-moving larvae.