Recording Multi-Neuronal Activity in Unrestrained Animals with 3D Random-Access 2-Photon Microscopy

To study the neural correlates of naturalistic behaviors, we seek to record neural activity in unrestrained animals. Due to behavior-induced motion artifacts, such recordings are particularly challenging for two-photon fluorescence microscopy. Studying multi-neuronal activity in the central nervous system in moving larval Drosophila, whose brain is translated, rotated, and deformed in 3D, has been a great challenge. To overcome the challenges, we developed a method capable of calcium recording from multiple neurons in a brain of a freely moving Drosophila larva with extremely low latency (100 μsec) while simultaneously recording the animal’s posture and locomotion. We combined a resonant ultrasonic lens as a fast axial scanner and two acousto-optic deflectors as a lateral scanner that allows inertia-free relocation of the excitation beam at constant time. This method allows continuous recording from fluorescently labeled neurons while correcting the scanning position to sub-micron precision in real-time. With this method, we recorded calcium activities from a diverse set of neurons: pre-motor neurons, visual interneurons, and descending neurons in the VNC and brain of unrestrained crawling Drosophila larvae. We showed a correlation between the natural activation of mooncrawler descending neuron (MDN) and the larva’s backward crawling behavior, confirming a previous study using optogenetic activation and CaMPARI (Calcium-Modulated PhotoActivatable Ratiometric Integrator) to establish causation and correlation (Carreira et al. 2018). Our multi-neuronal recording of pre-motor neurons also revealed sequential activation during forward crawling; a result essential to understand how timed activation for locomotion is generated in a moving animal. Our technique can also be extended beyond moving larvae to stabilize recordings in a variety of moving substrates and expand the repertoire of neural activity that can be studied in cellular-resolution.