Extraction of bouton-like structures from neuropil calcium imaging data

The neuropil, the plexus of axons and dendrites, plays a critical role in operating the circuit processing of the nervous system. Revealing the spatiotemporal activity pattern within the neuropil would clarify how the information flows throughout the nervous system. However, calcium imaging to examine the circuit dynamics has mainly focused on the soma population due to their discrete distribution. The development of a methodology to analyze the calcium imaging data of a densely packed neuropil would provide us with new insights into the circuit dynamics. Here, we propose a new method to decompose calcium imaging data of the neuropil into populations of bouton-like synaptic structures with a standard desktop computer. To extract bouton-like structures from calcium imaging data, we introduced a new type of modularity, a widely used quality measure in graph theory, and optimized the clustering configuration by a simulated annealing algorithm, which is established in statistical physics. To assess this method's performance, we conducted calcium imaging of the neuropil of Drosophila larvae. Based on the obtained data, we established artificial neuropil imaging datasets. We applied the decomposition procedure to the artificial and experimental calcium imaging data and extracted individual bouton-like structures successfully. Based on the extracted spatiotemporal data, we analyzed the network structure of the central nervous system of fly larvae and found it was scale-free. These results demonstrate that neuropil calcium imaging and its decomposition could provide new insight into our understanding of neural processing.