One of the fundamental challenges on neurosciences is whole-brain imaging at cellular resolution using a live animal. Simultaneous measurement of the multi-neuronal activities of the whole brain of a live animal will provide a new sight for exploring and understanding neural mechanisms for information processing. To realize this, we used the C. elegans CNS as a simple model brain and we designed a 4-D imaging system that can acquire time-sequential 3-D images at three wavelength. For image processing of acquired data, we developed a line of programs, which perform positional tracking and segmentation of each neuron.
Combining this imaging system and a ratio-metric fluorescent Ca2+ indicator and mCherry as a positional marker, we carried out 4-D Ca2+ imaging of the C. elegans CNS, and we succeeded in visualizing and measurement of neuronal activities of most neurons of the CNS without any anesthesia. Analysis of temporal changes in ratio of the individual neurons revealed that multiple neurons responded with positive and negative correlation to stimulation by such as an attractive odor and a repulsive metal ion. This result may suggest that these neurons are components of the neuronal circuit, which are involved in the sensory signaling pathway. On the other hand under non-stimulus condition, we observed synchronized rhythmic activity in multiple neurons, implying that pacemakers or pattern generators may exist in C. elegans.
Taken together, our 4-D Ca2+ imaging techniques provide a measurement method of neuronal activities in the CNS of C. elegans and revealed that multi-neuronal dynamics: 1) responding multi-neurons may involve in information transfer or/and processing, 2) rhythmic activity of other group of neurons may confer a pace or a pattern generation that correlates to behavior such as locomotion. We are now trying to reveal relationship between these neuronal dynamics by analysis of these multi-neuronal activities. Moreover, we plan to identify these neurons by combining neuronal specific promoters and fluorescent with distinguishable wavelength to adapt these neurons on the synaptic connectivity map. .
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