[
International Worm Meeting,
2021]
Measurement of neuronal activities in non-invasive and unanesthetized condition is important for understanding neuronal function in intact animals. Ca2+ imaging by fluorescent gene encoded calcium indicators (GECI) are a powerful way to measure neuronal activities in C. elegans. Although Ca2+ imaging revealed important aspects in neuronal functions, the measurement of neuronal membrane voltage is important to understand the neuronal functions. Furthermore, the relations of change of membrane voltages and changes of Ca2+ has not been fully understood. Recently, several types of gene encoded voltage indicators (GEVI) that are derived from 7TM proteins used for optogenetics has been developed to measure changes of membrane voltage in living animals. Even though the fluorescence of these GEVIs is dim, they showed fast time constants and relatively high fluorescent change depend on voltages. Among those GEVIs, we use paQuasAr3 for the voltage measurement, because it shows relatively higher fluorescence with other superior characteristics. Since AWA, one of the olfactory sensory neurons, which is responsible for diacetyl sensation, was reported to show all-or-none action potentials (Liu et al. 2018), we firstly analyzed AWA voltage changes induced by diacetyl. We found that fluorescence of paQuasAr3 expressed in AWA cell body is changed in response to diacetyl stimulation with high reproducibility. At the beginning of the stimulation, the transient increase and decrease of fluorescence intensity was observed, whereas the relatively higher fluorescence intensity was sustained during the stimulation. To elucidate relations between the Ca2+ responses and the voltage responses, we made wild-type animals expressing paQuasAr3 and GCaMP6f in AWA neurons, and measured both fluorescence at a cell body simultaneously. We found that the changes of paQuasAr3 started faster than the changes of GCaMP. These analyses will give insights on the neuronal functions in informational processing.
Yoshida, Ryo, Teramoto, Takayuki, Toyoshima, Yu, Ishihara, Takeshi, Hirose, Osamu, Iino, Yuichi, Tokunaga, Terumasa
[
International Worm Meeting,
2015]
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. .