Kuge, Sayuri et al. (2013) International Worm Meeting "Ca2+ dynamics of a whole single neuron."

Teramoto, Takayuki, Ishihara, Takeshi, Kuge, Sayuri

[International Worm Meeting, 2013]

The nematode C.elegans is an excellent model organism for studying how neuronal circuits activity transform sensory signals. Ca2+ imaging of neurons using Ca2+sensitive fluorescent proteins including GCaMP and yellow cameleon has revealed functions of the neuronal circuits. However, the mechanism of circuit function at single-neuron resolution is still unclear. To elucidate the functional characteristics of a single neuron, we analyzed Ca2+ dynamics of a whole single neuron by a 4-D imaging system based on a confocal microscope with a piezo lens positioner. The system enabled us to capture 3-D reconstructed images of a whole single neuron with more than 4 steric images per second. To analyze the Ca2+ dynamics of a whole neuron, we used G-GECO series of Ca2+ sensors, which were developed from G-CAMP. To observe the various basal Ca2+ concentrations, we developed G-GECO derivatives with higher affinity to Ca2+. We analyzed animals expressing Ca2+ indicators in AWCON neuron by str-2 promoter in the olfactory chip. We hope that our 4-D imaging system enables us to observe Ca2+ dynamics at dendrites, a cell body, and an axon independently and to analyze precise mechanisms of sequential rapid neuronal activation and inactivation in a whole single neuron.

Kuge, Sayuri et al. (2015) International Worm Meeting "A quenching-type of fluorescent Ca2+ indicator for detecting neuronal inhibition."

Nishihara, Tomonobu, Ishihara, Takeshi, Teramoto, Takayuki, Nagai, Takeharu, Matsuda, Tomoki, Kuge, Sayuri, Furuie, Hironobu

[International Worm Meeting, 2015]

Fluorescent Ca2+ indicators have been rapidly improved for the last several decades and now are indispensable tools for cell biology, especially neuronal science, because they enable us to monitor neuronal activities. However, these fluorescent Ca2+ indicators including GCaMPs are ideal only for monitoring the activation of neurons. To understand precise functions of the neuronal network, a fluorescent Ca2+ indicator that is suitable for monitoring the neuronal inhibition would be necessary. We previously reported various "pericam" types of indicators that are based on circularly permuted green fluorescent protein (cpGFP) fused to calmodulin and calmodulin-target peptide, M13. Among these, "inverse-pericam" has unique property; the fluorescence intensity gets 7-fold dimmer upon Ca2+ binding. Although the dynamic range is relatively big among that of the reported Ca2+ indicators, it should be enlarged to effectively monitor the neuronal inhibition. To this end, we first created a large mutant library of "inverse-pericam" by iterative error-prone PCR. The mutant libraries of genetic variants of inverse-pericam indicators were expressed in the E. coli periplasm and were screened by comparing fluorescence intensity between high- and low-Ca2+ conditions. After screening of about 20,000 bacterial colonies, we found a variant, which shows nearly 20-folds dynamic range in vitro. When the inverse-pericam variant, which we named IP2.0 was expressed in HeLa cells, the fluorescence of IP2.0 was greatly quenched responding to histamine stimulation, indicating that IP2.0 is useful to detect change of the intracellular concentration of Ca2+. Then we examined whether the decrease of Ca2+ concentration during the stimulation of isoamyl alcohol, as well as the increase of Ca2+ concentration after removing isoamyl alcohol can be monitored by expressing IP2.0 in AWCON neuron of C. elegans. While the fluorescence of GCaMPs has known to be reduced slightly during the stimulation, we observed the increase of fluorescence of IP2.0 during the stimulation. These results suggest that odor stimuli decreases Ca2+ concentration in AWCON neurons and so IP2.0 will be an indispensable tool for studying neuronal inhibition.

Usuyama, Mamoru et al. (2015) International Worm Meeting "Ca2+ dependent negative feedback loop in AWC olfactory neurons: from mathematical viewpoint."

Usuyama, Mamoru, Ishihara, Takeshi, Iwasaki, Yuishi, Kuge, Sayuri, Teramoto, Takayuki

[International Worm Meeting, 2015]

AWC receptor neurons show inhibitory response for odor stimulus and transient excitement for odor removal. This means that AWC neurons have mechanism which suppresses neural activity by external stimulation and activates by removal of the stimulation. AWC neurons change [Ca2+] via cGMP as second messenger. However, how AWC neurons change [cGMP] by stimulation is still unclear. In order to reproduce odor response in AWC neurons, we propose the negative feedback system to control [cGMP] in stimulation. The mathematical model consists of biochemical reactions and ionic transports through ion channels. Each biochemical molecules in the model, such as Ca2+, cGMP and so on, give concentrations and reaction rate constants to describe the intracellular biochemical reactions. The model is designed to easily mimic genetic assay such as gene over expression and its knock out experiments. We consider the two experimental features of odor response: (1) Peak [Ca2+] of off-response was linearly increased with duration of stimulus. (2)[Ca2+] was quickly decreased when neurons receive stimulus. Our previous work suggests that two or more Ca2+ dependent negative feedback loops for controlling cGMP production is necessary to reproduce odor responses in AWC (Usuyama, et. al 2012). In our study, however, further speculations in signaling pathway and parameter search show that only one negative feedback loop is enough to reproduce such experimental features. In addition to the experimental features, we consider the response of AWC neurons to successive stepwise stimulation. And we report "neural response" to odor stimulus in "wild type" and "mutant" in sillco, such as peak value and time constants of [Ca2+] change.