Miyanishi, Yosuke et al. (2011) International Worm Meeting "Functional imaging of neuronal activity for 2-nonanone stimulation."

Nakai, Junichi, Kimura, Kotaro, Miyanishi, Yosuke

[International Worm Meeting, 2011]

To better understand the neural basis that regulates a worm's sensory behavior and its modulation by learning, we are studying avoidance behavioral responses to 2-nonanone. We previously reported that the avoidance behavior to 2-nonanone is enhanced, rather than reduced, after preexposure to the odor, and this enhancement is acquired as a non-associative dopamine-dependent learning (Kimura et al., J. Neurosci., 2010; Fujita and Kimura, this abstract). In addition, we observed that worms respond to a spatial gradient of 2-nonanone (Yamazoe and Kimura, CE Neuro, 2010), which cannot be simply explained by the pirouette or weathervane strategies. 2-nonanone is mainly sensed by the AWB neurons, which have been shown to exhibit odor-OFF response in aqueous step stimulation with 2-nonanone (Troemel et al., Cell 1997; Ha et al., Neuron 2010). To understand how the neuronal circuits of worms regulate the characteristic 2-nonanone behavioral response, we are monitoring calcium changes in the AWB and downstream neurons using G-CaMP 4 (Shindo et al., PLoS ONE, 2010). We thank Drs. S. Oda, K. Yoshida, and Y. Iino (U. Tokyo) for suggestions on microfluidics; M. Hendricks and Y. Zhang (Harvard) for aqueous 2-nonanone stimulation; and E. Busch and M. de Bono (MRC) for gaseous microfluidic stimulation.

Tanimoto, Yuki et al. (2013) International Worm Meeting "A virtual reality running machine for worms-a highly integrated microscope system for olfactory behavior."

Nakai, Junichi, Fei, Xianfeng, Kawazoe, Yuya, Miyanishi, Yosuke, Fujita, Kosuke, Hashimoto, Koichi, Tanimoto, Yuki, Yamazaki, Shuhei, Kimura, Kotaro, Gengyo-Ando, Keiko, Busch, Karl Emanuel

[International Worm Meeting, 2013]

A major function of the nervous system is to transform sensory information into an appropriate behavioral response. The neural mechanisms that mediate sensorimotor transformation are commonly studied by quantifying the behavioral and neural responses to a controlled sensory stimulus. Presenting a controlled chemical stimulus to freely behaving animals under a high-power microscope, however, is challenging. Here, we present a novel integrated microscope system that stimulates a freely moving worm with a virtual odor gradient, tracks its behavioral response, and optically monitors or manipulates neural activity in the worm during this olfactory behavior. In this system, an unrestricted worm is maintained in the center of a bright field by an auto-tracking motorized stage that is regulated by a pattern-matching algorithm at 200 Hz [1]. In addition, the worm is stimulated continuously by an odor flowing from a tube, the concentration of which can be temporally controlled. The odor concentration used in this system is based on the concentration used in the traditional plate assay paradigm (Yamazoe et al., CeNeuro 2012), and can be monitored with a semiconductor sensor connected to the end of the tube when necessary. Using this system, we investigated the neural basis of behavioral responses to a repulsive odor 2-nonanone in worms. We monitored and modulated sensory neuron activity in behaving worms by using calcium imaging and optogenetics, respectively, and found that the avoidance behavior to 2-nonanone is achieved by two counteracting sensory pathways that respond to changes in temporal odor concentration as small as ~10 nM/s (Yamazoe et al., this meeting). Our integrated microscope system, therefore, will allow us to achieve a new level of understanding for sensorimotor transformation during chemosensory behaviors. [1] Maru et al., IEEE/SICE Int. Symp. Sys. Integr. Proc., 2011.

Tanimoto, Yuki et al. (2015) International Worm Meeting "Neuronal mechanisms for C. elegans olfactory navigation revealed by a highly integrated microscope system."

Miyanishi, Yosuke, Yamazoe, Akiko, Fei, Xianfeng, Iwasaki, Yuishi, Hashimoto, Koichi, Kawazoe, Yuya, Kimura, Kotaro, Fujita, Kosuke, Nakai, Junichi, Gengyo-Ando, Keiko, Yamazaki, Shuhei, Tanimoto, Yuki

[International Worm Meeting, 2015]

The nervous system of animals transforms dynamically changing sensory information from the environment into appropriate behavioral responses. In particular, olfactory information plays critical roles in adaptive behaviors in the form of long- and short-range chemical cues that encode spatiotemporal information and chemical identity. To elucidate the neuronal mechanisms underlying olfactory behavior, it is desirable to quantify behaviors and neural circuit activities under realistic olfactory stimulus. However, reproducing realistic spatiotemporal patterns in odor concentrations is challenging due to diffusion, turbulent flow, and measurability of odor signals. We have developed an integrated microscope system that produces a virtual odor environment to quantify behaviors and neural circuit activities of the nematode C. elegans. In this system, C. elegans is maintained in the view field of a calcium imaging microscope by an auto-tracking stage using a pattern-matching algorithm. Simultaneously, odor stimulus is controlled with sub-second and sub-muM precision to reproduce realistic temporal patterns. Using this system, we have found that two types of sensory neurons play significant roles to choose a proper migratory direction for navigation in a gradient of the repulsive odor 2-nonanone. Calcium imaging and optogenetic analysis revealed that temporal increments of repulsive odor trigger turns that randomize the migratory direction, while temporal decrements of the odor suppress turning for migration down the gradient. Further mathematical analysis indicated that these sensory neurons are not only antagonizing, but also responding to odor concentration changes at different time scales for the efficient migration. Using this method will lead to comprehensive understanding of cellular mechanisms of decision making in a simple neural circuit.

Yamazoe, Akiko et al. (2013) International Worm Meeting "A neuronal mechanism for navigation along a repulsive odor gradient."

Iwasaki, Yuishi, Hashimoto, Koichi, Kawazoe, Yuya, Fujita, Kosuke, Busch, Karl Emanuel, Iino, Yuichi, Gengyo-Ando, Keiko, Nakai, Junichi, Fei, Xianfeng, Yamazaki, Shuhei, Tanimoto, Yuki, Miyanishi, Yosuke, Yamazoe, Akiko, Kimura, Kotaro

[International Worm Meeting, 2013]

For survival and reproduction, animals navigate toward or away from certain stimuli, which requires the coordinated transformation of sensory information into motor responses. In worms, the pirouette and the weathervane strategies are considered the primary navigation strategies for responding chemosensory stimuli. We found, however, that worms use a novel navigation strategy in odor avoidance behavior: In a gradient of the repulsive odor 2-nonanone, worms efficiently avoid the odor, and ~80% of initiation of long, straight migrations ("runs") were away from the odor source, which cannot be simply explained by the two known major strategies. Direct measurement of local odor concentration suggested that pirouettes are efficiently switched to runs when worms sense negative dC/dt of 2-nonanone. To test whether runs are indeed caused by negative dC/dt, we established an integrated microscope system that tracks a freely moving worm during stimulation with a virtual odor gradient and simultaneously allows for calcium imaging and optogenetic manipulations of neuronal activity (Tanimoto et al., this meeting). Using this system, we found that a realistic temporal decrement in 2-nonanone concentration (~ 10 nM/sec) caused straight migration by suppressing turns. We also found that a pair of AWB sensory neurons were continuously activated during the odor decrement and that optogenetic activation or inactivation of AWB neurons suppressed or increased turning frequency, respectively. In addition, we found that ASH nociceptive neurons increased turning frequency during odor increment. Taken together, our data indicate that the counteracting turn-inducing and turn-suppressing sensory pathways can effectively transform temporal sensory information into spatial movement to select the right path leading away from potential hazards.

Wabnig, Sebastian et al. (2011) International Worm Meeting "Addition of the genetically encoded, red-shifted Ca2+ sensor RCaMP to the C. elegans optogenetic toolbox."

Akerboom, Jasper, Wabnig, Sebastian, Stirman, Jeffrey, Gottschalk, Alexander, Looger, Loren, Lu, Hang

[International Worm Meeting, 2011]

Calcium is a universal 2nd messenger affecting a broad variety of cellular events. To assess Ca2+ signalling in vivo, several groups developed genetically encoded calcium indicators (GECIs). These include GCaMPs (~470nm ex.) or Cameleons (~415nm ex.) to detect Ca2+ responses1,2,3. To stimulate neurons in live animals, we have introduced Channelrhodopsin-2 (ChR2), photoactivated adenylyl cyclase (PACa), and bacteriorhodopsin as optogenetic tools which are excited by blue light (~470nm)4,5,6. These are useful tools to investigate effects of stimulation of specific neurons, but until now, investigating C. elegans neural network responses depends more or less on behavior as a readout for network function4,5,6. Since all of the current GECI are based on GFP, CFP or YFP1,2,3, simultaneous PACa or ChR2 (~470 nm) based optogenetic stimulation combined with calcium imaging requires complicated setups to handle the overlapping excitation spectra of these tools7. To overcome these problems, we introduced a newly red-shifted GECI to the C. elegans optogenetic toolbox. RCaMP is a red fluorescent protein, coupled to calmodulin and M13 domain, which increases its fluorescence emission intensity in response to Ca2+ binding. We expressed RCaMP under the pmyo3 promoter in body wall muscles (BWMs) of animals expressing ChR2 from the punc-17 promoter in cholinergic motor neurons (transgene zxIs6). Measurements were done on an inverted fluorescence microscope equipped with two high power light-emitting diodes (LEDs; 590 and 470nm) coupled with a beam splitter that allowed simultaneous illumination at two wavelengths. Separating the excitation wavelengths 470nm (for ChR2) and 590nm (for RCaMP) allowed Ca2+ measurements in the BWMs without excitation of ChR2. Here we show RCaMP based Ca2+ measurements of BWM activation caused by ChR2-mediated photostimulation of cholinergic motor neurons. We currently test the possibility to analyze neuronal networks using both tools in neurons. The RCaMP/ChR2 combination promises a wide range of applications to investigate cell-cell interactions and network function in live animals, even if no behavior is evoked, on a simple imaging setup. 1 Tian et al. (2009), Nat Meth 6, 875-81 2 Nakai et al. (2001) Nat Biotech 19, 137-41 3 Miyawaki (1997) Nature 388, 882-7 4 Nagel et al. (2005) Curr Biol 15, 2279-84 5 Weissenberger et al (2011) J Neurochem 116, 616-25 6 Stirman et al. (2011) Nat Meth 8, 153-8 7 Guo et al. (2009) Nat Meth 6, 891-896.