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.

Ikejiri, Yosuke et al. (2021) International Worm Meeting "Learning-dependent gain control by asymmetric modulation of the first- and second-order time-differential of stimulus in sensory neurons"

Ikejiri, Yosuke, Kimura, Kotaro, Yamazaki, Shuhei, Tanimoto, Yuki, Iwatani, Yasushi

[International Worm Meeting, 2021]

Animals respond to environmental stimuli whose intensity varies by approximately 1010-fold, although neural responses can only change by 102-fold, which requires proper adjustment of the relationship between the environmental stimuli and the neural response. One example of this adjustment is neural gain control, defined as the change in the slope of a neural response to a stimulus, instead of a general reduction (adaptation) or enhancement (sensitization) of the response. However, these mechanisms are poorly elucidated. Here, we report that the neural gain control in the ASH nociceptive neuron occurs by asymmetric modulation of the first- and second-order time-differentials of sensory stimulus. Previously, we showed that the worm's avoidance behavior to the repulsive odor 2-nonanone is enhanced by pre-exposure to the odor as a type of non-associative learning (Kimura et al., J Neurosci 2010). We now found that the ASH responses, which are activated by increasing the 2-nonanone concentrations (Tanimoto et al., eLife 2017), are modulated by the odor learning. Quantitative odor stimuli analysis revealed that the naive ASH neurons respond similarly to small and large linear increases in odor concentration, whereas the pre-exposed ASH neurons only respond to large increases. Analysis of the stimulus-response relationships suggested that this learning-dependent change is a neural gain control of response. Interestingly, mathematical analysis revealed that the ASH response is approximated by the sum of the first- and second-order time-differentials of odor concentration, and the second-order time-differential is greatly suppressed by learning. We found that the terms of the first- and second-order time differential are expressed by the variable coefficients. To test the validity of this model, we compared it with the first-order time-differential only model and second-order time-differential only model using the Bayesian information criterion (BIC). As predicted, in naive ASH neurons, the model of the sum of the first- and second-order time-differentials of odor concentration was the best fit, and the first-order time-differential only model was the best fit in the pre-exposed condition. These results may suggest that the ASH response is mediated by the long (corresponding to the first-order term) and transiently (the second-order term) activated voltage-gated calcium channels and that the contribution of these channels are modulated by the odor stimulus.

Zheng, Ying Grace et al. (2015) International Worm Meeting "Optophysiological dissection of individual dopaminergic neuron pairs during food-dependent behavioral modulation."

Kimura, Kotaro, Zheng, Ying Grace, Tanimoto, Yuki, Fei, Xianfeng, Hashimoto, Koichi

[International Worm Meeting, 2015]

Dopamine (DA) functions as an important neuromodulator responsible for regulating various behaviors in animals. While DA signaling has been shown to play a significant role in behavioral regulation, the mechanism by which DA-releasing (DAergic) neurons are activated in response to environmental stimuli remains unclear. In C. elegans, DA functions as a "presence of food" signal responsible for modulating various food-dependent behaviors (Chase and Koelle, Wormbook, 2007). Four pairs of DAergic neurons, CEPD, CEPV, ADE, and PDE neurons, work redundantly for behavioral modulation in response to a bacterial lawn, which is sensed as a mechanical stimulus (Sawin et al., Neuron, 2000). A previous calcium imaging study revealed that one class of DAergic neurons, the CEP neurons, are indeed activated by mechanical stimuli (Kindt et al., Neuron, 2007). However, the timing and manner of activation of specific DAergic neuron pairs in response to the bacterial lawn have not yet been revealed since functional dissection of DAergic neurons by calcium imaging and/or optogenetics is difficult. All the known DAergic neuron-specific promoters express genes in all DAergic neurons-not in just one or a subset of them. Therefore, sophisticated machine vision techniques are necessary to target one of the many DAergic neurons in a worm migrating upon an agar surface to enter a bacterial lawn. In an attempt to understand how individual DAergic neuron pairs are activated by food stimuli and how this activation affects worm behavior, we conducted optophysiological analyses using our original auto-tracking integrated microscope system (Tanimoto et al., this meeting). First, we monitored the activation patterns of each DA neuron pair in freely moving worms by calcium imaging; we found that CEPD, CEPV, and PDE pairs were differently activated upon food-entry (The ADE pair was not analyzed because of dim fluorescence from the cells). At present, we are optogenetically stimulating individual DAergic neuron pairs in freely moving worms to test whether these differences in activation patterns reflect functional differences in the neurons. Our analysis will shed light on activities and functions of each of the genetically indistinguishable neurons in this small model organism.

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.

Yamazaki, Shuhei et al. (2013) International Worm Meeting "Genetic analysis of dopamine signaling for repulsive odor learning."

Kimura, Kotaro, Yamazaki, Shuhei

[International Worm Meeting, 2013]

Through learning, an animal can optimize its chances for survival and reproduction by modifying its behavior based on prior experiences. Multiple types of learning, and the molecular mechanisms that mediate learning, have been studied in both vertebrates and invertebrates. Nevertheless, How sensory behavior is modulated by learning and how specific molecules are involved in this process are not well understood. Previously, we have shown that dopamine signaling is required for non-associative learning of odor avoidance behavior of worms. Worms exhibit an enhanced avoidance behavior to 2-nonanone after preexposure to the odor, and this enhancement is regulated in RIC neurons by dopamine signaling via the D2-like dopamine receptor DOP-3 (Kimura et al., 2010, J. Neurosci.). Currently, we are working towards identifying new genes that can genetically interact with the dopamine-signaling pathway to regulate and/or enhance 2-nonanone avoidance. We have found some mutant strains that exhibit behavioral defects that are similar to those exhibited by dopamine mutants. We plan to identify these mutations with whole-genome sequencing, and reveal the physiological role of their gene products by using our integrated microscope system (Tanimoto et al., this meeting).

Yamazaki, Shuhei et al. (2015) International Worm Meeting "Identifying neural signaling that cooperates with dopamine signaling in repulsive odor learning."

Kimura, Kotaro, Yamazoe, Akiko, Yamazaki, Shuhei

[International Worm Meeting, 2015]

Dopamine in association with other neural signals plays crucial roles in various brain functions such as locomotory regulation, reward, emotion, learning and memory. However, the mechanism by which multiple neural signals cooperatively regulate brain functions is not well understood because of neural circuit complexity. To address this issue, we are studying repulsive odor learning regulated by dopamine signaling in C. elegans (Kimura et al., 2010, J. Neurosci.). Upon preexposure to 2-nonanone, the animals exhibit enhanced avoidance behavior to this odorant as a type of non-associative learning. This enhancement is regulated by dopamine signaling via the D2-like dopamine receptor, DOP-3, in a pair of RIC interneurons. Currently, we are working towards identifying genes that genetically interact with dopamine signaling for repulsive odor learning. We have identified several mutant strains that exhibit behavioral defects similar to those seen in dopamine mutants. We first plan to identify the site-of-action of identified genes by cell-specific rescue experiments. The physiological role of the gene will then be analyzed by our integrated microscope system to quantify the relationship between odor stimuli, neural responses, and behavior (Tanimoto et al., this meeting). These analyses will help us understand the interaction between the newly identified neural signaling and dopamine signaling in modulation of learning.

Yamazaki, S. et al. (2017) International Worm Meeting "Learning-dependent behavioral modulation of sensory behavior revealed by machine learning and optophysiological analysis in a virtual environment."

Ikejiri, Y., Hiramatsu, F., Yamazaki, S., Fujita, K., Kimura, K., Tanimoto, Y., Hashimoto, K., Maekawa, T., Yamazoe-Umemoto, A.

[International Worm Meeting, 2017]

Animals modify their behavior based on experiences as learning, although identifying component(s) of behavior modulated by learning has been difficult. In contrast to neural activities, which can be monitored in large numbers of cells simultaneously recently, behavior in general is still analyzed in classic ways and insufficiently studied using simple measures, such as velocity, migratory distance, and/or the probability of selecting a particular goal. Comprehensive classifications of animals' behavior by using machine vision and machine learning methods have been achieved (Gomez-Marin et al., Nat Neurosci, 2014; Brown et al., PNAS, 2013). However, these methods have not been applied to animals' sensory behavior because of technical limitations in measuring sensory signals that animals receive during the behavior. To overcome this problem and effectively identify behavioral components modulated by learning, we used machine learning aiming to detect changes in navigation of worms in a measured odor gradient. We have previously reported that, after experiencing the repulsive odor 2-nonanone for 1 h, worm's odor avoidance behavior is enhanced, and that they move away from the odor source more efficiently (Kimura et al., J Neurosci, 2010). We have also quantified the dynamic changes in the odor concentration during odor avoidance behavior (Yamazoe-Umemoto et al., Neurosci Res, 2015). In the present study, we used decision tree, a machine learning algorithm, to extract features of the animal's sensorimotor response during navigation modified by odor learning. During the migration down the odor gradient, naive worms responded to slight increases in the repulsive odor concentration by stopping forward movements and initiating turns. In contrast, the probability of response was lowered after learning, suggesting that the learned worms ignore "a yellow light". Consistently, by calcium imaging of ASH neurons, whose activation causes turns under a virtual odor gradient (Tanimoto et al., this meeting), we found that the ASH response to a small increase in the odor concentration was reduced after learning. Furthermore, by applying the decision tree analysis, multiple mutant strains were categorized into several groups based on behavioral features. Thus, the integrative machine learning analysis of sensory information and behavioral response is a powerful tool to obtain comprehensive understanding of dynamic activities of neural circuits and its modulation by learning.

Brissette, B. et al. (2019) International Worm Meeting "Using the calcium integrator CaMPARI to map circuits in C. elegans."

Brissette, B., Ringstad, N.

[International Worm Meeting, 2019]

Although an anatomical connectome for the C. elegans nervous system was determined more than 30 years ago [White et al., 1986], it remains challenging to define the neural circuits that generate specific behaviors. Circuit mapping has relied on a combination of functional imaging - e.g. with calcium reporters such as GCaMP - and targeted ablation or silencing of specific neurons. An alternative approach to mapping circuits uses CaMPARI, a fluorophore that was engineered to irreversibly convert from green to red fluorescence upon coincident irradiation with 405 nm light and high calcium [Fosque et al., 2015]. This probe, therefore, converts neural activity into a color-change. We sought to determine whether it is possible to use CaMPARI for circuit mapping in freely behaving C. elegans. Towards this aim, we have generated transgenics that express CaMPARI in specific neurons and demonstrated that CaMPARI in these neurons can be robustly converted by widefield irradiation of unrestrained animals. Using a hypomorphic mutation of the EGL-19 L-type calcium channel, we show that in vivo conversion of CaMPARI is calcium-dependent. Most importantly, with CaMPARI expressed in the mechanosensory dopamine neurons, we have shown that CaMPARI conversion in these neurons is accelerated by interactions with a bacterial lawn, which is known to activate dopaminergic neurons to control foraging behavior. In addition to validating the CaMPARI probe, these experiments also revealed functional heterogeneity in dopamine neurons. CaMPARI in the lateral ADE neurons was robustly converted regardless of the presence or absence of bacteria. By contrast, CEP neurons were more rapidly converted in the presence of bacteria, and ventral CEPs were more robustly converted than their dorsal counterparts. It was previously suggested by Kimura and colleagues that dopamine neurons are not equally sensitive to food-stimuli [Tanimoto et al., 2016]; our CaMPARI data also indicate functional differences between these putatively homologous cells. We have generated other CaMPARI transgenics that mark distinct populations of sensory neurons and interneurons. Our preliminary data indicate that these transgenics report differential activation of neural circuits by food-stimuli. Our next goal is to use these transgenics to map in an unbiased way the neural circuits that generate food-response behaviors.