Yamazoe-Umemoto A et al. (2018) Bio Protoc "Measuring Spatiotemporal Dynamics of Odor Gradient for Small Animals by Gas Chromatography."

Kimura KD, Iwasaki Y, Yamazoe-Umemoto A

[Bio Protoc, 2018]

Odor is the most fundamental chemical stimulus that delivers information regarding food, mating partners, enemies, and danger in the surrounding environment. Research on odor response in animals is widespread, although studies on experimental systems in which the gradient of odor concentration is quantitatively measured has been quite limited. Here, we describe a method for measuring a gradient of odor concentration established by volatilization and diffusion in a relatively small enclosed space, which has been used widely in laboratories to analyze small model animals such as the nematode <i>Caenorhabditis elegans</i> and the fruit fly <i>Drosophila melanogaster</i>. We first vaporized known amounts of a liquid odorant 2-nonanone in a tank and subjected them to gas chromatographic analysis to obtain a calibration curve. Then, we aspirated a small amount of gas phase from a small hole on an agar plate and measured the odor concentration. By repeating this at different spatial and temporal points, we were able to detect a gradient of the odor concentration that increased over time. Furthermore, by applying these measured values to mathematical models of volatilization and diffusion, we were able to visualize an estimated dynamic change in odor concentration over an agar plate. Combining monitoring of odor concentration change in an agar plate with behavioral monitoring by machine vision will allow us to estimate how the brain computes information regarding odor concentration change in order to regulate behavior.

Yamazoe-Umemoto A et al. (2015) Neurosci Res "Modulation of different behavioral components by neuropeptide and dopamine signalings in non-associative odor learning of Caenorhabditis elegans."

Yamazoe-Umemoto A, Iwasaki Y, Iino Y, Fujita K, Kimura KD

[Neurosci Res, 2015]

An animal's behavior is modulated by learning; however, the behavioral component modulated by learning and the mechanisms of this modulation have not been fully understood. We show here that two types of neural signalings are required for the modulation of different behavioral components in non-associative odor learning in the nematode Caenorhabditis elegans. We have previously found that C. elegans avoid the repulsive odor 2-nonanone, and preexposure to the odor for 1h enhances the avoidance behavior as a type of non-associative learning. Systematic quantitative analyses of behavioral components revealed that the odor preexposure caused increases in average duration of straight migration (&quot;runs&quot;) only when the animals were migrating away from the odor source within a certain range of bearing, which likely corresponds to odor decrement. Further, genetic analyses revealed that the genes for neuropeptide or dopamine signalings are both required for the enhanced odor avoidance. Neuropeptide signaling genes were required for the preexposure-dependent increase in run duration. In contrast, dopamine signaling genes were required not for the increase in run duration but likely for maintenance of run direction. Our results suggests that multiple behavioral components are regulated by different neuromodulators even in non-associative learning in C. elegans.

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.

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 Y et al. (2017) Elife "Calcium dynamics regulating the timing of decision-making in C. elegans."

Hashimoto K, Nakai J, Gengyo-Ando K, Iino Y, Kimura KD, Fei X, Tanimoto Y, Busch KE, Iwasaki Y, Fujita K, Yamazaki SJ, Kawazoe Y, Miyanishi Y, Yamazoe-Umemoto A

[Elife, 2017]

Brains regulate behavioral responses with distinct timings. Here we investigate the cellular and molecular mechanisms underlying the timing of decision-making during olfactory navigation in Caenorhabditis elegans. We find that, based on subtle changes in odor concentrations, the animals appear to choose the appropriate migratory direction from multiple trials as a form of behavioral decision-making. Through optophysiological, mathematical and genetic analyses of neural activity under virtual odor gradients, we further find that odor concentration information is temporally integrated for a decision by a gradual increase in intracellular calcium concentration ([Ca(2+)]i), which occurs via L-type voltage-gated calcium channels in a pair of olfactory neurons. In contrast, for a reflex-like behavioral response, [Ca(2+)]i rapidly increases via multiple types of calcium channels in a pair of nociceptive neurons. Thus, the timing of neuronal responses is determined by cell type-dependent involvement of calcium channels, which may serve as a cellular basis for decision-making.

Tanimoto, Y. et al. (2017) International Worm Meeting "Calcium dynamics regulates the timing of decision-making in worms."

Iwasaki, Y., Tanimoto, Y., Hashimoto, K., Nakai, J., Miyanishi, Y., Kawazoe, Y., Fujita, K., Kimura, K., Gengyo-Ando, K., Iino, Y., Yamazoe-Umemoto, A., Busch, K. E., Fei, X., Yamazaki, S.

[International Worm Meeting, 2017]

The brain processes sensory information to generate various physiological responses with different timing (i.e., with different response latencies). In decision-making, for example, animals choose one from multiple behavioral options based on environmental sensory information, with a temporal delay associated with the certainty of sensory information. The neural mechanism of timing, however, is largely unclear. We report the cellular and molecular mechanisms underlying the timing of decision-making during olfactory navigation in worms. Based on subtle changes in concentrations of the repulsive odor 2-nonanone, worms efficiently choose the appropriate migratory direction after multiple trials as a form of behavioral decision-making, which is different from the typical biased random walk. From simultaneous monitoring of behavior and neural activity in virtual odor gradients, we found that two pairs of sensory neurons regulate this behavioral response in an opposing manner with different temporal dynamics. ASH nociceptive neurons exhibit a time-differential response to an increase in the 2-nonanone concentration, which leads to an immediate turning response similar to a "reflex." In contrast, AWB olfactory neurons exhibit a time-integral response to a decrease in the odor concentration, which leads to turn suppression with a temporal delay resembling "deliberation." We further found that the AWB response is independent of synaptic connections and is mediated by a gradual calcium influx, mainly via L-type voltage-gated calcium channel (VGCC) EGL-19, whereas the ASH response is mediated by rapid calcium influx via multiple types of calcium channels. Thus, the timing of neuronal responses, such as deliberate decision-making or rapid reflex, is determined by cell type-dependent involvement of calcium channels. Interestingly, such time-integral neural responses have also been observed in decision-making by primates and rodents, and are considered to be mediated by recurrent neural circuits, although intracellular mechanisms have also been proposed. We suggest that a single-cell temporal integrator with L-type VGCCs, such as the AWB neuron, may be the evolutionarily conserved molecular basis for decision-making.

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.

Yamazoe, Akiko et al. (2011) International Worm Meeting "Worms maintain their response to the repulsive odor 2-nonanone during avoidance behavior."

Yamazoe, Akiko, Kimura, Kotaro, Iwasaki, Yuishi

[International Worm Meeting, 2011]

Animals can maintain their behavioral response to environmental stimuli even under unstable environmental conditions and during various animal movements. To investigate neural mechanisms for such robust behavioral responses, it is necessary to quantitatively analyze the time-course changes in the correlation between the stimulus and behavioral response. For this, we quantitatively analyzed stimulus as well as behavior of worms' avoidance response to repulsive odor 2-nonanone. When animals migrate away from a source of repulsive signal, their avoidance response is likely weakened. In a previous study, however, we have shown that worms exhibited a constant average velocity of avoidance from 2-nonanone for 10 min (Kimura et al., J. Neurosci., 2010), suggesting a neural mechanism for such constant avoidance. In addition to the quantitative analysis of avoidance response to 2-nonanone (Yamazoe & Kimura, CeNeuro, 2010), we recently developed a technique to measure the concentration of 2-nonanone at specific spatial and temporal points of gas phase in the assay plate. By using a highly sensitive gas chromatograph, we observed a clear gradient of 2-nonanone, of which concentration increased with time. Based on this measured gradient of 2-nonanone, we determined the 2-nonanone concentration that each worm experienced during the avoidance assay (Cworm) and observed the following: (1) During the first 2 min of the assay worms did not initiate avoidance response and migrated randomly, and Cworm increased continuously up to the order of mM at 2 min. (2) After 2 min, worms started to migrate farther away from the odor source, and Cworm was maintained around the concentration, despite increase in the concentration gradient. (3) Cworm decreased effectively during runs, while it increased and decreased largely during pirouettes. (4) When compared between the early and late phases of the assay, the maximum dCworm/dt in each run decreased several fold along with the avoidance behavior, even though the orientation directions did not change considerably; that is, even when the gradient of 2-nonanone became shallower, the accuracy of worm orientation appeared maintained. These results suggest that worms may increase sensitivity to dC/dt during exposure to a certain concentration of 2-nonanone. We are currently conducting computer simulation to test this hypothesis. Further analysis may help us uncover the mechanism of maintaining proper behavioral responses.

Quartey MO et al. (2021) Sci Rep "The A(1-38) peptide is a negative regulator of the A(1-42) peptide implicated in Alzheimer disease progression."

Pennington PR, Heistad RM, Nyarko JNK, Barnes JR, Bolanos MAC, Parsons MP, Knudsen KJ, De Carvalho CE, Leary SC, Mousseau DD, Buttigieg J, Maley JM, Quartey MO

[Sci Rep, 2021]

The pool of -Amyloid (A) length variants detected in preclinical and clinical Alzheimer disease (AD) samples suggests a diversity of roles for A peptides. We examined how a naturally occurring variant, e.g. A(1-38), interacts with the AD-related variant, A(1-42), and the predominant physiological variant, A(1-40). Atomic force microscopy, Thioflavin T fluorescence, circular dichroism, dynamic light scattering, and surface plasmon resonance reveal that A(1-38) interacts differently with A(1-40) and A(1-42) and, in general, A(1-38) interferes with the conversion of A(1-42) to a -sheet-rich aggregate. Functionally, A(1-38) reverses the negative impact of A(1-42) on long-term potentiation in acute hippocampal slices and on membrane conductance in primary neurons, and mitigates an A(1-42) phenotype in Caenorhabditis elegans. A(1-38) also reverses any loss of MTT conversion induced by A(1-40) and A(1-42) in HT-22 hippocampal neurons and APOE 4-positive human fibroblasts, although the combination of A(1-38) and A(1-42) inhibits MTT conversion in APOE 4-negative fibroblasts. A greater ratio of soluble A(1-42)/A(1-38) [and A(1-42)/A(1-40)] in autopsied brain extracts correlates with an earlier age-at-death in males (but not females) with a diagnosis of AD. These results suggest that A(1-38) is capable of physically counteracting, potentially in a sex-dependent manner, the neuropathological effects of the AD-relevant A(1-42).

Danielle Thierry-Mieg et al. (2003) Worm Breeder's Gazette "www.wormgenes.org , a new gene centered database"

Vahan Simonyan, Michel Potdevin, Mark Sienkiewicz, Yuji KOHARA, Jean Thierry-Mieg, Danielle Thierry-Mieg, Tadasu SHIN-I

[Worm Breeder's Gazette, 2003]

Wormgenes is a new resource for C.elegans offering a detailed summary about each gene and a powerful query system.