The Large-Scale Cultivation of Nematodes to Study Their Collective Behaviors.

Animals exhibit dynamic collective behaviors, as observed in flocks of birds, schools of fish, and crowds of humans. The collective behaviors of animals have been investigated in the fields of both biology and physics. In the laboratory, researchers have used various model animals such as the fruit fly and zebrafish for approximately a century, but it has remained a major challenge to study large-scale complex collective behavior orchestrated by these genetically tractable model animals. This paper presents a protocol to create an experimental system of collective behaviors in Caenorhabditis elegans. The propagated worms climb on the lid of the Petri plate and show collective swarming behavior. The system also controls worm interactions and behaviors by changing the humidity and light stimulation. This system allows us to examine the mechanisms underlying collective behaviors by changing environmental conditions and examining the effects of individual-level locomotion on collective behaviors using mutants. Thus, the system is useful for future research in both physics and biology.

Caenorhabditis elegans transfers across a gap under an electric field as dispersal behavior.

Interactions between different animal species are a critical determinant of each species' evolution and range expansion. Chemical, visual, and mechanical interactions have been abundantly reported, but the importance of electric interactions is not well understood. Here, we report the discovery that the nematode Caenorhabditis elegans transfers across electric fields to achieve phoretic attachment to insects. First, we found that dauer larvae of C. elegans nictating on a substrate in a Petri dish moved directly to the lid through the air due to the electrostatic force from the lid. To more systematically investigate the transfer behavior, we constructed an assay system with well-controlled electric fields: the worms flew up regardless of whether a positive or negative electric field was applied, suggesting that an induced charge within the worm is related to this transfer. The mean take-off speed is 0.86 m/s, and the worm flies up under an electric field exceeding 200 kV/m. This worm transfer occurs even when the worms form a nictation column composed of up to 100 worms; we term this behavior "multiworm transfer." These observations led us to conclude that C. elegans can transfer and attach to the bumblebee Bombus terrestris, which was charged by rubbing with flower pollen in the lab. The charge on the bumblebee was measured with a coulomb-meter to be 806 pC, which was within the range of bumblebee charges and of the same order of flying insect charges observed in nature, suggesting that electrical interactions occur among different species.

Genome Editing of C. elegans.

Caenorhabditis elegans, a 1 mm long free-living nematode, is a traditional model animal for genetic investigations of various biological processes. Characteristic features that make C. elegans a powerful model of choice for eukaryotic genetic studies include its rapid life cycle, well-annotated genome, simple morphology, and transparency. Recently, genome editing technologies have been increasingly used in C. elegans, thereby facilitating their genetic analyses. Here, I introduce a protocol frequently used in C. elegans genome editing.

Human FAM3C restores memory-based thermotaxis of Caenorhabditis elegans famp-1/m70.4 loss-of-function mutants.

The family with sequence similarity 3 (FAM3) superfamily represents a distinct class of signaling molecules that share a characteristic structural feature. Mammalian FAM3 member C (FAM3C) is abundantly expressed in neuronal cells and released from the synaptic vesicle to the extracellular milieu in an activity-dependent manner. However, the neural function of FAM3C has yet to be fully clarified. We found that the protein sequence of human FAM3C is similar to that of the N-terminal tandem domains of Caenorhabditis elegans FAMP-1 (formerly named M70.4), which has been recognized as a tentative ortholog of mammalian FAM3 members or protein-O-mannose ß-1,2-N-acetylglucosaminyltransferase 1 (POMGnT1). Missense mutations in the N-terminal domain, named Fam3L2, caused defects in memory-based thermotaxis but not in chemotaxis behaviors; these defects could be restored by AFD neuron-specific exogenous expression of a polypeptide corresponding to the Fam3L2 domain but not that corresponding to the Fam3L1. Moreover, human FAM3C could also rescue defective thermotaxis behavior in famp-1 mutant worms. An in vitro assay revealed that the Fam3L2 and FAM3C can bind with carbohydrates, similar to the stem domain of POMGnT1. The athermotactic mutations in the Fam3L2 domain caused a partial loss-of-function of FAMP-1, whereas the C-terminal truncation mutations led to more severe neural dysfunction that reduced locomotor activity. Overall, we show that the Fam3L2 domain-dependent function of FAMP-1 in AFD neurons is required for the thermotaxis migration of C. elegans and that human FAM3C can act as a substitute for the Fam3L2 domain in thermotaxis behaviors.

Collective pattern formations of animals in active matter physics.

Active matter refers to systems composed of elements that are self-propelled by the dissipation of energy, in which dynamical patterns emerge, as is the case of flocks of birds and schools of fish. Some researchers in active matter physics seek to identify unified descriptions of such collective motions through interdisciplinary approaches by biologists and physicists. Through such collaborations, experimental studies pertaining to active matter physics have been developing recently, which allow us to verify the proposed mathematical models. Here, we review collective pattern formations and behaviors of animals from the perspective of active matter physics.

All-Optical Wide-Field Selective Imaging of Fluorescent Nanodiamonds in Cells, In Vivo and Ex Vivo.

Fluorescence imaging is a critical tool to understand the spatial distribution of biomacromolecules in cells and in vivo, providing information on molecular dynamics and interactions. Numerous valuable insights into biological systems have been provided by the specific detection of various molecular species. However, molecule-selective detection is often hampered by background fluorescence, such as cell autofluorescence and fluorescence leakage from molecules stained by other dyes. Here we describe a method for all-optical selective imaging of fluorescent nanodiamonds containing nitrogen-vacancy centers (NVCs) for wide-field fluorescence bioimaging. The method is based on the fact that the fluorescence intensity of NVCs strictly depends on the configuration of ground-state electron spins, which can be controlled by changing the pulse recurrence intervals of microsecond excitation laser pulses. Therefore, by using regulated laser pulses, we can oscillate the fluorescence from NVCs in a nanodiamond, while oscillating other optical signals in the opposite phase to NVCs. As a result, we can reconstruct a selective image of a nanodiamond by using a series of oscillated fluorescence images. We demonstrate application of the method to the selective imaging of nanodiamonds in live cells, in microanimals, and on a hippocampal slice culture obtained from a rat. Our approach potentially enables us to achieve high-contrast images of nanodiamond-labeled biomolecules with a signal-to-background ratio improved by up to 100-fold over the standard fluorescence image, thereby providing a more powerful tool for the investigation of molecular dynamics in cells and in vivo.

Calcium Imaging in Freely Behaving Caenorhabditis elegans with Well-Controlled, Nonlocalized Vibration.

Nonlocalized mechanical forces, such as vibrations and acoustic waves, influence a wide variety of biological processes from development to homeostasis. Animals cope with these stimuli by modifying their behavior. Understanding the mechanisms underlying such behavioral modification requires quantification of neural activity during the behavior of interest. Here, we report a method for calcium imaging in freely behaving Caenorhabditis elegans with nonlocalized vibration of specific frequency, displacement, and duration. This method allows the production of well-controlled, nonlocalized vibration using an acoustic transducer and quantification of evoked calcium responses at single-cell resolution. As a proof of principle, the calcium response of a single interneuron, AVA, during the escape response of C. elegans to vibration is demonstrated. This system will facilitate understanding of neural mechanisms underlying behavioral responses to mechanical stimuli.

Tracking the 3D Rotational Dynamics in Nanoscopic Biological Systems.

The rotation of an object cannot be fully tracked without understanding a set of three angles, namely, roll, pitch, and yaw. Tracking these angles as a three-degrees-of-freedom (3-DoF) rotation is a fundamental measurement, facilitating, for example, attitude control of a ship, image stabilization to reduce camera shake, and self-driving cars. Until now, however, there has been no method to track 3-DoF rotation to measure nanometer-scale dynamics in biomolecules and live cells. Here we show that 3-DoF rotation of biomolecules can be visualized via nitrogen-vacancy centers in a fluorescent nanodiamond using a tomographic vector magnetometry technique. We demonstrate application of the method to three different types of biological systems. First, we tracked the rotation of a single molecule of the motor protein F1-ATPase by attaching a nanodiamond to the γ-subunit. We visualized the 3-step rotation of the motor in 3D space and, moreover, a delay of ATP binding or ADP release step in the catalytic reaction. Second, we attached a nanodiamond to a membrane protein in live cells to report on cellular membrane dynamics, showing that 3D rotational motion of the membrane protein correlates with intracellular cytoskeletal density. Last, we used the method to track nonrandom motions in the intestine of Caenorhabditis elegans. Collectively, our findings show that the method can record nanoscale 3-DoF rotation in vitro, in cells, and even in vivo. 3-DoF rotation tracking introduces a new perspective on microscopic biological samples, revealing in greater detail the functional mechanisms due to nanoscale dynamics in molecules and cells.

Lipid class composition of membrane and raft fractions from brains of individuals with Alzheimer's disease.

Perturbation of the homeostasis of brain membrane lipids has been implicated in the pathomechanism of Alzheimer's disease (AD). The ε4 allele of the apolipoprotein E gene (APOE) confers an increased risk, in a dosage-dependent manner, for brain amyloid-ß accumulation and the development of sporadic AD. An effect of the APOE genotype on brain lipid homeostasis may underlie the AD risk associated with the ε4 allele. In this research, we examined an effect of APOE ε4 on the lipid class composition of crude membranes and raft-enriched fractions of brains. We applied enzymatic reaction-based methods for the quantification of phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidic acid, and sphingomyelin. Our results indicate that brain lipid class composition was neither significantly altered in AD subjects nor affected by the presence of the APOE ε4 allele.

C. elegans collectively forms dynamical networks.

Understanding physical rules underlying collective motions requires perturbation of controllable parameters in self-propelled particles. However, controlling parameters in animals is generally not easy, which makes collective behaviours of animals elusive. Here, we report an experimental system in which a conventional model animal, Caenorhabditis elegans, collectively forms dynamical networks of bundle-shaped aggregates. We investigate the dependence of our experimental system on various extrinsic parameters (material of substrate, ambient humidity and density of worms). Taking advantage of well-established C. elegans genetics, we also control intrinsic parameters (genetically determined motility) by mutations and by forced neural activation via optogenetics. Furthermore, we develop a minimal agent-based model that reproduces the dynamical network formation and its dependence on the parameters, suggesting that the key factors are alignment of worms after collision and smooth turning. Our findings imply that the concepts of active matter physics may help us to understand biological functions of animal groups.

Noninvasive Mechanochemical Imaging in Unconstrained Caenorhabditis elegans.

Physical forces are transduced into chemical reactions, thereby ultimately making a large impact on the whole-animal level phenotypes such as homeostasis, development and behavior. To understand mechano-chemical transduction, mechanical input should be quantitatively delivered with controllable vibration properties⁻frequency, amplitude and duration, and its chemical output should be noninvasively quantified in an unconstrained animal. However, such an experimental system has not been established so far. Here, we develop a noninvasive and unconstrained mechanochemical imaging microscopy. This microscopy enables us to evoke nano-scale nonlocalized vibrations with controllable vibration properties using a piezoelectric acoustic transducer system and quantify calcium response of a freely moving C. elegans at a single cell resolution. Using this microscopy, we clearly detected the calcium response of a single interneuron during C. elegans escape response to nano-scale vibration. Thus, this microscopy will facilitate understanding of in vivo mechanochemical physiology in the future.

Genome Editing of C. elegans.

Caenorhabditis elegans, a 1 mm long free-living nematode, is a traditional model animal for genetic investigations of various biological processes. Characteristic features that make C. elegans a powerful model of choice for eukaryotic genetic studies include its rapid life cycle, well-annotated genome, simple morphology, and transparency. Recently, genome editing technologies have been increasingly used in C. elegans, thereby facilitating their genetic analyses. Here, I introduce a protocol frequently used in C. elegans genome editing.

Nanoscale Mechanical Stimulation Method for Quantifying C. elegans Mechanosensory Behavior and Memory.

Withdrawal escape response of C. elegans to nonlocalized vibration is a useful behavioral paradigm to examine mechanisms underlying mechanosensory behavior and its memory-dependent change. However, there are very few methods for investigating the degree of vibration frequency, amplitude and duration needed to induce behavior and memory. Here, we establish a new system to quantify C. elegans mechanosensory behavior and memory using a piezoelectric sheet speaker. In the system, we can flexibly change the vibration properties at a nanoscale displacement level and quantify behavioral responses under each vibration property. This system is an economic setup and easily replicated in other laboratories. By using the system, we clearly detected withdrawal escape responses and confirmed habituation memory. This system will facilitate the understanding of physiological aspects of C. elegans mechanosensory behavior in the future.

Genome Editing in C. elegans and Other Nematode Species.

Caenorhabditis elegans, a 1 mm long free-living nematode, is a popular model animal that has been widely utilized for genetic investigations of various biological processes. Characteristic features that make C. elegans a powerful model of choice for eukaryotic genetic studies include its rapid life cycle (development from egg to adult in 3.5 days at 20 °C), well-annotated genome, simple morphology (comprising only 959 somatic cells in the hermaphrodite), and transparency (which facilitates non-invasive fluorescence observations). However, early approaches to introducing mutations in the C. elegans genome, such as chemical mutagenesis and imprecise excision of transposons, have required large-scale mutagenesis screens. To avoid this laborious and time-consuming procedure, genome editing technologies have been increasingly used in nematodes including C. briggsae and Pristionchus pacificus, thereby facilitating their genetic analyses. Here, I review the recent progress in genome editing technologies using zinc-finger nucleases (ZFNs), transcriptional activator-like nucleases (TALENs), and clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 in nematodes and offer perspectives on their use in the future.

Optically Detected Magnetic Resonance of Nanodiamonds In Vivo; Implementation of Selective Imaging and Fast Sampling.

Single-molecule fluorescence measurements of biological samples frequently suffer from background autofluorescence originating from fluorescent materials pre-existing in living samples, and from unstable photo-physical properties of fluorescent labeling molecules. In this study, we first describe our method of selective imaging of nanodiamonds containing nitrogen-vacancy centers, promising fluorescent color centers, by a combination of optically detected magnetic resonance. The resultant images exhibit perfect elimination of extraneous fluorescence in real-time microscope observations. As the practical example applied to an in vivo system, we measured the resonance spectrum of nanodiamonds introduced into the intestine of Caenorhabditis elegans in the clear background and compared the spectral profile over time. The observed evolution strongly suggests that the rotation of the nanodiamond was detected. We also report our recent progress in the development of a spectrometer equipped with an avalanche photo-diode for fast sampling of photons, which can be used while observing the selective image of a field of view in a real-time manner. This apparatus is suitable for exploring dynamics through the measurement of fluctuation in fluorescence intensity caused by a rotating nanodiamond.

High-throughput optical quantification of mechanosensory habituation reveals neurons encoding memory in Caenorhabditis elegans.

A major goal of neuroscience studies is to identify the neurons and molecules responsible for memory. Mechanosensory habituation in Caenorhabditis elegans is a simple form of learning and memory, in which a circuit of several sensory neurons and interneurons governs behavior. However, despite the usefulness of this paradigm, there are hardly any systems for rapid and accurate behavioral genetic analysis. Here, we developed a multiplexed optical system to genetically analyze C. elegans mechanosensory habituation, and identified two interneurons involved in memory formation. The system automatically trains large populations of animals and simultaneously quantifies the behaviors of various strains by optically discriminating between transgenic and nontransgenic animals. Biochemical and cell-specific behavioral analyses indicated that phosphorylation of cyclic AMP response element-binding protein (CREB), a factor known to regulate memory allocation, was facilitated during training and this phosphorylation in AVA and AVD interneurons was required for habituation. These interneurons are a potential target for cell-specific exploration of the molecular substrates of memory.

Simplified method for cell-specific gene expression analysis in Caenorhabditis elegans.

In the neural circuit functional identities of individual neurons are mainly specified by their differential gene expression patterns. Unveiling functional roles of each neuron requires cell-specific interrogation of neural circuitry in the context of gene expressions. The mRNA tagging strategy in Caenorhabditis elegans is a powerful technique, in which cell-specific transcripts can be isolated by co-immunoprecipitating the complexes of mRNAs and epitope-tagged poly(A) binding protein (3× FLAG-PAB-1), expressed in target neurons. However, the conventional protocol requires laborious and time-consuming procedures; chromosomal integration of gene encoding 3× FLAG-PAB-1 and bleaching of obtained integrant animals for the isolation of huge amounts of synchronized animals. In this paper, we have presented a simplified methodology for cell-specific mRNA tagging analysis in C. elegans. We show that mRNA tagging was achieved using transgenic animals expressing 3× FLAG-PAB-1 as an extrachromosomal array under the control of the flp-18 promoter, without the chromosomal integration procedure. Furthermore, we successfully isolated cell-specific mRNAs from adult transgenic animals synchronously grown from eggs laid by gravid adults during a time window of 3h. This simplification facilitates the implementation of cell-specific gene expression analysis of C. elegans, which contributes to the understanding of neural circuitry at a cell-specific resolution.

Versatile strategy for isolating transcription activator-like effector nuclease-mediated knockout mutants in Caenorhabditis elegans.

Targeted genome editing using transcription activator-like effector nuclease (TALEN) and clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 systems has recently emerged as a potentially powerful method for creating locus-specific mutations in Caenorhabditis elegans. Due to the low mutation frequencies, one of the crucial steps in using these technologies is screening animals that harbor a targeted mutation. In previous studies, identifying targeted mutations in C. elegans usually depended on observations of fluorescent markers such as a green fluorescent protein or visible phenotypes such as dumpy and uncoordinated phenotypes. However, this strategy is limited in practice because the phenotypes caused by targeted mutations such as defects in sensory behaviors are often apparently invisible. Here, we describe a versatile strategy for isolating C. elegans knockout mutants by TALEN-mediated genome editing and a heteroduplex mobility assay. We applied TALENs to engineer the locus of the neural gene glr-1, which is a C. elegans AMPA-type receptor orthologue that is known to have crucial roles in various sensory behaviors. Knockout mutations in the glr-1 locus, which caused defective mechanosensory behaviors, were efficiently identified by the heteroduplex mobility assay. Thus, we demonstrated the utility of a TALEN-based knockout strategy for creating C. elegans with mutations that cause invisible phenotypes.

Real-time background-free selective imaging of fluorescent nanodiamonds in vivo.

Recent developments of imaging techniques have enabled fluorescence microscopy to investigate the localization and dynamics of intracellular substances of interest even at the single-molecule level. However, such sensitive detection is often hampered by autofluorescence arising from endogenous molecules. Those unwanted signals are generally reduced by utilizing differences in either wavelength or fluorescence lifetime; nevertheless, extraction of the signal of interest is often insufficient, particularly for in vivo imaging. Here, we describe a potential method for the selective imaging of nitrogen-vacancy centers (NVCs) in nanodiamonds. This method is based on the property of NVCs that the fluorescence intensity sensitively depends on the ground state spin configuration which can be regulated by electron spin magnetic resonance. Because the NVC fluorescence exhibits neither photobleaching nor photoblinking, this protocol allowed us to conduct long-term tracking of a single nanodiamond in both Caenorhabditis elegans and mice, with excellent imaging contrast even in the presence of strong background autofluorescence.

Identification of the AFD neuron as the site of action of the CREB protein in Caenorhabditis elegans thermotaxis.

Behaviour is a consequence of computation in neural circuits composed of massive synaptic connections among sensory neurons and interneurons. The cyclic AMP response element-binding protein (CREB) responsible for learning and memory is expressed in almost all neurons. Nevertheless, we find that the Caenorhabditis elegans CREB orthologue, CRH-1, is only required in the single bilateral thermosensory neuron AFD, for a memory-related behaviour. Restoration of CRH-1 in AFD of CREB-depleted crh-1 mutants rescues its thermotactic defect, whereas restorations in other neurons do not. In calcium-imaging analyses, the AFD neurons of CREB-depleted crh-1 mutants exhibit an abnormal response to temperature increase. We present a new platform for analysing the mechanism of behavioural memory at single-cellular resolution within the neural circuit.