Takuma Sugi

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.

Regulation of behavioral plasticity by systemic temperature signaling in Caenorhabditis elegans

Animals cope with environmental changes by altering behavioral strategy. Environmental information is generally received by sensory neurons in the neural circuit that generates behavior. However, although environmental temperature inevitably influences an animals entire body, the mechanism of systemic temperature perception remains largely unknown. We show here that systemic temperature signaling induces a change in a memory-based behavior in C. elegans. During behavioral conditioning, non-neuronal cells as well as neuronal cells respond to cultivation temperature through a heat-shock transcription factor that drives newly identified gene expression dynamics. This systemic temperature signaling regulates thermosensory neurons non-cell-autonomously through the estrogen signaling pathway, producing thermotactic behavior. We provide a link between systemic environmental recognition and behavioral plasticity in the nervous system.

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.

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

Significance A central aim of neuroscience studies is to locate physical substrates of memory, namely neurons and molecules encoding memory. Achieving this goal requires cell-specific interrogations of neural circuitry. However, it has remained difficult to rapidly and accurately quantify the memory of animals expressing a transgene in a cell-specific manner. Our study presents a powerful optical system to perform cell-specific behavioral genetic analysis in a high-throughput manner, and demonstrates its utility by identifying two interneurons as the neural substrates of mechanosensory memory in C. elegans. We propose that our identified interneurons can be novel targets for cell-specific exploration of the molecular substrates of memory. 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.

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.

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.