Sugi, Takuma et al. (2011) International Worm Meeting "Regulation of behavioral plasticity by systemic temperature perception in Caenorhabditis elegans."

Mori, Ikue, Nishida, Yukuo, Sugi, Takuma

[International Worm Meeting, 2011]

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, environmental temperature inevitably influences animals' entire body, although systemic temperature perception remains largely unknown. We show here that systemic temperature perception induces change of a memory-based behavior. During behavioral conditioning, somatic cells such as body wall muscles and intestine respond to cultivation temperatures through heat-shock transcription factor that drives expression changes of genes involved in estrogen synthesis. The systemic signaling, via an estrogen signaling pathway, regulates cell non-autonomously the AFD thermosensory neuron by modulating the downstream of the cGMP-dependent temperature signaling for thermotactic behavior. We provide a link between systemic environmental recognition and behavioral plasticity by the nervous system. We further identified AFD as a unit of CREB-dependent temperature memory within a neural circuit. CREB is expressed in nearly all cells and best known for its involvement in learning and memory via interactions with the estrogen receptor. We however found that C. elegans CREB ortholog, CRH-1, acts only in the one thermosensory neuron for thermotactic behavior. Surprisingly, restoration of crh-1 in AFD of CRH-1-depleted C. elegans completely rescues its behavioral defect, whereas restorations in other neurons do not. Calcium imaging indicated that CRH-1 is responsible for the responsiveness of AFD neuron to the temperature above a threshold that is set by temperature memory. This presents a novel platform to analyze the mechanism for behavioral memory at the single cellular resolution within the neural network.

Sugi, Takuma (2013) International Worm Meeting "High-throughput behavioral imaging reveals the neurons responsible for mechanosensory memory in C. elegans."

Sugi, Takuma

[International Worm Meeting, 2013]

Behavioral genetic studies in C. elegans have provide a new paradigm by unveiling a role of genes and neurons in a neural circuit. The greatest advantage in C. elegans behavioral studies is that a variety of transgenic experiments, such as rescue experiments and RNAi-mediated gene disruption, can be done in a cell-specific manner. The throughput of these transgenic studies has been increased by developments of image processing software (Swierczek et al., Nat. Mathods., 2011), which accelerate analysis of acquired data. However, the throughput remains to be limited by behavioral assay itself, because discriminations of transgenic worms from nontransgenic worms are required for each strain before or during data acquisition, due to semi-stable inheritance of extrachromosomal transgenes. Here, I developed a multiplexed behavioral imaging system, which simultaneously quantifies behaviors of various different transgenic strains. In order to illuminate transgenic strains labeled by a fluorescent injection marker on the four assay plates placed side by side, I constructed the optical system by spatially multiplexing the beam from a high-power excitation laser and navigating them to all assay plates. Through the USB-based CCD cameras with the emission filters on each assay plate, I succeeded to record movements of fluorescence as behavior of transgenic strains in a wide-field of view (>30 worms), discriminating them from nontransgenic worms. I thus demonstrated high-throughput behavioral imaging by combining this system with the tapping system that delivers mechanical stimuli simultaneously to all assay plates in a temporally controlled manner and with machine-vision software. I tried to identify a site of action of CRH-1/CREB responsible for memory formation, on mechanosensory behavior. I created a variety of transgenic strains that each express the dominant negative form of CRH-1 under the various cell-specific promoters, and quantified their mechanosensory responses. The behavioral imaging indicated that CRH-1 acts in AVA and AVD neurons, suggesting that these neurons are responsible for mechanosensory memory. These results demonstrated that my system can be applied to high-throughput behavioral genetic studies.

Hiroyuki Sasakura et al. (2007) International Worm Meeting "Analysis and screening of genes involved in thermosensory signaling."

Keita Suzuki, Hiroko Ito, Hiroyuki Sasakura, Ikue Mori, Takuma Sugi

[International Worm Meeting, 2007]

C. elegans senses the environmental temperature by AFD and AWC sensory neurons. AFD neurons are specialized sensory neurons for temperature reception. AWC, known as olfactory neurons, also senses the temperature (Kuhara et al., this meeting). Genetic analysis suggested that signal transduction of temperature is similar to that of olfactory and visual system in C. elegans and other animals. Namely, cGMP produced by guanylate cyclases is used as second messenger and cGMP dependent cation channel TAX-2 and TAX-4 are crucial for activation of AFD and AWC. Although thermoreceptor itself is not identified, it is plausible that GPCRs sense the environmental temperature based on analogy to olfactory and visual system. Consistent with this possibility, G alpla ODR-3 and negative regulator of G protein EAT-16 are important for thermosensation in AWC sensory neurons (Kuhara et al., this meeting). GPCRs expressed in AFD and/or AWC become good candiates for thermoreceptors. It has been reported that B0496.5 encodes GPCR and expressed in sensory ending of both AFD and AWC (Colosimo et al., 2004). To examine B0496.5 gene function, we isolated two deletion mutants, nj62 and nj63. From deletion sites, both alleles are likely to be null. When nj62 and nj63 mutants are cultivated in 20 degree, they exhibited significantly smaller isothermal tracking than wild type. We also tested these mutants on linear thermal gradient. When nj62 and nj63 mutants are cultivated in 20 or 23 degree, they migrated to the lower temperature than wild type animals. From behavioral tests, B0496.5 gene likely has an important role on thermosensation. Overexpression experiments and physiological analysis are in progress. We also performed forward genetic screening to isolate new genes involved in AFD thermosensory signaling. The gene expression of nhr-38 is AFD specific and regulated by temperature stimulus. For example, the mutations of tax-4 or cmk-1 genes induce the downregulation of nhr-38::GFP (Satterlee et al., 2004; H.S., I.M., A.Kuhara, unpublished results). We utilized this phenomenon and isolated 12 suppressor strains that recover the gene expression of nhr-38::GFP in cmk-1 mutants. The behaviors of 11 strains are identical to athermotactic phenotype of cmk-1 mutants, while one suppressor strain IK673 exhibited neomorpholic, cryophilic phenotype. The gene responsible for this mutation was mapped on Ch I -0.69 to + 2.98. We thank T. Ishihara for nhr-38::GFP(H13::GFP), H. Inada for TMV-UV library, and CGC for cmk-1 mutants.

Maruo, Ayana et al. (2017) International Worm Meeting "Single cell transcriptome analysis of ASJ thermosensory neuron regulating cold tolerance."

Takagaki, Natsune, Maruo, Ayana, Ohta, Akane, Kuhara, Atsushi

[International Worm Meeting, 2017]

ASJ sensory neuron that has been known as a light and pheromone sensing neuron senses temperature and regulates cold tolerance through insulin signaling (Ohta, Ujisawa et al., Nature commun, 2014). Cold tolerance in C. elegans is a phenomenon that 20 deg C-cultivated animals cannot survive at 2 deg C, whereas 15 deg C-cultivated animals can survive at 2 deg C. Our previous analyses suggest that cold tolerance is regulated by tissues network containing neurons, intestine and sperm, in which a feedback regulation from sperm to ASJ thermosensory neuron is essential (Sonoda, et al., Cell Reports, 2016). However, we have not known thermo-sensing molecules on ASJ and how ASJ is controlled by sperm signaling remain poorly understood. To identify new genes involved in temperature sensation and temperature tolerance, we conducted RNA-sequencing analysis comparing mRNA expression levels between ASJ and ASE gustatory neuron by using single cell RNA-sequencing analysis (Sugi & Ohtani, BBRC 2014). mRNAs were extracted from animals that were cultivated at constant temperature (15 deg C, 20 deg C, or 25 deg C) or cultivated under temperature-shifted conditions (15 deg C to 25 deg C or 25 deg C to 15 deg C). By comparing gene expression levels between ASJ and ASE in 20 deg C cultivated-animals, we found the expression levels of ~600 genes such as neuropeptides, neuropeptide receptors, insulin were altered. We measured cold tolerance phenotypes of about fifty mutants defective in those genes, and found that mutants defective in avr-15 encoding glutamate-gated chloride channel and clh-1 encoding CLC-type chloride channels showed abnormal cold tolerance.

Nishida, Yukuo et al. (2009) International Worm Meeting "Genome-wide analysis of thermotactic behavior controlled by CREB and its downstream genes in Caenorhabditis elegans."

Sugi, Takuma, Mori, Ikue, Nishida, Yukuo

[International Worm Meeting, 2009]

Animals memorize environmental information for optimal behavior. In order to understand the molecular basis of memory formation, we took advantage of C. elegans thermotactic behavior, which was designated with the simple neural circuit, including processes of thermosensation and memory formation (Mori et al., 2007). In the latest study, we revealed that heat-shock transcription factor acts as a body thermal sensor, thereby controlling thermotactic behavior (Sugi et al., unpublished). This work shed light on the requirement of transcriptional event for thermotactic behavior. We then focused on the transcriptional factor crh-1, the ortholog of cAMP responsible element binding protein (CREB), because CREB is a representative example as a memory-regulated transcriptional factor. Yet, little is known about the molecular identities of CREB downstream genes, although the expectant role of CREB is to control the activation of the genes that are critical for memory formation. We first examined the importance of crh-1 in thermotaxis of C. elegans. When wild-type animals and crh-1 mutants were cultivated at 17 deg C, both animals migrated to lower temperature regions on the thermal gradient. In the case of cultivation at 23 deg C, although wild-type animals migrated to higher temperature regions, the mutants dispersed on the thermal gradient. We further conducted the temperature-shifted thermotaxis assay. At 3 hours after shifting the cultivation temperature from 17 deg C to 23 deg C, although wild-type animals changed to migrate to the new temperature 23 deg C, the mutants changed to disperse on the thermal gradient. These results suggest that crh-1 mutants exhibit defect in acquisition of higher temperature memory, further implying that transcriptional activity of CRH-1 is essential for memorizing a higher temperature. To isolate the genes generating the temperature memory, we proceed to conduct the genome-wide microarray analysis in combination with the temperature-shifted thermotaxis assay. Then we plan to compare the gene expression changes between wild-type animals and crh-1 mutants during the higher temperature acquisition process. In addition, cell-specific rescue experiments are underway to identify cells in which transcriptional activity of CRH-1 is required for thermotaxis. We will then examine the relationship between the known molecular pathway within neurons identified by the cell-specific rescue experiments of crh-1 mutants and the pathway regulated by the isolated CRH-1 downstream genes. We conceive that further analyses of crh-1 and its downstream genes provide comprehensive insights into the molecular basis for temperature memory of thermotactic behavior.