Screening for cold tolerance genes in C. elegans, whose expressions are affected by anticancer drugs camptothecin and leptomycin B.

Temperature is a vital environmental factor affecting organisms' survival as they determine the mechanisms to tolerate rapid temperature changes. We demonstrate an experimental system for screening chemicals that affect cold tolerance in Caenorhabditis elegans. The anticancer drugs leptomycin B and camptothecin were among the 4000 chemicals that were screened as those affecting cold tolerance. Genes whose expression was affected by leptomycin B or camptothecin under cold stimuli were investigated by transcriptome analysis. Abnormal cold tolerance was detected in several mutants possessing genes that were rendered defective and whose expression altered after exposure to either leptomycin B or camptothecin. The genetic epistasis analysis revealed that leptomycin B or camptothecin may increase cold tolerance by affecting a pathway upstream of the insulin receptor DAF-2 that regulates cold tolerance in the intestine. Our experimental system combining drug and cold tolerance could be used for a comprehensive screening of genes that control cold tolerance at a low cost and in a short time period.

G protein-coupled receptor-based thermosensation determines temperature acclimatization of Caenorhabditis elegans.

Animals must sense and acclimatize to environmental temperatures for survival, yet their thermosensing mechanisms other than transient receptor potential (TRP) channels remain poorly understood. We identify a trimeric G protein-coupled receptor (GPCR), SRH-40, which confers thermosensitivity in sensory neurons regulating temperature acclimatization in Caenorhabditis elegans. Systematic knockdown of 1000 GPCRs by RNAi reveals GPCRs involved in temperature acclimatization, among which srh-40 is highly expressed in the ADL sensory neuron, a temperature-responsive chemosensory neuron, where TRP channels act as accessorial thermoreceptors. In vivo Ca2+ imaging demonstrates that an srh-40 mutation reduced the temperature sensitivity of ADL, resulting in supranormal temperature acclimatization. Ectopically expressing SRH-40 in a non-warmth-sensing gustatory neuron confers temperature responses. Moreover, temperature-dependent SRH-40 activation is reconstituted in Drosophila S2R+ cells. Overall, SRH-40 may be involved in thermosensory signaling underlying temperature acclimatization. We propose a dual thermosensing machinery through a GPCR and TRP channels in a single sensory neuron.

Temperature acclimation: Temperature shift induces system conversion to cold tolerance in C. elegans.

Acclimation to temperature is one of the survival strategies used by organisms to adapt to changing environmental temperatures. Caenorhabditis elegans' cold tolerance is altered by previous cultivation temperature, and similarly, past low-temperature induces a longer lifespan. Temperature is thought to cause a large shift in homeostasis, lipid metabolism, and reproduction in the organism because it is a direct physiological factor during chemical events. This paper will share and discuss what we know so far about the neural and molecular mechanisms that control cold tolerance and lifespan by altering lipid metabolism and physiological characteristics. We hope that this will contribute to a better understanding of how organisms respond to temperature changes.

Head-tail-head neural wiring underlies gut fat storage in Caenorhabditis elegans temperature acclimation.

Animals maintain the ability to survive and reproduce by acclimating to environmental temperatures. We showed here that Caenorhabditis elegans exhibited temperature acclimation plasticity, which was regulated by a head-tail-head neural circuitry coupled with gut fat storage. After experiencing cold, C. elegans individuals memorized the experience and were prepared against subsequent cold stimuli. The cyclic adenosine monophosphate (cAMP) response element-binding protein (CREB) regulated temperature acclimation in the ASJ thermosensory neurons and RMG head interneurons, where it modulated ASJ thermosensitivity in response to past cultivation temperature. The PVQ tail interneurons mediated the communication between ASJ and RMG via glutamatergic signaling. Temperature acclimation occurred via gut fat storage regulation by the triglyceride lipase ATGL-1, which was activated by a neuropeptide, FLP-7, downstream of CREB. Thus, a head-tail-head neural circuit coordinated with gut fat influenced experience-dependent temperature acclimation.

Molecular physiology regulating cold tolerance and acclimation of Caenorhabditis elegans.

Many organisms can survive and proliferate in changing environmental temperatures. Here, we introduce a molecular physiological mechanism for cold tolerance and acclimation of the nematode Caenorhabditis elegans on the basis of previous reports and a new result. Three types of thermosensory neurons located in the head, ASJ, ASG, and ADL, regulate cold tolerance and acclimation. In ASJ, components of the light-signaling pathway are involved in thermosensation. In ASG, mechanoreceptor DEG-1 acts as thermoreceptor. In ADL, transient receptor potential channels are thermoreceptors; however, the presence of an additional unidentified thermoreceptor is also speculated. ADL thermoresponsivity is modulated by oxygen sensory signaling from URX oxygen sensory neurons via hub interneurons. ASJ releases insulin and steroid hormones that are received by the intestine, which results in lipid composition changing with cold tolerance. Additionally, the intestinal transcriptional alteration affects sperm functions, which in turn affects the thermosensitivity of ASJ; thus, the neuron-intestine-sperm-neuron tissue circuit is essential for cold tolerance.

Cellular identity and Ca2+ signaling activity of the non-reproductive GnRH system in the Ciona intestinalis type A (Ciona robusta) larva.

Tunicate larvae have a non-reproductive gonadotropin-releasing hormone (GnRH) system with multiple ligands and receptor heterodimerization enabling complex regulation. In Ciona intestinalis type A larvae, one of the gnrh genes, gnrh2, is conspicuously expressed in the motor ganglion and nerve cord, which are homologous structures to the hindbrain and spinal cord, respectively, of vertebrates. The gnrh2 gene is also expressed in the proto-placodal sensory neurons, which are the proposed homologue of vertebrate olfactory neurons. Tunicate larvae occupy a non-reproductive dispersal stage, yet the role of their GnRH system remains elusive. In this study, we investigated neuronal types of gnrh2-expressing cells in Ciona larvae and visualized the activity of these cells by fluorescence imaging using a calcium sensor protein. Some cholinergic neurons and dopaminergic cells express gnrh2, suggesting that GnRH plays a role in controlling swimming behavior. However, none of the gnrh2-expressing cells overlap with glycinergic or GABAergic neurons. A role in motor control is also suggested by a relationship between the activity of gnrh2-expressing cells and tail movements. Interestingly, gnrh2-positive ependymal cells in the nerve cord, known as a kind of glia cells, actively produced Ca2+ transients, suggesting that active intercellular signaling occurs in the glia cells of the nerve cord.

OSM-9 and OCR-2 TRPV channels are accessorial warm receptors in Caenorhabditis elegans temperature acclimatisation.

Caenorhabditis elegans (C. elegans) exhibits cold tolerance and temperature acclimatisation regulated by a small number of head sensory neurons, such as the ADL temperature-sensing neurons that express three transient receptor potential vanilloid (TRPV) channel subunits, OSM-9, OCR-2, and OCR-1. Here, we show that an OSM-9/OCR-2 regulates temperature acclimatisation and acts as an accessorial warmth-sensing receptor in ADL neurons. Caenorhabditis elegans TRPV channel mutants showed abnormal temperature acclimatisation. Ectopic expression of OSM-9 and OCR-2 in non-warming-responsive gustatory neurons in C. elegans and Xenopus oocytes revealed that OSM-9 and OCR-2 cooperatively responded to warming; however, neither TRPV subunit alone was responsive to warming. A warming-induced OSM-9/OCR-2-mediated current was detectable in Xenopus oocytes, yet ADL in osm-9 ocr-2 double mutant responds to warming; therefore, an OSM-9/OCR-2 TRPV channel and as yet unidentified temperature receptor might coordinate transmission of temperature signalling in ADL temperature-sensing neurons. This study demonstrates direct sensation of warming by TRPV channels in C. elegans.

Temperature signaling underlying thermotaxis and cold tolerance in Caenorhabditis elegans.

Caenorhabditis elegans has a simple nervous system of 302 neurons. It however senses environmental cues incredibly precisely and produces various behaviors by processing information in the neural circuit. In addition to classical genetic analysis, fluorescent proteins and calcium indicators enable in vivo monitoring of protein dynamics and neural activity on either fixed or free-moving worms. These analyses have provided the detailed molecular mechanisms of neuronal and systemic signaling that regulate worm responses. Here, we focus on responses of C. elegans against temperature and review key findings that regulate thermotaxis and cold tolerance. Thermotaxis of C. elegans has been studied extensively for almost 50 years, and cold tolerance is a relatively recent concept in C. elegans. Although both thermotaxis and cold tolerance require temperature sensation, the responsible neurons and molecular pathways are different, and C. elegans uses the proper mechanisms depending on its situation. We summarize the molecular mechanisms of the major thermosensory circuit as well as the modulatory strategy through neural and tissue communication that enables fine tuning of thermotaxis and cold tolerance.

The mechanoreceptor DEG-1 regulates cold tolerance in Caenorhabditis elegans.

Caenorhabditis elegans mechanoreceptors located in ASG sensory neurons have been found to sense ambient temperature, which is a key trait for animal survival. Here, we show that experimental loss of xanthine dehydrogenase (XDH-1) function in AIN and AVJ interneurons results in reduced cold tolerance and atypical neuronal response to changes in temperature. These interneurons connect with upstream neurons such as the mechanoreceptor-expressing ASG. Ca2+ imaging revealed that ASG neurons respond to warm temperature via the mechanoreceptor DEG-1, a degenerin/epithelial Na+ channel (DEG/ENaC), which in turn affects downstream AIN and AVJ circuits. Ectopic expression of DEG-1 in the ASE gustatory neuron results in the acquisition of warm sensitivity, while electrophysiological analysis revealed that DEG-1 and human MDEG1 were involved in warm sensation. Taken together, these results suggest that cold tolerance is regulated by mechanoreceptor-mediated circuit calculation.

daf-16/FOXO isoform b in AIY neurons is involved in low preference for Bifidobacterium infantis in Caenorhabditis elegans.

The neural and molecular mechanisms underlying food preference have been poorly understood. We previously showed that Bifidobacterium infantis (B. infantis), a well-known probiotic bacterium, extends the lifespan of Caenorhabditis elegans (C. elegans) compared with a standard food, Escherichia coli (E. coli) OP50. In this study, we characterized C. elegans behavior against B. infantis and examined the neural and molecular mechanisms governing that behavior. The majority of the wild-type animals were outside of the B. infantis lawn 10 min after transfer. Although worms did not prefer B. infantis compared to E. coli OP50, they preferred the B. infantis lawn over a lawn containing M9 buffer alone, in which there was no food. Mutant analyses suggested that leaving the B. infantis lawn required daf-16/FOXO. Isoform-specific mutant phenotypes suggested that daf-16 isoform b seemed to be associated with leaving. Genetic rescue experiments demonstrated that the function of daf-16b in AIY interneurons was involved in leaving the B. infantis lawn. The daf-18/PTEN mutants were also defective in leaving. In conclusion, C. elegans showed a low preference for B. infantis, and daf-16b in AIY interneurons and daf-18 had roles in leaving B. infantis.

Cold acclimation via the KQT-2 potassium channel is modulated by oxygen in Caenorhabditis elegans.

Adaptive responses to external temperatures are essential for survival in changing environments. We show here that environmental oxygen concentration affects cold acclimation in Caenorhabditis elegans and that this response is regulated by a KCNQ-type potassium channel, KQT-2. Depending on culture conditions, kqt-2 mutants showed supranormal cold acclimation, caused by abnormal thermosensation in ADL chemosensory neurons. ADL neurons are responsive to temperature via transient receptor potential channels-OSM-9, OCR-2, and OCR-1-with OCR-1 negatively regulating ADL function. Similarly, KQT-2 and KQT-3 regulate ADL activity, with KQT-2 positively regulating ADL function. Abnormal cold acclimation and acute temperature responses of ADL neurons in kqt-2 mutants were suppressed by an oxygen-receptor mutation in URX coelomic sensory neurons, which are electrically connected to ADL via RMG interneurons. Likewise, low oxygen suppressed supranormal kqt-2 cold acclimation. These data thus demonstrate a simple neuronal circuit integrating two different sensory modalities, temperature and oxygen, that determines cold acclimation.

Endoribonuclease ENDU-2 regulates multiple traits including cold tolerance via cell autonomous and nonautonomous controls in Caenorhabditis elegans.

Environmental temperature acclimation is essential to animal survival, yet thermoregulation mechanisms remain poorly understood. We demonstrate cold tolerance in Caenorhabditis elegans as regulated by paired ADL chemosensory neurons via Ca2+-dependent endoribonuclease (EndoU) ENDU-2. Loss of ENDU-2 function results in life span, brood size, and synaptic remodeling abnormalities in addition to enhanced cold tolerance. Enzymatic ENDU-2 defects localized in the ADL and certain muscle cells led to increased cold tolerance in endu-2 mutants. Ca2+ imaging revealed ADL neurons were responsive to temperature stimuli through transient receptor potential (TRP) channels, concluding that ADL function requires ENDU-2 action in both cell-autonomous and cell-nonautonomous mechanisms. ENDU-2 is involved in caspase expression, which is central to cold tolerance and synaptic remodeling in dorsal nerve cord. We therefore conclude that ENDU-2 regulates cell type-dependent, cell-autonomous, and cell-nonautonomous cold tolerance.

Diverse Regulation of Temperature Sensation by Trimeric G-Protein Signaling in Caenorhabditis elegans.

Temperature sensation by the nervous system is essential for life and proliferation of animals. The molecular-physiological mechanisms underlying temperature signaling have not been fully elucidated. We show here that diverse regulatory machinery underlies temperature sensation through trimeric G-protein signaling in the nematode Caenorhabditis elegans. Molecular-genetic studies demonstrated that cold tolerance is regulated by additive functions of three Gα proteins in a temperature-sensing neuron, ASJ, which is also known to be a light-sensing neuron. Optical recording of calcium concentration in ASJ upon temperature-changes demonstrated that three Gα proteins act in different aspects of temperature signaling. Calcium concentration changes in ASJ upon temperature change were unexpectedly decreased in a mutant defective in phosphodiesterase, which is well known as a negative regulator of calcium increase. Together, these data demonstrate commonalities and differences in the molecular components concerned with light and temperature signaling in a single sensory neuron.

Caenorhabditis elegans homologue of Prox1/Prospero is expressed in the glia and is required for sensory behavior and cold tolerance.

The Caenorhabditis elegans (C. elegans) amphid sensory organ contains only 4 glia-like cells and 24 sensory neurons, providing a simple model for analyzing glia or neuron-glia interactions. To better characterize glial development and function, we carried out RNA interference screening for transcription factors that regulate the expression of an amphid sheath glial cell marker and identified pros-1, which encodes a homeodomain transcription factor homologous to Drosophila prospero/mammalian Prox1, as a positive regulator. The functional PROS-1::EGFP fusion protein was localized in the nuclei of the glia and the excretory cell but not in the amphid sensory neurons. pros-1 deletion mutants exhibited larval lethality, and rescue experiments showed that pros-1 and human Prox1 transgenes were able to rescue the larval lethal phenotype, suggesting that pros-1 is a functional homologue of mammalian Prox1, at least partially. We further found that the structure and functions of sensory neurons, such as the morphology of sensory endings, sensory behavior and sensory-mediated cold tolerance, appeared to be affected by the pros-1 RNAi. Together, our results show that the C. elegans PROS-1 is a transcriptional regulator in the glia but is involved not only in sensory behavior but also in sensory-mediated physiological tolerance.

Natural variations of cold tolerance and temperature acclimation in Caenorhabditis elegans.

Temperature is critical for the survival and proliferation of animals, which must be adapted to cope with environmental temperature changes. In this study, we demonstrated natural variations in the phenotypes of temperature tolerance and temperature acclimation of the nematode Caenorhabditis elegans, and we decoded whole genome sequence of six natural variations, which enabled us to map responsible gene polymorphisms onto specific chromosomal regions. The C. elegans laboratory strain, N2, survives at 2 °C after cultivation at 15 °C but is unable to survive at 2 °C after cultivation at 20 or 25 °C. This cultivation-temperature-dependent cold tolerance occurs within a few hours after the temperature shift and is termed cold acclimation. We measured the cold tolerance and cold acclimation phenotypes of many natural variants isolated from various areas. CB4854 showed weaker cold tolerance associated with gene polymorphisms on the sex chromosome decoded by whole genome sequencing. Variable cold acclimation phenotypes were exhibited in twelve natural isolates and the large difference was seen between CB4856 and AB1 strains. CB4856, isolated from Hawaii, acclimated slowly to a new temperature, whereas AB1, isolated from Australia, acclimated rapidly. By the whole genome sequencing analysis, two different polymorphisms responsible for the accelerated cold acclimation in AB1 were mapped to specific chromosomal regions.

Sperm Affects Head Sensory Neuron in Temperature Tolerance of Caenorhabditis elegans.

Tolerance to environmental temperature change is essential for the survival and proliferation of animals. The process is controlled by various body tissues, but the orchestration of activity within the tissue network has not been elucidated in detail. Here, we show that sperm affects the activity of temperature-sensing neurons (ASJ) that control cold tolerance in Caenorhabditis elegans. Genetic impairment of sperm caused abnormal cold tolerance, which was unexpectedly restored by impairment of temperature signaling in ASJ neurons. Calcium imaging revealed that ASJ neuronal activity in response to temperature was decreased in sperm mutant gsp-4 with impaired protein phosphatase 1 and rescued by expressing gsp-4 in sperm. Genetic analysis revealed a feedback network in which ASJ neuronal activity regulates the intestine through insulin and a steroid hormone, which then affects sperm and, in turn, controls ASJ neuronal activity. Thus, we propose that feedback between sperm and a sensory neuron mediating temperature tolerance.

Reconstruction of Spatial Thermal Gradient Encoded in Thermosensory Neuron AFD in Caenorhabditis elegans.

During navigation, animals process temporal sequences of sensory inputs to evaluate the surrounding environment. Thermotaxis of Caenorhabditis elegans is a favorable sensory behavior to elucidate how navigating animals process sensory signals from the environment. Sensation and storage of temperature information by a bilaterally symmetric pair of thermosensory neurons, AFD, is essential for the animals to migrate toward the memorized temperature on a thermal gradient. However, the encoding mechanisms of the spatial environment with the temporal AFD activity during navigation remain to be elucidated. Here, we show how the AFD neuron encodes sequences of sensory inputs to perceive spatial thermal environment. We used simultaneous calcium imaging and tracking system for a freely moving animal and characterized the response property of AFD to the thermal stimulus during thermotaxis. We show that AFD neurons respond to shallow temperature increases with intermittent calcium pulses and detect temperature differences with a critical time window of 20 s, which is similar to the timescale of behavioral elements of C. elegans, such as turning. Convolution of a thermal stimulus and the identified response property successfully reconstructs AFD activity. Conversely, deconvolution of the identified response kernel and AFD activity reconstructs the shallow thermal gradient with migration trajectory, indicating that AFD activity and the migration trajectory are sufficient as the encoded signals for thermal environment. Our study demonstrates bidirectional transformation between environmental thermal information and encoded neural activity. SIGNIFICANCE STATEMENT: Deciphering how information is encoded in the nervous system is an important challenge for understanding the principles of information processing in neural circuits. During navigation behavior, animals transform spatial information to temporal patterns of neural activity. To elucidate how a sensory system achieves this transformation, we focused on a thermosensory neuron in Caenorhabditis elegans called AFD, which plays a major role in a sensory behavior. Using tracking and calcium imaging system for freely moving animals, we identified the response property of the AFD. The identified response property enabled us to reconstruct both neural activity from a temperature stimulus and a spatial thermal environment from neural activity. These results shed light on how a sensory system encodes the environment.

Light and pheromone-sensing neurons regulates cold habituation through insulin signalling in Caenorhabditis elegans.

Temperature is a critical environmental stimulus that has a strong impact on an organism's biochemistry. Animals can respond to changes in ambient temperature through behaviour or altered physiology. However, how animals habituate to temperature is poorly understood. The nematode C. elegans stores temperature experiences and can induce temperature habituation-linked cold tolerance. Here we show that light and pheromone-sensing neurons (ASJ) regulate cold habituation through insulin signalling. Calcium imaging reveals that ASJ neurons respond to temperature. Cold habituation is abnormal in a mutant with impaired cGMP signalling in ASJ neurons. Insulin released from ASJ neurons is received by the intestine and neurons regulating gene expression for cold habituation. Thus, temperature sensation in a light and pheromone-sensing neuron produces a robust effect on insulin signalling that controls experience-dependent temperature habituation.

Molecular mechanism for trimetric G protein-coupled thermosensation and synaptic regulation in the temperature response circuit of Caenorhabditis elegans.

How the nervous system controls the sensation and memory of information from the environment is an essential question. The nematode Caenorhabditis elegans is a useful model for elucidating neural information processing that mediates sensation and memory. The entire nervous system of C. elegans consists of only 302 neurons, and their wiring diagram has been revealed by electron microscopy analysis. Here, we review the molecular and physiological mechanisms responsible for the neural circuit-mediated temperature-seeking behavior (thermotaxis) in C. elegans. Recent molecular biology studies and optogenetic analyses, such as the optical manipulation of neural activity, and neural imaging have revealed the novel concept of neural calculation. Most significantly, trimetric G proteincoupled thermosensation, single sensory neuron-based memory, and the orchestrated synaptic transmission system have been elucidated.

A novel and conserved protein AHO-3 is required for thermotactic plasticity associated with feeding states in Caenorhabditis elegans.

Although a large proportion of molecules expressed in the nervous system are conserved from invertebrate to vertebrate, functional properties of such molecules are less characterized. Here, we show that highly conserved hydrolase AHO-3 acts as a novel regulator of starvation-induced thermotactic plasticity in Caenorhabditis elegans. As wild-type animals, aho-3 mutants migrated to the cultivation temperature on a linear thermal gradient after cultivation at a particular temperature with food. Whereas wild-type animals cultivated under food-deprived condition showed dispersed distribution on the gradient, aho-3 mutants exhibited tendency to migrate toward higher temperature. Such an abnormal behavior was completely rescued by the expression of human homologue of AHO-3, indicating that the molecular function of AHO-3 is highly conserved between nematode and human. The behavioral regulation by AHO-3 requires the N-terminal cysteine cluster, which ensures the proper subcellular localization of AHO-3 to sensory endings. Double-mutant analysis suggested that AHO-3 acts in the same pathway with ODR-3, a heterotrimeric G protein alpha subunit. Our results unveiled a novel neural protein in C. elegans, confirming its conserved role in behavioral regulation.