Kuhara A et al. (2024) Adv Exp Med Biol "Cold Tolerance in the Nematode Caenorhabditis elegans."

Ohta A, Kuhara A, Takagaki N, Okahata M

[Adv Exp Med Biol, 2024]

Organisms receive environmental information and respond accordingly in order to survive and proliferate. Temperature is the environmental factor of most immediate importance, as exceeding its life-supporting range renders essential biochemical reactions impossible. In this chapter, we introduce the mechanisms underlying cold tolerance and temperature acclimation in a model organism-the nematode Caenorhabditis elegans, at molecular and physiological levels. Recent investigations utilizing molecular genetics and neural calcium imaging have unveiled a novel perspective on cold tolerance within the nematode worm. Notably, the ASJ neuron, previously known to possess photosensitive properties, has been found to sense temperature and regulate the sperm and gut cell-mediated pathway underlying cold tolerance. We will also explore C. elegans' cold tolerance and cold acclimation at the molecular and tissue levels.

Kuhara A et al. (2008) Tanpakushitsu Kakusan Koso "[Neural circuit mechanism underlying thermotaxis behavior in C. elegans]."

Sasakura H, Mori I, Kuhara A, Kimata T

[Tanpakushitsu Kakusan Koso, 2008]

Kuhara A et al. (2006) J Neurosci "Molecular physiology of the neural circuit for calcineurin-dependent associative learning in Caenorhabditis elegans."

Kuhara A, Mori I

[J Neurosci, 2006]

How learning and memory is controlled at the neural circuit level is a fundamental question in neuroscience. However, molecular and cellular dissection of the neural circuits underlying learning and memory is extremely complicated in higher animals. Here, we report a simple neural circuit for learning behavior in Caenorhabditis elegans, where the calcium-activated phosphatase, calcineurin, acts as an essential modulator. The calcineurin mutant tax-6 showed defective feeding state-dependent learning behavior for temperature and salt. Surprisingly, defective associative learning between temperature and feeding state was caused by malfunctions of two pairs of directly connected interneurons, AIZ and RIA, in the mature nervous system. Monitoring temperature-evoked Ca2+ concentration changes in the AIZ-RIA neural pathway revealed that starvation, a conditioning factor, downregulated AIZ activity through calcineurin during associative learning between temperature and feeding state. Our results demonstrate the molecular and physiological mechanisms of a simple neural circuit for calcineurin-mediated associative learning behavior.

Kuhara A et al. (2008) Science "Temperature sensing by an olfactory neuron in a circuit controlling behavior of C. elegans."

Kimata T, Okumura M, Kimura KD, Inada H, Tanizawa Y, Takano R, Kuhara A, Matsumoto K, Mori I

[Science, 2008]

Temperature is an unavoidable environmental cue that affects metabolism and behavior of any creature on earth, yet how animals perceive temperature is poorly understood. The nematode Caenorhabditis elegans memorizes temperatures, and this stored information modifies subsequent migration in temperature gradient. We show that the olfactory neuron designated AWC senses temperature. Ca(2+) imaging revealed that AWC responds to temperature changes and that response thresholds differ depending on the temperature to which the animal was previously exposed. In the mutant with impairment of a heteromeric guanine nucleotide-binding protein (G protein) mediated signaling, AWC was hyperresponsive to temperature, whereas the AIY interneuron postsynaptic to AWC was hyporesponsive to temperature. Thus, temperature sensation exhibits a robust influence on a neural circuit controlling a memory-regulated behavior.

Kuhara A et al. (2011) Nat Commun "Neural coding in a single sensory neuron controlling opposite seeking behaviours in Caenorhabditis elegans."

Shimowada T, Mori I, Kuhara A, Ohnishi N

[Nat Commun, 2011]

Unveiling the neural codes for intricate behaviours is a major challenge in neuroscience. The neural circuit for the temperature-seeking behaviour of Caenorhabditis elegans is an ideal system to dissect how neurons encode sensory information for the execution of behavioural output. Here we show that the temperature-sensing neuron AFD transmits both stimulatory and inhibitory neural signals to a single interneuron AIY. In this circuit, a calcium concentration threshold in AFD acts as a switch for opposing neural signals that direct the opposite behaviours. Remote control of AFD activity, using a light-driven ion pump and channel, reveals that diverse reduction levels of AFD activity can generate warm- or cold-seeking behaviour. Calcium imaging shows that AFD uses either stimulatory or inhibitory neuronal signalling onto AIY, depending on the calcium concentration threshold in AFD. Thus, dual neural regulation in opposite directions is directly coupled to behavioural inversion in the simple neural circuit.

Kuhara A et al. (2002) Neuron "Negative regulation and gain control of sensory neurons by the C. elegans calcineurin TAX-6."

Mori I, Katsura I, Inada H, Kuhara A

[Neuron, 2002]

Animals sense and adapt to variable environments by regulating appropriate sensory signal transduction pathways. Here, we show that calcineurin plays a key role in regulating the gain of sensory neuron responsiveness across multiple modalities. C. elegans animals bearing a loss-of-function mutation in TAX-6, a calcineurin A subunit, exhibit pleiotropic abnormalities, including many aberrant sensory behaviors. The tax-6 mutant defect in thermosensation is consistent with hyperactivation of the AFD thermosensory neurons. Conversely, constitutive activation of TAX-6 causes a behavioral phenotype consistent with inactivation of AFD neurons. In olfactory neurons, the impaired olfactory response of tax-6 mutants to an AWC-sensed odorant is caused by hyperadaptation, which is suppressible by a mutation causing defective olfactory adaptation. Taken together, our results suggest that stimulus-evoked calcium entry activates calcineurin, which in turn negatively regulates multiple aspects of sensory signaling.

Kuhara, Atsushi et al. (2009) International Worm Meeting "Exploring the neural code in the neural circuit for thermotaxis behavior."

Mori, Ikue, Shimowada, Tomoyasu, Kuhara, Atsushi, Ohnishi, Noriyuki

[International Worm Meeting, 2009]

Behavior is an ultimate consequence of orchestrated neural calculation. Thermotaxis of C. elegans is an ideal system for comprehensively understanding how a neural circuit encodes a behavioral output (1). In thermotaxis neural circuit, temperature is mainly sensed by AFD neuron, and its information is conveyed to AIY interneuron (1, 2). To elucidate neural calculation, we aimed to inactivate neuronal activity by employing light-activated chloride pump halorhodopsin (HR) (3). Millisecond scale pulsed-light system, which allows Hz-controllable deliveries of light, was developed and equipped in both calcium imaging microscope and C. elegans auto-tracking microscope. Calcium imaging analysis demonstrated that pulsed-excitation of HR in AFD partially reduced calcium influx in AFD for thermal stimuli. Since AFD-ablated animals move randomly or migrate to lower temperature than the cultivation temperature on a thermal gradient (1), we speculated that exciting HR in AFD induces cryophilic or athermotactic abnormalities. We unexpectedly found that pulsed-excitation of HR in AFD induced thermophlic abnormality. Similar abnormality was observed in the mutant exhibiting an abnormal glutamate synaptic transmission in AFD due to AFD-specific defect in EAT-4 (vesicular glutamate transporter) (N. O., A. K. and I. M., this meeting). Previous report (4) and our calcium imaging analysis revealed that calcium influx in AFD for thermal stimuli induced calcium influx in AIY. This suggests that temperature information of AFD is conveyed to AIY through "excitatory" connection. We however found that pulsed-excitation of HR in AFD notably enhanced calcium influx of AIY for thermal stimuli, despite AFD activity itself was partially reduced. Similar abnormal thermal responses of both AFD and AIY were observed in thermophilic mutant tax-6 (calcineurin) that is defective in AFD thermosensory signaling (5, 6), implicating "inhibitory" connection from AFD to AIY. Altogether, these physiological and behavioral results are consistent with the notion that temperature signal in AFD dynamically affects AIY activity through both "inhibitory" and "excitatory" transmissions, which as a consequence likely generates opposite thermotactic behaviors, thermophilic and cryophlic migration on a temperature gradient. (1) Mori and Ohshima, Nature, 1995 (2) Kuhara, Okumura, et al., Science, 2008 (3) Zhang et al., Nature, 2007 (4) Biron et al., Nature Neuroscience, 2006 (5) Kuhara et al., Neuron, 2002 (6) Kuhara and Mori, J. Neurosci, 2006.

Atsushi Kuhara et al. (2005) International Worm Meeting "Essential neural circuit for associative learning between temperature and starvation"

Ikue Mori, Atsushi Kuhara

[International Worm Meeting, 2005]

Neural circuit regulating learning and memory is highly complicated in mammalian nervous system. How learning and memory is controlled at neural circuit level? Here we show the simplest neural circuit regulating associative learning in C. elegans, where calcineurin, the calcium-activated protein phosphatase, plays a critical role as a modulator. The calcineurin encoded by tax-6 gene is expressed in nearly all neurons including interneurons (1), which should be the essential site for learning processes. To investigate the function of TAX-6 calcineurin in the aspect of learning, we made use of the interneuronal TAX-6 deficient transgenic mutants, tax-6;Ex[sensory+, inter-], where TAX-6 is expressed only in sensory neurons of tax-6 null mutants. We found that tax-6;Ex[sensory+, inter-] animals showed defects in two associative learning paradigms including associative learning between temperature and starvation (2), although their capability of sensation were normal. TAX-6 expression in tax-6 adult animals using heat shock promoter did rescue abnormal associative learning, indicating that TAX-6 calcineurin is necessary for neural function in the mature neural circuit. Defective associative learning between temperature and starvation of tax-6;Ex[sensory+, inter-] mutants were rescued by the expression of tax-6 cDNA in both AIZ and RIA interneurons. The AIZ-RIA mediated neural pathway is a critical wiring for thermotaxis neural circuit (3), suggesting that associative learning is regulated at neural circuit level. In order to monitor the physiological responses of AIZ and RIA interneurons to thermal stimuli under the conditioned (starved) and unconditioned (fed) state, we used a genetically encoded calcium indicator, cameleon. We found that AIZ interneuron responded to both warming and cooling in well-fed wild type animals, while the response of other interneuron not directly connected to major thermotaxis-related neurons was minimum, if not undetectable. Thermal responses of AIZ neurons in wild-type animals were down-regulated at starved condition. These results suggest that the conditioning factor, starvation, negatively regulates the neuronal activity of AIZ interneurons. We are also measuring the activities of AIZ and RIA interneurons in tax-6 mutants. Through the analysis of this neural circuit at molecular and physiological level, we are hoping to identify the fundamental neuronal mechanism of learning and memory conserved throughout C. elegans to human. (1) Kuhara et. al., 2002, Neuron 33, 751. (2) Mohri et. al., 2005, Genetics, in press. (3) Mori and Ohshima, 1995, Nature 376, 344.

Atsushi Kuhara et al. (2007) International Worm Meeting "Olfactory neuron senses temperature through the G protein-coupled signaling."

Atsushi Kuhara, Masatoshi Okumura, Kunihiro Matsumoto, Ikue Mori, Koutarou D Kimura

[International Worm Meeting, 2007]

Animal can sense environmental stimuli such as light, smell, and temperature by using sensory neurons. While light and smell are received by G protein-coupled receptors, temperature is received by a channel type receptor known as TRP channel. Here we show novel temperature sensing mechanism through G protein-coupled signaling. We found that loss-of-function mutation in inhibitor of G protein, RGS EAT-16 (1), led to cryophilic abnormality, which was rescued by expressing eat-16 cDNA in AWC sensory neuron known as an olfactory neuron (2). We investigated whether AWC is capable of sensing temperature using Ca2+imaging with Ca2+indicator cameleon. Ca2+concentration in AWC of wild-type was increased by warming, and decreased by cooling, whereas the response of other sensory neuron was minimal. Thermal response of AWC in eat-16 was hyper sensitive, suggesting that RGS EAT-16 acts as negative regulator for AWC activation, and that hyper activation of AWC causes cryophilic abnormality. Olfactory signals in AWC are transmitted through G protein-coupled signaling (3). Interestingly, cryophilic abnormality of RGS eat-16 mutant was suppressed by the mutations in the genes involved in G protein-coupled olfactory signaling, such as odr-3 G<font face=symbol>a</font> and tax-4 cGMP-gated channel. Thermal responses of AWC in odr-3 and tax-4 mutants were also strongly decreased. These results indicate that G protein-coupled signaling is essential for thermosensation, and that both temperature and olfactory signaling are transmitted thorough the similar molecular pathway. TRP-channel acts as a thermo receptor in vertebrate. OSM-9 is the TRP channel localizing at AWC cell body (4), which is involved in olfactory adaptation. We found that cryophilic abnormality of eat-16 was partially suppressed by osm-9 mutation. AWC neuronal activity in osm-9 mutant was however increased by warming same response as wild-type. After two minutes from warming, Ca2+concentration in AWC of wild-type was continuously increased. Interestungly, osm-9 mutant showed defect in the continuous activation, proposing that OSM-9 TRP channel is involved in continuity of neural response. Altogether, our results demonstrated that AWC known as an olfactory neuron, senses temperature through the G protein signaling, and that TRP channel is involved in continuity of neural response. We are identifying new genes involved in thermosemsation by cloning the responsible genes for suppressors of thermotaxis defect in eat-16. 1)Hajdu-Cronin et al., Gen Dev, 1999, 2)Bargmann et al., Cell, 1993, 3)Roayaie et al., Neuron, 1998, 4)Tobin et al., Neuron, 2002.

Astushi Kuhara et al. (2006) East Asia C. elegans Meeting "G protein-coupled signaling pathway is required for thermosensation"

Ikue Mori, Astushi Kuhara, Masatoshi Okumura, Koutarou Kimura

[East Asia C. elegans Meeting, 2006]

Animal can sense environmental stimuli such as light, smell, and temperature by using specialized sensory neurons. While light and smell are received by G protein-coupled receptors, temperature is thought to be received by a channel type receptor known as TRP channel. Here we show novel temperature sensing mechanism through G protein-mediated pathway. We found that loss-of-function mutation in eat-16 gene encoding RGS, a negative regulator of G protein, causes hyper activation of AWC sensory neuron, thereby leading to abnormal thermotaxis. The AWC neuron is well known as an olfactory neuron, where olfactory signaling is regulated through G protein-coupled signaling pathway. Interestingly, thermotactic defect in eat-16 mutant was suppressed by the mutations in genes involved in G protein-coupled olfactory signaling pathway, such as odr-3 Ga and tax-4 cGMP gated channel (CNGC). These results indicate that G protein-mediated pathway is essential for thermosensation, and that AWC sensory neuron discriminates between temperature and olfactory signaling using the same molecular pathway. In order to monitor the physiological responses of AWC to thermal stimuli, we used a genetically encoded calcium indicator, cameleon. Calcium concentration in AWC of wild-type was increased by warming, and decreased by cooling. By contrast, AWC in Ga odr-3 and CNGC tax-4 mutants were less responsive to warming. These results suggest that thermal stimuli physiologically activates AWC thorough G protein-mediated pathway. Responses of AWC to warming were also partially reduced in osm-9 mutants lacking OSM-9 TRP channel, which is a key molecule for olfactory plasticity known as olfactory adaptation. To observe plastic property of AWC for temperature sensation, we investigated whether AWC activity is plastically controlled dependent on previous cultivation temperature. We found that AWC responded to step-like warming above a threshold temperature that is set by cultivation temperature, suggesting that AWC activity is modulated dependent on temperature memory. We are trying to test whether OSM-9 TRP channel is involved in the modulation of thermosensory signaling. Altogether, our results demonstrate the molecular and physiological mechanisms for G protein-mediated thermosensation.