Nishijima, Saori et al. (2011) International Worm Meeting "Appetitive olfactory learning and associative long-term memory in C. elegans ."

MARUYAMA, Ichiro, Nishijima, Saori

[International Worm Meeting, 2011]

C. elegans is an excellent model organism for the study of learning and memory at cellular and molecular levels because of the relative simplicity of the nervous system, which is invariant from animal to animal. Here we developed a paradigm for the study of associative learning and memory by classical (Pavlovian) conditioning of worms with nonanol, as a conditioned stimulus (CS), and potassium chloride (KCl) as an unconditioned stimulus (US). Before the conditioning, worms avoided nonanol, an aversive olfactory stimulus, and were attracted by KCl, an attractive gustatory stimulus, in chemotaxis assays. After massed training without an intertrial interval (ITI) or spaced training with 10-min ITI, by contrast, trained worms were attracted to nonanol. Memory induced by the massed training was extinguished within an hour, while that induced by the spaced training was retained for 24 hours. Worms treated with cycloheximide or actinomycin D failed to form the memory induced by the spaced training, whereas the memory induced by the massed training was not significantly affected by the mRNA and protein synthesis inhibitors. Furthermore, the memory induced by the massed training, but not that induced by the spaced training, was sensitive to experimental disruption by cold-shock anesthesia. By definition, therefore, the memories induced by the massed and spaced training are classified as short-term and long-term memories, respectively. Hence this appetitive olfactory conditioning with nonanol and KCl as CS and US, respectively, shares many of the defining features of associative learning as exemplified by classical conditioning: stimulus- and paring specificity, contiguity and contingency learning, and both short- as well as long-term retention. In support of this, C. elegans mutants defective in nmr-1 encoding an NMDA receptor subunit failed to form both of the short-term and long-term memory, while mutations in crh-1 encoding the CREB transcription factor affected only on the formation of the long-term memory.

Lisicovas, Viktoras et al. (2015) International Worm Meeting "Long-term olfactory associative memory induction by optogenetic stimulation of ASH neuron."

MARUYAMA, Ichiro, Lisicovas, Viktoras

[International Worm Meeting, 2015]

We investigated whether associative memory can be formed through activation of sensory neurons with channelrhodopsin-2. Previously, a conditioning paradigm, using acidic solution as an unconditioned stimulus (US) and 1-propanol as a conditioned stimulus (CS), was shown to induce odor associations lasting as long as 24 hours. A tracking confocal microscope was used to design an aversive conditioning paradigm using optical stimulation in ASH neurons as a US and exposure to vapor of 100% 1-propanol as a CS. A closer investigation of the tracking data obtained from single individuals revealed that conditioned animals responded to 1-propanol with deeper bends than naive animals. To quantify this phenomenon we wrote a custom script for assessing the probability of observing the animal in that behavior. The learning index was calculated as the change in this probability before and after training. Wild type animals showed no predictable increase in learning index, while transgenic animals did. We then examined whether this behavior could persist for at least 6 hr. Two training regimens were used: with an inter-trial period (ITI) of 3 and 10 min. The first regimen was expected to induce short-term memory and the latter long-term memory. When conditioned animals were assayed 6 hr after training, the increase in learning index for 10-min ITI, but not for 3-min ITI was observed. Our findings demonstrate that olfactory conditioning can, at least in part, be achieved using optogenetic stimulation to induce a long-term associative memory.

Murayama, Takashi et al. (2021) International Worm Meeting "Electrophysiological properties of amphid sensory neurons in C. elegans"

Murayama, Takashi, MARUYAMA, Ichiro

[International Worm Meeting, 2021]

Since the lack of voltage-gated sodium channels in C. elegans neurons, it has long been believed that C. elegans neurons transmit electrical signals by passive propagation. However, we have recently reported regenerative depolarization in a major gustatory sensory neuron, ASEL, upon stimulation with an increase of environmental NaCl concentration. This all-or-none membrane depolarization may set the threshold for the sensory neuron to respond significant environmental changes. To test this possibility, therefore, we examined electrophysiological properties of other amphid sensory neurons. Membrane depolarization of amphid sensory neurons were recorded upon current injection by whole-cell patch-clamp techniques. All 14 types of amphid neurons, which consist of 10 pairs of symmetrical neurons and two pairs of asymmetrical neurons, AWCon/AWCoff and ASEL/ASER, showed supralinear depolarization upon current injection. In this meeting, we will report membrane depolarization of several amphid neurons upon puff stimulation of natural substances.

Murayama, Takashi et al. (2009) International Worm Meeting "Cellular and molecular mechanism underlying C. elegans chemotaxis toward mild alkaline pH."

FUJIWARA, Mayuki, Murayama, Takashi, MARUYAMA, Ichiro

[International Worm Meeting, 2009]

Wild-type C. elegans is attracted to mild alkaline pH in environment. To investigate cellular and molecular modes underlying this chemosensory behavior, we have used agar plate assays with a linear pH gradient. Along pH gradients from pH 6.8 to pH8.5, wild-type worms were attracted toward higher pH regions, whereas che-1 mutants defective in chemosensory ASE neurons were not. Laser ablation of ASEL and ASER indicated that ASEL is essential for the chemotaxis. Consistently, ASEL- or ASER-specific rescue of dyf-3, a mutant defective in sensory cilium structures, also indicated that ASEL is responsible for the chemotactic attraction. Furthermore, it was found that pH shifts from low to high, but not from high to low, activated ASEL when the neuronal activity was monitored by using a voltage-sensitive fluorescent protein. ASEL-specific rescue of tax-4 improved the defect of the mutant in the chemotaxis toward mild alkaline pH. This result suggests that the TAX-2/TAX-4 cyclic-nucleotide gated ion channel is involved in the chemotaxis. When we searched candidate genes that regulate the TAX-2/TAX-4 ion channels by RNAi, knock-down of gcy-14, which encodes a cell-surface receptor-type guanylyl cyclase, significantly impaired the chemotaxis toward mild alkaline pH. The gcy-14 gene is preferentially expressed in ASEL, and GCY-14 fused with GFP was localized to the tip of ASEL sensory endings, suggesting that GCY-14 acts as a sensor molecule in ASEL for mild alkaline pH.

Sassa, Toshihiro et al. (2011) International Worm Meeting "High alkaline pH sensation in C. elegans."

Murayama, Takashi, MARUYAMA, Ichiro, Sassa, Toshihiro

[International Worm Meeting, 2011]

High alkaline pH is harmful to organisms including C. elegans. Wild-type worms avoid higher alkaline pH than pH 11 as a noxious stimulus. C. elegans mutants defective in gustatory sensory neurons, che-2, dyf-3, osm-1 and osm-3, showed abnormal behaviors in high alkaline pH avoidance assay. Mutants in osm-9 and ocr-2 genes encoding transient receptor potential (TRP) channels also had abnormal behaviors in the assay. The expression of OSM-9 in the ASH sensory neurons rescued the defect in osm-9 mutants. Upon stimulation with high alkaline pH, Ca2+ concentration increase was observed in wild-type worms by Ca2+ imaging using G-CaMP as a Ca2+ indicator, while no Ca2+ increase was observed in osm-9 and ocr-2 mutants. This demonstrates that ASH is responsible for the high alkaline pH sensation. Furthermore, the following C. elegans mutants also showed abnormal behaviors in the high alkaline pH avoidance: goa-1 and egl-30 defective in G-protein coupled receptor (GPCR)-mediated signaling cascades, and egl-3 and egl-21 defective in vesicular neuropeptide processing. The increase of Ca2+ concentrations in ASH neurons of goa-1 and egl-3 mutants was normal upon high alkaline pH stimulation, indicating that Ca2+ influx into ASH is not affected by these mutations. These results suggest that signaling cascades that include GPCR and neuropeptides are responsible for the processing of high alkaline pH avoidance.

Ahamed, Tosif et al. (2017) International Worm Meeting "The deterministic dynamics of random search in C. elegans."

MARUYAMA, Ichiro, Ahamed, Tosif, Stephens, Greg

[International Worm Meeting, 2017]

Foraging C. elegans actively modulate their search strategy to effectively sample their environment. These decisions are based on statistical distributions of food patches in the environment, sensory cues and their recent experience. Examples of C. elegans search behaviors include area restricted search, salt chemotaxis and roaming-dwelling. Studies based on centroid measures have suggested a random search framework to explain these behaviors. Within this framework, worms sample an environment by executing straight runs punctuated randomly by sharp turns made by reversals, omega turns or a combination of both. When the frequency of sharp turns is high, worms perform a diffusive search, densely sampling a small area. On the other hand when the frequency of sharp turns is small, they perform a ballistic search, going straight for a long period of time. In this study we take another look at random search in C. elegans by analyzing the continuous dynamics in the low dimensional space of C. elegans postures given by the eigenworm representation. To our surprise, we find that the randomness in random search is largely a result of deterministic chaos instead of sensory or motor noise. Thus, the chaotic nature of the dynamics implies that the behavior of the worms is not random, but predictable for a finite period of time. This prediction window is governed by a quantity called the Maximal Lyapunov Exponent (MLE), which is positive for chaotic systems. Highly chaotic systems have a small prediction window due to a large MLE, while, weakly chaotic systems have a big prediction window resulting from a small MLE. We find that for worms engaged in an area restricted search, the MLE gradually reduces with time as the worms transition from a diffusive search strategy to a ballistic search strategy. Moreover, the reversal frequency of these worms is positively correlated with the MLE, consistent with the observation that worms get more predictable as they reduce their reversal frequency. Furthermore, the variation in MLE enough to describe most of the random search behavior, suggesting the intriguing possibility that only one degree of freedom underlies control of C. elegans foraging behavior. Finally, we use this theoretical framework to probe the biology of random search by studying the roles of monoamines dopamine and serotonin in foraging. In summary, we extend past theoretical work on C.elegans random search by including detailed postural dynamics and find that deterministic chaotic dynamics underlie the apparent randomness of C.elegans search strategies. Our analysis also suggests that the MLE of the dynamics is potentially the only parameter that the worm controls to modulate its search strategy and decide how predictable or unpredictable it wants to be.

Ito, Satomi et al. (2015) International Worm Meeting "Long-term associative memory in Caenorhabditis elegans."

Ito, Satomi, Nishigima, Saori, MARUYAMA, Ichiro

[International Worm Meeting, 2015]

Sensory neurons perceive information from the environment, which affects animal's behavior. C. elegans is an excellent model organism for the study of neuronal circuits that regulate the behavior, because of its relatively simple nervous system. The animal can also learn and store memory of non-associative memory like habituation and associative memory of two environmental stimuli. We are interested in molecular mechanisms and cellular networks underlying the associative memory, and have developed protocols for induction of short-term and long-term olfactory appetitive memories, STM and LTM, respectively, in C. elegans. We used 1-nonanol, a weak, volatile aversive chemical to C. elegans, as a conditioned stimulus (CS) and potassium chloride (KCl), a strong attractive substance, as an unconditioned stimulus (US). We conditioned animals with massed and spaced trainings to induce STM and LTM, respectively. Young adult animals in a small plastic tube sealed the bottom with a 30 um-nylon mesh sheet were stimulated with 1-nonanol vapor in a beaker, and then with KCl solution. For the massed training, animals were repeatedly conditioned as described above eight times without an inter-trial interval (ITI) between the conditionings. For the spaced training, on the other hand, animals were conditioned in the similar way to the massed training with ITI, during which animals were rested on an NGM plate. Immediately or hours later after the conditioning, animals were analyzed by chemotaxis assay on an agar plate spotted with 1-nonanol. As results, the trained animals successfully learned and retained the memory for 6 hours by the massed training and for 48 hours after the spaced training. We have also examined whether the training induces CS-specific memory by using 2-nonanone as a CS, instead of 1-nonanol. After training with 2-nonanone and KCl, animals formed 2-nonanone-specific, but not 1-nonanol-specific, associative memory. These results demonstrate that the protocols can successfully induce associative STM and LTM in C. elegans. We are currently trying to elucidate neuronal circuits responsible for the learning and memory.

Sanehisa, Shigeki et al. (2009) International Worm Meeting "C. elegans mutants defective in high-alkaline pH avoidance."

FUJIWARA, Mayuki, Sanehisa, Shigeki, Murayama, Takashi, MARUYAMA, Ichiro

[International Worm Meeting, 2009]

Cellular and molecular bases underlying nociception of high alkaline pH still remains to be explored. C. elegans senses higher alkaline pH ranges than ~pH 10.5 as a noxious stimulus. On an agar plate with a pH gradient, wild-type worms avoided higher pH regions, and moved toward lower pH ranges. To understand the molecular and cellular basis of the high-alkaline pH avoidance, we have screened mutants defective in high-alkaline pH avoidance from animals treated with ethylmethanesulfonate (EMS), using the plate assay. Among five mutants isolated that could not avoid the high-alkaline pH, three of them showed normal chemotaxis behaviors toward water-soluble attractants such as NaCl and mild-alkaline pH, as well as normal osmotic avoidance behaviors. We will discuss about molecules and neurons responsible for the nociception of high-alkaline pH.

Shindou, Tomomi et al. (2015) International Worm Meeting "Electrophysiological properties of ASE neurons."

Wickens, Jeffery, MARUYAMA, Ichiro, Shindou, Tomomi, Shindou, Mayumi, Murayama, Takashi

[International Worm Meeting, 2015]

A pair of ASE chemosensory neurons, ASEL and ASER, are major salt sensors, and play critical roles in chemotaxis to NaCl. Calcium imaging has previously revealed that ASEL and ASER are activated by an increase and decrease in NaCl concentrations, respectively (Suzuki et al., 2008; Ortiz et al., 2009). These asymmetric responses by ASEL and ASER to changes of NaCl concentrations are crucial to efficient chemotaxis of C. elegans toward higher concentrations of NaCl. While Goodman et al. (1998) reported in situ whole-cell patch-clump recording of ASER, electrophysiological characterisation of ASE neurons is still required to understand how the neurons respond to the NaCl concentration changes.Toward the goal, we have investigated electrophysiological properties of ASE neurons in wild-type C. elegans by in vivo whole-cell patch-clamp recordings, and have found that both of ASE neurons showed resting membrane potentials of approximately -60 mV and membrane resistances of about 2 Gomega. In both of ASE neurons, voltage responses to current injections showed solitary action potentials. Depolarization of wild-type ASEL was observed when a puff of 150 mM NaCl was applied to the animal's nose in bath solution containing 50 mM NaCl. On the other hand, a puff of NaCl-free buffer induced ASER depolarization. These results are consistent with those of calcium imaging. To understand roles of the action potentials in ASE, we are currently trying to analyse electrophysiological properties of ASE neurons in various mutants.References1. Suzuki et al., Nature 454: 114-118 (2008)2. Ortiz et al., Current Biology 19: 996-1004 (2009)3. Goodman et al., Neuron 20: 763-772 (1998).

RJ Eustance et al. (1999) International C. elegans Meeting "UNC-13 and Synaptic Neurotransmission"

J Duerr, RJ Eustance, I Maruyama, J Rand, A Duke, H Maruyama, J McManus

[International C. elegans Meeting, 1999]

unc-13 is essential for normal neuronal function and is involved in neurotransmitter release; C. elegans mutants are uncoordinated and resistant to the anti-cholinesterase aldicarb. UNC-13 has several regions homologous to PKC regulatory domains conferring it with calcium, phorbol ester and phospholipid binding properties (Maruyama and Brenner, PNAS 88 , 1991). These binding properties indicate that UNC-13 may regulate neurotransmitter release in response to calcium and as part of a signal transduction pathway. A 5.9kb transcript coding for a 200kDa protein was initially identified (Maruyama and Brenner, PNAS 88, 1991). We identified two additional types of transcripts using cDNA library screens and RACE. One transcript includes a 1kb alternatively spliced exon and another transcript lacks the 5' region included in the other two transcripts. All three transcripts are identical at the 3' end. 21 of the 40 unc-13 alleles in our collection are sequenced. Mutations in the 5' end alter two types of transcripts resulting in an uncoordinated coily phenotype. A 2.8kb deletion at the 3' end removes all unc-13 transcripts, resulting in a lethal phenotype. This 2.8kb deletion was identified by Bob Barstead using PCR analysis. Antibodies recognizing different regions of UNC-13 indicate that protein products from the three transcripts have different expression and localization patterns. Antibodies recognizing the N-terminal region label synapses, but not synaptic vesicles, of most or all neurons. Many unc-13 alleles have decreased staining with this antibody. Antibodies against the region coded for by the alternatively spliced exon stain some sensory neurons and some neuronal processes. Antibodies recognizing the C-terminal region, common to all predicted UNC-13 proteins, label synapses and neuronal processes of most or all neurons. This staining pattern decreases in only 3 alleles, all of which are lethal or partially lethal. The existence of several unc-13 transcripts, the alternate antibody staining patterns, and the phenotypes resulting from elimination of some or all transcripts suggest that unc-13 protein products have different expression and localization patterns and possibly different functions.