Navigational choice between reversal and curve during acidic pH avoidance behavior in Caenorhabditis elegans.

BACKGROUND: Under experimental conditions, virtually all behaviors of Caenorhabditis elegans are achieved by combinations of simple locomotion, including forward, reversal movement, turning by deep body bending, and gradual shallow turning. To study how worms regulate these locomotion in response to sensory information, acidic pH avoidance behavior was analyzed by using worm tracking system. RESULTS: In the acidic pH avoidance, we characterized two types of behavioral maneuvers that have similar behavioral sequences in chemotaxis and thermotaxis. A stereotypic reversal-turn-forward sequence of reversal avoidance caused an abrupt random reorientation, and a shallow gradual turn in curve avoidance caused non-random reorientation in a less acidic direction to avoid the acidic pH. Our results suggest that these two maneuvers were each triggered by a distinct threshold pH. A simulation study using the two-distinct-threshold model reproduced the avoidance behavior of the real worm, supporting the presence of the threshold. Threshold pH for both reversal and curve avoidance was altered in mutants with reduced or enhanced glutamatergic signaling from acid-sensing neurons. CONCLUSIONS: C. elegans employ two behavioral maneuvers, reversal (klinokinesis) and curve (klinotaxis) to avoid acidic pH. Unlike the chemotaxis in C. elegans, reversal and curve avoidances were triggered by absolute pH rather than temporal derivative of stimulus concentration in this behavior. The pH threshold is different between reversal and curve avoidance. Mutant studies suggested that the difference results from a differential amount of glutamate released from ASH and ASK chemosensory neurons.

The Caenorhabditis elegans R13A5.9 gene plays a role in synaptic vesicle exocytosis.

The Caenorhabditis elegans R13A5.9 gene encodes a putative membrane protein with homologs in mammals. When the R13A5.9 protein was fused to different fluorescent proteins, signal was observed in or near synaptic vesicles; thus, we sought to determine whether this gene plays a role in synaptic vesicle formation, function, or exocytosis. R13A5.9 mutant worms exhibited low sensitivity to aldicarb (an acetylcholinesterase inhibitor), which suggested that vesicular loading or release, or acetylcholine synthesis, was disrupted in these organisms. This was supported by the observation that an R13A5.9 mutant strain exhibited an excessive accumulation of synaptic vesicles. Collectively, these results suggest a functional role for R13A5.9 in synaptic vesicle exocytosis.

Chemotaxis behavior toward an odor is regulated by constant sodium chloride stimulus in Caenorhabditis elegans.

We studied the chemotaxis behavior of Caenorhabditis elegans toward a chemoattractant in the presence of background sensory stimulus. Chemotaxis toward an odor butanone was greater in the presence of sodium chloride (NaCl) than that without NaCl. By contrast, chemotaxis toward NaCl was not affected by a butanone background. The salt-sensing ASE neuron-deficient che-1(p674) mutants and worms with ASE genetically ablated showed high chemotaxis toward butanone, regardless of the presence of a NaCl background. Therefore, in wild-type worms, information from ASE in the absence of NaCl suppresses butanone chemotaxis, while the suppression is removed in the presence of NaCl.

Evaluation of Head Movement Periodicity and Irregularity during Locomotion of Caenorhabditis elegans.

Caenorhabditis elegans is suitable for studying the nervous system, which controls behavior. C. elegans shows sinusoidal locomotion on an agar plate. The head moves not only sinusoidally but also more complexly, which reflects regulation of the head muscles by the nervous system. The head movement becomes more irregular with senescence. To date, the head movement complexity has not been quantitatively analyzed. We propose two simple methods for evaluation of the head movement regularity on an agar plate using image analysis. The methods calculate metrics that are a measure of how the head end movement is correlated with body movement. In the first method, the length along the trace of the head end on the agar plate between adjacent intersecting points of the head trace and the quasi-midline of the head trace, which was made by sliding an averaging window of 1/2 the body wavelength, was obtained. Histograms of the lengths showed periodic movement of the head and deviation from it. In the second method, the intersections between the trace of the head end and the trace of the 5 (near the pharynx) or 50% (the mid-body) point from the head end in the centerline length of the worm image were marked. The length of the head trace between adjacent intersections was measured, and a histogram of the lengths was produced. The histogram for the 5% point showed deviation of the head end movement from the movement near the pharynx. The histogram for the 50% point showed deviation of the head movement from the sinusoidal movement of the body center. Application of these methods to wild type and several mutant strains enabled evaluation of their head movement periodicity and irregularity, and revealed a difference in the age-dependence of head movement irregularity between the strains. A set of five parameters obtained from the histograms reliably identifies differences in head movement between strains.

A model of the intracellular response of an olfactory neuron in Caenorhabditis elegans to odor stimulation.

We developed a mathematical model of a hypothetical neuronal signal transduction pathway to better understand olfactory perception in Caenorhabditis elegans. This worm has only three pairs of olfactory receptor neurons. Intracellular Ca(2+) decreases in one pair of olfactory neurons in C. elegans, the AWC neurons, following application of an attractive odor and there is a transient increase in intracellular Ca(2+) following removal of odor. The magnitude of this increase is positively correlated with the duration of odor stimulation. Additionally, this Ca(2+) transient is induced by a cGMP second messenger system. We identified likely candidates for the signal transduction molecules functioning in this system based on available gene expression and physiological data from AWCs. Our model incorporated a G-protein-coupled odor receptor, a G-protein, a guanylate cyclase as the G-protein effector, and a single phosphodiesterase. Additionally, a cyclic-nucleotide-gated ion channel and a voltage-gated ion channel that mediated calcium influx were incorporated into the model. We posited that, upon odor stimulation, guanylate cyclase was suppressed by the G-protein and that, upon cessation of the stimulus, the G-protein-induced suppression ceased and cGMP synthesis was restored. A key element of our model was a Ca(2+)-dependent negative feedback loop that ensured that the calcium increases were transient. Two guanylate cyclase-activating proteins acted on the effector guanylate cyclase to negatively regulate cGMP signaling and the resulting calcium influx. Our model was able to closely replicate in silico three important features of the calcium dynamics of AWCs. Specifically, in our simulations, [Ca(2+)] increased rapidly and reached its peak within 10 s after the odor stimulus was removed, peak [Ca(2+)] increased with longer odor exposure, and [Ca(2+)] decreased during a second stimulus that closely followed an initial stimulus. However, application of random background signal ('noise') showed that certain components of the pathway were particularly sensitive to this noise.

Evolution of vertebrate transient receptor potential vanilloid 3 channels: opposite temperature sensitivity between mammals and western clawed frogs.

Transient Receptor Potential (TRP) channels serve as temperature receptors in a wide variety of animals and must have played crucial roles in thermal adaptation. The TRP vanilloid (TRPV) subfamily contains several temperature receptors with different temperature sensitivities. The TRPV3 channel is known to be highly expressed in skin, where it is activated by warm temperatures and serves as a sensor to detect ambient temperatures near the body temperature of homeothermic animals such as mammals. Here we performed comprehensive comparative analyses of the TRPV subfamily in order to understand the evolutionary process; we identified novel TRPV genes and also characterized the evolutionary flexibility of TRPV3 during vertebrate evolution. We cloned the TRPV3 channel from the western clawed frog Xenopus tropicalis to understand the functional evolution of the TRPV3 channel. The amino acid sequences of the N- and C-terminal regions of the TRPV3 channel were highly diversified from those of other terrestrial vertebrate TRPV3 channels, although central portions were well conserved. In a heterologous expression system, several mammalian TRPV3 agonists did not activate the TRPV3 channel of the western clawed frog. Moreover, the frog TRPV3 channel did not respond to heat stimuli, instead it was activated by cold temperatures. Temperature thresholds for activation were about 16 °C, slightly below the lower temperature limit for the western clawed frog. Given that the TRPV3 channel is expressed in skin, its likely role is to detect noxious cold temperatures. Thus, the western clawed frog and mammals acquired opposite temperature sensitivity of the TRPV3 channel in order to detect environmental temperatures suitable for their respective species, indicating that temperature receptors can dynamically change properties to adapt to different thermal environments during evolution.

A small nucleolar RNA functions in rRNA processing in Caenorhabditis elegans.

CeR-2 RNA is one of the newly identified Caenorhabditis elegans noncoding RNAs (ncRNAs). The characterization of CeR-2 by RNomic studies has failed to classify it into any known ncRNA family. In this study, we examined the spatiotemporal expression patterns of CeR-2 to gain insight into its function. CeR-2 is expressed in most cells from the early embryo to adult stages. The subcellular localization of this RNA is analogous to that of fibrillarin, a major protein of the nucleolus. It was observed that knockdown of C/D small nucleolar ribonucleoproteins (snoRNPs), but not of H/ACA snoRNPs, resulted in the aberrant nucleolar localization of CeR-2 RNA. A mutant worm with a reduced amount of cellular CeR-2 RNA showed changes in its pre-rRNA processing pattern compared with that of the wild-type strain N2. These results suggest that CeR-2 RNA is a C/D snoRNA involved in the processing of rRNAs.

In vivo calcium imaging of OFF-responding ASK chemosensory neurons in C. elegans.

BACKGROUND: How neurons and neuronal circuits transform sensory input into behavior is not well understood. Because of its well-described, simple nervous system, Caenorhabditis elegans is an ideal model organism to study this issue. Transformation of sensory signals into neural activity is a crucial first step in the sensory-motor transformation pathway in an animal's nervous system. We examined the properties of chemosensory ASK neurons of C. elegans during sensory stimulation. METHOD: A genetically encoded calcium sensor protein, G-CaMP, was expressed in ASK neurons of C. elegans, and the intracellular calcium dynamics of the neurons were observed. RESULTS: After application of the attractants l-lysine or food-related stimuli, the level of calcium in ASK neurons decreased. In contrast, responses increased upon stimulus removal. Opposite responses were observed after application and removal of a repellent. CONCLUSION: The observed changes in response to external stimuli suggest that the activity of ASK neurons may impact stimulus-evoked worm behavior. The stimulus-ON/activity-OFF properties of ASK neurons are similar to those of vertebrate retinal photoreceptors. GENERAL SIGNIFICANCE: Analysis of sensory-motor transformation pathways based on the activity and structure of neuronal circuits is an important goal in neurobiology and is practical in C. elegans. Our study provides insights into the mechanism of such transformation in the animal.

Phase-dependent preference of thermosensation and chemosensation during simultaneous presentation assay in Caenorhabditis elegans.

BACKGROUND: Multi-sensory integration is necessary for organisms to discriminate different environmental stimuli and thus determine behavior. Caenorhabditis elegans has 12 pairs of amphid sensory neurons, which are involved in generating behaviors such as thermotaxis toward cultivation temperature, and chemotaxis toward chemical stimuli. This arrangement of known sensory neurons and measurable behavioral output makes C. elegans suitable for addressing questions of multi-sensory integration in the nervous system. Previous studies have suggested that C. elegans can process different chemoattractants simultaneously. However, little is known about how these organisms can integrate information from stimuli of different modality, such as thermal and chemical stimuli. RESULTS: We studied the behavior of a population of C. elegans during simultaneous presentation of thermal and chemical stimuli. First, we examined thermotaxis within the radial temperature gradient produced by a feedback-controlled thermoregulator. Separately, we examined chemotaxis toward sodium chloride or isoamyl alcohol. Then, assays for simultaneous presentations of 15 degrees C (colder temperature than 20 degrees C room temperature) and chemoattractant were performed with 15 degrees C-cultivated wild-type worms. Unlike the sum of behavioral indices for each separate behavior, simultaneous presentation resulted in a biased migration to cold regions in the first 10 min of the assay, and sodium chloride-regions in the last 40 min. However, when sodium chloride was replaced with isoamyl alcohol in the simultaneous presentation, the behavioral index was very similar to the sum of separate single presentation indices. We then recorded tracks of single worms and analyzed their behavior. For behavior toward sodium chloride, frequencies of forward and backward movements in simultaneous presentation were significantly different from those in single presentation. Also, migration toward 15 degrees C in simultaneous presentation was faster than that in 15 degrees C-single presentation. CONCLUSION: We conclude that worms preferred temperature to chemoattractant at first, but preferred the chemoattractant sodium chloride thereafter. This preference was not seen for isoamyl alcohol presentation. We attribute this phase-dependent preference to the result of integration of thermosensory and chemosensory signals received by distinct sensory neurons.

Modulation of Caenorhabditis elegans chemotaxis by cultivation and assay temperatures.

The chemotaxis behaviors of the nematode Caenorhabditis elegans cultivated at various temperatures (15 degrees C, 20 degrees C and 25 degrees C) were examined at various temperatures (10 degrees C, 15 degrees C, 20 degrees C and 25 degrees C) to determine the multi-sensory integration of physical (thermal) and chemical sensory information within its nervous system. Chemotaxis behavior toward sodium acetate and ammonium chloride were differently affected by both assay and cultivation temperatures, suggesting that the temperature effect on chemotaxis is not general, but rather distinctive for each chemosensory pathway. Since thermosensory cues are likely encountered constantly in C. elegans, we supposed that the chemotaxis behaviors of worms are achieved by the integration of chemo- and thermosensory information. To verify the possible contribution of thermosensory function in chemotaxis, we examined the chemotaxis behaviors of ttx-1(p767) mutant worms with defective AFD thermosensory neurons. The chemotaxis behaviors toward sodium acetate or ammonium chloride of mutant worms cultivated at 20 degrees C and 25 degrees C were reduced relative to those of wild-type worms. These results indicate the important role of multi-sensory integration of chemosensory and thermosensory information in chemotaxis behavior of the C. elegans.

Adaptive evolution of the uncoupling protein 1 gene contributed to the acquisition of novel nonshivering thermogenesis in ancestral eutherian mammals.

Homeotherms possess various physiological mechanisms to maintain their body temperature, thus allowing them to adapt to various environments. Under cold conditions, most eutherian mammals upregulate heat production in brown adipose tissue (BAT), and uncoupling protein (UCP) 1 is an essential factor in BAT thermogenesis. The evolutionary origin of UCP1 was believed to have been a specific event occurring in eutherian lineages. Recently, however, the UCP1 ortholog was found in fishes, which uncovers a more ancient origin of this gene than previously believed. Here we investigate the evolutionary process of UCP1 by comparative genomic approach. We found that UCP1 evolved rapidly by positive Darwinian selection in the common ancestor of eutherians, although this gene arose in the ancestral vertebrate, since the orthologous genes were shared among most of the vertebrate species. Adaptive evolution occurred after the divergence between eutherians and marsupials, which is consistent with the fact that BAT has been found only in eutherians. Our findings indicate that positive Darwinian selection acted on UCP1 contributed to the acquisition of an efficient mechanism for body temperature regulation in primitive eutherians. Phylogenetic reconstruction of UCP1 with two paralogs (UCP2 and UCP3) among vertebrate species revealed that the gene duplication events which produced these three genes occurred in the common ancestor of vertebrates much earlier than the emergence of eutherians. Thus, our data demonstrate that novel gene function can evolve without de novo gene duplication event.

Simulation of C. elegans thermotactic behavior in a linear thermal gradient using a simple phenomenological motility model.

The nematode Caenorhabditis elegans has been reported to exhibit thermotaxis, a sophisticated behavioral response to temperature. However, there appears to be some inconsistency among previous reports. The results of population-level thermotaxis investigations suggest that C. elegans can navigate to the region of its cultivation temperature from nearby regions of higher or lower temperature. However, individual C. elegans nematodes appear to show only cryophilic tendencies above their cultivation temperature. A Monte-Carlo style simulation using a simple individual model of C. elegans provides insight into clarifying apparent inconsistencies among previous findings. The simulation using the thermotaxis model that includes the cryophilic tendencies, isothermal tracking and thermal adaptation was conducted. As a result of the random walk property of locomotion of C. elegans, only cryophilic tendencies above the cultivation temperature result in population-level thermophilic tendencies. Isothermal tracking, a period of active pursuit of an isotherm around regions of temperature near prior cultivation temperature, can strengthen the tendencies of these worms to gather around near-cultivation-temperature regions. A statistical index, the thermotaxis (TTX) L-skewness, was introduced and was useful in analyzing the population-level thermotaxis of model worms.

Caenorhabditis elegans mutants having altered preference of chemotaxis behavior during simultaneous presentation of two chemoattractants.

Upon presentation of two distinct chemoattractants such as sodium acetate and diacetyl simultaneously, the nematode Caenorhabditis elegans was preferentially attracted by one of these chemoattractants. We isolated two mutants having altered preference of chemotaxis behavior toward simultaneous presentation of sodium acetate and diacetyl. The chep-1(qr1) (CHEmosensory Preference) mutant preferred sodium acetate to diacetyl, while the chep-2(qr2) mutant preferred diacetyl to sodium acetate in simultaneous presentation of these chemoattractants. The chemotaxis behavior of chep-2(qr2) mutant in simultaneous presentation suggests a function of chep-2 gene products within the chemosensory informational integration pathway as well as in the chemosensory pathway.

Evolution of thermoTRP ion channel homologs in vertebrates.

In mammalian thermosensation, nine temperature-sensitive ion channels that are activated by distinct temperature thresholds have been identified as thermosensors. These ion channels belong to the transient receptor potential (TRP) superfamily and are referred to as "thermoTRPs" (TRPV1, TRPV2, TRPV3, TRPV4, TRPM2, TRPM4, TRPM5, TRPM8, and TRPA1). To elucidate the evolutionary processes of thermoTRPs, we conducted comprehensive searches for mammalian thermoTRP gene homologs in the draft genome sequences of chicken (Gallus gallus), western clawed frog (Xenopus tropicalis), zebrafish (Danio rerio), and pufferfish (Fugu rubripes). Newly identified homologs were compared with known thermoTRPs, and phylogenetic analyses were conducted. Our comparative analyses revealed that most of the mammalian thermo-TRP members already existed in the common ancestor of fishes and tetrapods. Tetrapods shared almost the same repertoire, except that the western clawed frog expanded TRPV4s (six copies) and TRPM8s (two copies), which were diversified considerably. Comparisons of nonsynonymous and synonymous substitution rates among TRPV4s suggested that one copy of the TRPV4 channel in the western clawed frog retained its original function, while the other copies diversified and obtained slightly different properties. In fish lineages, several members of thermo-TRPs have duplicated in the whole genome duplication occurred in the ancestral ray-finned fish; however, some of the copies have subsequently been lost. Furthermore, fishes do not possess the three members of thermoTRPs existed in mammals, e.g., thermoTRPs activated by noxious heat, warm, and cool temperatures. Our results suggest that thermosensation mechanisms have changed through vertebrate evolution with respect to thermosensor repertoires.

Computer-driven automatic identification of locomotion states in Caenorhabditis elegans.

We developed a computer-driven tracking system for the automated analysis of the locomotion of Caenorhabditis elegans. The algorithm for the identification of locomotion states on agar plates (forward movement, backward movement, rest, and curl) includes the identification of the worm's head and tail. The head and tail are first assigned, by using three criteria, based on time-sequential binary images of the worm, and the determination is made based on the majority of the three criteria. By using the majority of the criteria, the robustness was improved. The system allowed us to identify locomotion states and to reconstruct the path of a worm using more than 1h data. Based on 5-min image sequences from a total of 230 individual wild-type worms and 22 mutants, the average error of identification of the head/tail for all strains was 0.20%. The system was used to analyze 70 min of locomotion for wild-type and two mutant strains after a worm was transferred from a seeded plate to a bacteria-free assay plate. The error of identifying the state was less than 1%, which is sufficiently accurate for locomotion studies.

Interactions between Caenorhabditis elegans individuals during chemotactic response.

The chemotactic response of the nematode Caenorhabditis elegans is known to be affected by the population density on an assay plate, suggesting the existence of interactions between individual animals. To clarify the interactions between individuals during chemotaxis, we investigated the effect of population density at an attractant area on the chemotactic response to water-soluble sodium acetate and odorant diacetyl using wild-type N2 animals and daf-22 (m130) mutants, which have defective pheromone secretion but can sense pheromone. The chemotaxis index of N2 animals at 90 min of the assay negatively correlated with the number of animals on the assay plate regardless of the type of attractant used (p<0.01). On the other hand, there was no significant difference in the chemotaxis indices of daf-22 (m130) mutants for either of the attractants between the low-and high-population groups. When daf-22 (m130) mutants of a high population density were placed at the attractant location in advance, the chemotaxis index of N2 animals was almost the same as that in the control assay in which no animals were placed at the attractant location in advance. When N2 animals of a high population density were placed at the attractant location in advance, the chemotaxis indices of N2 animals and daf-22 (m130) mutants were significantly smaller than those obtained in the control assay (p<0.05). In the absence of an attractant, we observed a decline in the fraction of animals in the neighborhood of N2 animals of a high population density, although the nematodes were not influenced by daf-22 (m130) mutants of a high population density. These results suggest that the attraction of nematodes to chemicals is inhibited by an increase in the concentration of the pheromone generated by N2 animals at the attractant location.

Serotonin deficiency shortens the duration of forward movement in Caenorhabditis elegans.

Serotonin has been implicated in numerous behaviors in a wide variety of animals. We examined the effect of serotonin deficiency, induced by genetic perturbations and cell ablations, on the duration of Caenorhabditis elegans forward movement. Mutants with defective serotonin biosynthesis or worms with ablated serotonergic neurons showed a markedly decreased duration of forward movement, suggesting involvement of this neuromodulator in the regulation of the duration of worm locomotion.

Chemotaxis of Caenorhabditis elegans during simultaneous presentation of two water-soluble attractants, l-lysine and chloride ions.

Lysine and chloride ions are water-soluble attractants for Caenorhabditis elegans. When chemotaxis behavior to either of these attractants was assayed separately, the radial concentration gradients of 3 M lysine and 0.1 M ammonium chloride had similar potencies for attracting worms. However, when the concentration gradients of lysine and ammonium chloride at these concentrations were presented simultaneously, worms preferred lysine to ammonium chloride more than expected from the results obtained in separate experiments, suggesting the presence of an interaction between these two sensory information pathways within the nervous system. Chemotaxis behavior toward the radial concentration gradient of one of these attractants superimposed on a uniform concentration of the other attractant showed that the chemotaxis was augmented or attenuated by the ammonium chloride background depending on the background concentration, and attenuated by the lysine background, further supporting the interaction between the two sensory information pathways.

Neural network model to generate head swing in locomotion of Caenorhabditis elegans.

Computer simulation of the neural network composed of the head neurons of Caenorhabditis elegans was performed to reconstruct the realistic changes in the membrane potential of motoneurons in swinging the head for coordinated forward locomotion. The model neuron had ion channels for calcium and potassium, whose parameters were obtained by fitting the experimental data. Transmission properties of the chemical synapses were set as graded. The neural network involved in forward movement was extracted by tracing the neuronal activity flow upstream from the motoneurons connected to the head muscles. Simulations were performed with datasets, which included all combinations of the excitatory and inhibitory properties of the neurons. In this model, a pulse input entered only from motoneuron VB1, and activation of the stretch receptors on SAA neurons was necessary for the periodic bending. The synaptic output property of each neuron was estimated for the alternate contraction of the dorsal and ventral muscles. The AIB neuron was excitatory, RIV and SMD neurons seemed to be excitatory and RMD and SAA neurons seemed to be inhibitory. With datasets violating Dale's principle for the SMB neuron, AIB neuron was excitatory and RMD neuron was inhibitory. RIA, RIV and SMD neurons seemed to be excitatory.

Neurons regulating the duration of forward locomotion in Caenorhabditis elegans.

The locomotory behavior of Caenorhabditis elegans consists of four simple events, forward and backward movements, omega-shaped turns and rests. The wide variety of behaviors of this worm is achieved through a combination of these simple locomotions. To gain insight into the neuronal mechanisms regulating this locomotion, we analyzed the locomotory behavior of C. elegans over a long time period. By using an automatic worm tracking system, we revealed the existence of at least two distinct behavioral states -- pivoting and traveling -- in the forward locomotion of C. elegans in the absence of food. Pivoting is characterized by pronounced directional switching and resulting in short-duration forward movement, whereas in the traveling state forward movement is of longer duration. Pivoting occurred when we transferred a well-fed worm to an unseeded plate, and then the transition to traveling occurred, successively. We showed that, by laser ablation, antagonistic neuronal pathways consisting of nine classes of sensory neurons and four classes of interneurons were involved in this regulation. Loss of any one of these neurons altered the locomotory behavior.