Ryuzo Shingai

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.

Cloning of a putative γ-aminobutyric acid (GABA) receptor subunit ϱ3 cDNA

Abstract Cloned cDNA encoding a putative member of GABA receptor ϱ-subunit class was isolated from rat-retina-mRNA-derived libraries. The cDNA encodes a signal peptide of 21 amino acids followed by the mature ϱ3 subunit sequence of 443 amino acids. The proposed amino acid sequence exhibits 63 and 61% homology to the previously-reported human ϱ1 and rat ϱ2 sequences, respectively. Northern blot analysis demonstrated the expression of mRNA for ϱ3 subunit in retina.

Identification of GABAA Receptor Subunits in Rat Retina: Cloning of the Rat GABAA Receptor ρ2-Subunit cDNA

Abstract: We identified GABAA receptor subunits in rat retina using PCR. The high degree of conservation among previously described members of ligand‐gated anion channels in transmembrane domains was used to design degenerate sense and antisense oligonucleotides. These oligonucleotides were used as primers for PCR, which was applied to the rat retina cDNA. Analysis of clones derived from the PCR amplification identified the GABAAα1, β1, β3, and γ2 subunits and the glycine α1 subunit. In addition, two clones closely related to the human GABAAρ‐subunit class were obtained. Molecular cloning revealed one of them as the rat counterpart of the human ρ2 subunit. Northern blot analysis demonstrated the expression of mRNAs for ρ subunits in retina. These results further support the hypothesis that bicuculline‐insensitive GABA channels in rat retina are comprised of ρ subunits.

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.

Functional expression of GABA ϱ 3 receptors in Xenopus oocytes

Abstract Homomeric rat GABA ϱ 3 receptors were expressed in Xenopus oocytes, and their pharmacological profile was investigated electrophysiologically. GABA activated the ϱ 3 receptors with an EC 50 value of 7.5 μM and a Hill coefficient of 1.6. The GABA-induced current was not antagonized by bicuculline (100 μM), but was blocked by picrotoxin (IC 50 : 0.68 μ M for 100 μM GABA). The current was almost insensitive to pentobarbital, diazepam and a neurosteroid, 3α-OH-DHP. Many of the pharmacological properties of the p 3 subunit were similar to those of the previously reported rat ϱ 1 and ϱ 2 subunits and GABA c receptors.

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 worms 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.

Durations and frequencies of free locomotion in wild type and GABAergic mutants of Caenorhabditis elegans.

We investigated how much time wild-type Caenorhabditis elegans (Bristol N2) nematodes and the GABA-deficient unc25 mutant and the vesicular GABA transporter-deficient unc47 mutant spent moving. The worms were allowed to move freely on the surface of agarose plates either with or without the food bacterium OP50. We identified forward movement, backward movement, resting and turns by watching images on video and computer displays. Forward movement lasted longer and rests were briefer without, than with, bacteria. Frequency distributions except for backward movement fitted a sum of two exponential functions. The duration of backward movement was not strongly influenced by exposure to bacteria, whereas the frequency of backward movements increased in their presence. The duration of forward movement of unc25 nematodes had no long component, thus differing from that of N2 and unc47 strain nematodes in treatments with and without bacteria. The durations of resting in these mutants were much longer than in the N2 strain, especially in the absence of bacteria. The turn frequency of unc47 nematodes had a higher short component than that of the wild type N2 and unc25 nematodes, in the absence of bacteria. A neural network model is discussed in conjunction with the features of mutants and current knowledge of GABAergic neural transmission.

Localization of γ-aminobutyric acid (gaba) receptor ρ3 subunit in rat retina

WE used digoxigenin-labelled single strand DNA probes to examine the expression of the mRNA encoding γ-aminobutyric acid receptor ρ3 subunit in sections of the adult rat retina. Transcript for the ρ3 subunit was found in cell somata of a portion of cells lying in the ganglion cell layer.

trans-4-Amino-2-methylbut-2-enoic acid (2-MeTACA) and (±)-trans-2-aminomethylcyclopropanecarboxylic acid ((±)-TAMP) can differentiate rat ρ3 from human ρ1 and ρ2 recombinant GABAC receptors

This study investigated the effects of a number of GABA analogues on rat ρ3 GABAC receptors expressed in Xenopus oocytes using 2‐electrode voltage clamp methods. The potency order of agonists was muscimol (EC50=1.9±0.1 μM) (+)‐trans‐3‐aminocyclopentanecarboxylic acids ((+)‐TACP; EC50=2.7±0.9 μM) trans‐4‐aminocrotonic acid (TACA; EC50=3.8±0.3 μM) GABA (EC50=4.0±0.3 μM) > thiomuscimol (EC50=24.8±2.6 μM) > (±)‐cis‐2‐aminomethylcyclopropane‐carboxylic acid ((±)‐CAMP; EC50=52.6±8.7 μM) > cis‐4‐aminocrotonic acid (CACA; EC50=139.4±5.2 μM). The potency order of antagonists was (±)‐trans‐2‐aminomethylcyclopropanecarboxylic acid ((±)‐TAMP; KB=4.8±1.8 μM) (1,2,5,6‐tetrahydropyridin‐4‐yl)methylphosphinic acid (TPMPA; KB=4.8±0.8 μM) > (piperidin‐4‐yl)methylphosphinic acid (P4MPA; KB=10.2±2.3 μM) 4,5,6,7‐tetrahydroisoxazolo[5,4‐c]pyridin‐3‐ol (THIP; KB=10.2±0.3  μM) imidazole‐4‐acetic acid (I4AA; KB=12.6±2.7 μM) > 3‐aminopropylphosphonic acid (3‐APA; KB=35.8±13.5 μM). trans‐4‐Amino‐2‐methylbut‐2‐enoic acid (2‐MeTACA; 300 μM) had no effect as an agonist or an antagonist indicating that the C2 methyl substituent is sterically interacting with the ligand‐binding site of rat ρ3 GABAC receptors. 2‐MeTACA affects ρ1 and ρ2 but not ρ3 GABAC receptors. In contrast, (±)‐TAMP is a partial agonist at ρ1 and ρ2 GABAC receptors, while at rat ρ3 GABAC receptors it is an antagonist. Thus, 2‐MeTACA and (±)‐TAMP could be important pharmacological tools because they may functionally differentiate between ρ1, ρ2 and ρ3 GABAC receptors in vitro.

Effect of simultaneous presentation of multiple attractants on chemotactic response of the nematode Caenorhabditis elegans.

Chemotactic behaviors of the nematode, Caenorhabditis elegans in response to chemical attractants, such as water-soluble sodium acetate and an odorant diacetyl, which were sensed by different sensory neurons, were investigated using various concentrations of these chemical attractants. In the presence of only sodium acetate attractant, the fraction of animals that were roaming around the outside of the attractant and original locations correlated negatively with the chemotaxis index for sodium acetate (P < 0.01). In contrast, the fraction of animals that remained in the original location correlated negatively with the chemotaxis index in the presence of only diacetyl attractant (P < 0.05). These results indicate that the manner of chemotaxis responses differs between sodium acetate and diacetyl. In order to investigate the effect of multiple attractants on chemotactic behaviors, the chemotactic responses to simultaneous presentation of sodium acetate and diacetyl were examined. The fraction of animals that gathered at the 0.7 M sodium acetate location was greater than that at the 0.1% diacetyl location in the presence of both attractants (P < 0.05), although the chemotaxis indexes for 0.7 M sodium acetate and 0.1% diacetyl were similar in the presence of a single attractant. On the other hand, the fraction of animals that gathered at the 0.02% diacetyl location was greater than that at the 0.1M sodium acetate location in the presence of both attractants (P < 0.05), although the chemotaxis indexes for 0.02% diacetyl and 0.1M sodium acetate were similar in the presence of a single attractant. These results suggest the existence of excitatory and/or inhibitory connections in the neuronal circuit for attractant selection, and that the efficacy of these connections may change according to the concentrations of both attractants.