Hisashi Shidara

A method for selective ablation of neurons in C. elegans using the phototoxic fluorescent protein, KillerRed

Specific neuron ablation with laser microbeam has been used in behavioral analysis of Caenorhabditis elegans. However, this method is hard to acquire many ablated worms, and is unable to compare behavioral changes just before and after ablation. Here, we developed an ablation method by using genetically encoded photosensitizer protein, KillerRed, which produces reactive oxygen species by green light irradiation. Ablation of AWA sensory neurons abolished the chemotaxis to AWA specific sensitive attractant, diacetyl, and no functional effect on the other sensory neuron, AWC, which senses benzaldehyde. This ablation method can be useful for analyzing neural in situ.

Odorant-induced membrane potential depolarization of AIY interneuron in Caenorhabditis elegans

Although some interneurons in C. elegans have been shown to have unusual region-specific Ca(2+) dynamics, the region-specific Ca(2+) and membrane potential response properties of these neurons are largely unknown due to technical limitations. In this report, we focused on one of these neurons, AIY interneuron, where Ca(2+) dynamics have been detected only in neurites, and not the soma, during odor and temperature stimulation to determine whether membrane potential and Ca(2+) are region-specific dynamics and distinct from one another. To visualize voltage change both in the soma and neurites of AIY, we used voltage-sensitive fluorescent protein (VSFP) 2.42. First, we confirmed that the sensor protein worked correctly in C. elegans by depolarizing AIY interneuron with high concentrations of KCl. Next, we observed membrane potential depolarization during odor (isoamyl alcohol) stimulation in both neurites and the soma. Additionally, depolarization of membrane potential with direct application of high KCl induced a Ca(2+) increase in the soma. From these results, we conclude that membrane potential behavior and Ca(2+) dynamics in AIY differ in its subcellular regions and that VSFP2.42 can be a useful tool for studying information processing in single neurons.

Effect of interactions among individuals on the chemotaxis behaviours of Caenorhabditis elegans

ABSTRACT In many species, individual social animals interact with others in their group and change their collective behaviours. For the solitary nematode Caenorhabditis elegans strain N2, previous research suggests that individuals can change the behaviour of other worms via pheromones and mechanosensory interactions. In particular, pheromones affect foraging behaviour, so that the chemotactic behaviours of individuals in a group (population) can be modulated by interactions with other individuals in the population. To investigate this, we directly compared the chemotactic behaviours of isolated (single) worms with those of individual animals within a population. We found that worms approached an odour source in a distinct manner depending on whether they were alone or in a population. Analysis of behaviours of the N2 worm and a pheromone production-defective mutant revealed that the ‘pirouette’ strategy was modulated by interaction of the worms via pheromones. Thus, pheromones play an important role in the characteristic collective behaviours seen in the population condition. Summary: Chemotactic behaviours of Caenorhabditis elegans in a population are modulated by pheromones, leading to changes in collective behaviours.

Compartmentalized cGMP responses of olfactory sensory neurons in Caenorhabditis elegans

Cyclic guanosine monophosphate (cGMP) plays a crucial role as a second messenger in the regulation of sensory signal transduction in many organisms. In AWC olfactory sensory neurons of Caenorhabditis elegans, cGMP also has essential and distinctive functions in olfactory sensation and adaptation. According to molecular genetic studies, when nematodes are exposed to odorants, a decrease in cGMP regulates cGMP-gated channels for olfactory sensation. Conversely, for olfactory adaptation, an increase in cGMP activates protein kinase G to modulate cellular physiological functions. Although these opposing cGMP responses in single neurons may occur at the same time, it is unclear how cGMP actually behaves in AWC sensory neurons. A hypothetical explanation for opposing cGMP responses is region-specific behaviors in AWC: for odor sensation, cGMP levels in cilia could decrease, whereas odor adaptation is mediated by increased cGMP levels in soma. Therefore, we visualized intracellular cGMP in AWC with a genetically encoded cGMP indicator, cGi500, and examined spatiotemporal cGMP responses in AWC neurons. The cGMP imaging showed that, after odor exposure, cGMP levels in AWC cilia decreased transiently, whereas levels in dendrites and soma gradually increased. These region-specific responses indicated that the cGMP responses in AWC neurons are explicitly compartmentalized. In addition, we performed Ca2+ imaging to examine the relationship between cGMP and Ca2+. These results suggested that AWC sensory neurons are in fact analogous to vertebrate photoreceptor neurons. SIGNIFICANCE STATEMENT Cyclic guanosine monophosphate (cGMP) plays crucial roles in the regulation of sensory signal transduction in many animals. In AWC olfactory sensory neurons of Caenorhabditis elegans, cGMP also has essential and distinctive functions involving olfactory sensation and adaptation. Here, we visualized intracellular cGMP in AWC neurons with a genetically encoded cGMP indicator and examined how these different functions could be regulated by the same second messenger in single neurons. cGMP imaging showed that, after odor application, cGMP levels in cilia decreased transiently, whereas levels in dendrites and soma gradually increased. These region-specific responses indicated that the responses in AWC neurons are explicitly compartmentalized. In addition, by combining cGMP and Ca2+ imaging, we observed that AWC neurons are analogous to vertebrate photoreceptor neurons.

3P230 Sensory stimulation from a specific modality adapts a different modality in Caenorhabditis elegans(15. Neuroscience & Sensory systems,Poster)

In study on C. elegans, the neural functions and circuits have been revealed at just single sensory modality, but it is unknown how a specific modality influences others. Here, we showed that chemotaxis to specific volatile odorants decreased by 5 min pre-exposure to gustatory cue (Mg2+) by behavior sassy, suggesting that the cross modality adaptation is observed in C. elegans. A specific neuron ablation with a phototaxis protein, KillerRed, revealed that AIY interneuron, which is projected by olfactory and gustatory sensory neurons, required for this adaptation. The function of AIY was also confirmed by intracellular Ca2+ measurement. The Ca2+ response to odor stimulation was diminished by the adaptation, also indicating that AIY is necessary for the adaptation.