In this thesis we first present the theoretical and practical background of the femtosecond laser ablation technique. We describe the origin of the intensity threshold and its contribution to localization of damage as well as the impact of the repetition rate on resolution. We present the experimental setup and some practical considerations, including the pulse energy dependence of damage, various surgery types and their associated survival rates, and different capabilities of neuronal regeneration. The remainder of the thesis describes our experiments, findings, and interpretations. We used femtosecond laser ablation within the ASJ neuron to ablate the cell bodies, sever dendrites, and sever axons to determine their contributions to the locomotion, dauer entry, and dauer exit behaviors. We find that ablation of the ASJ cell bodies in L1 and L4 rescues the unc-1(e580) locomotory defect. Because mutation of ssu-1(fc73) rescues unc-1(e580) locomotory and volatile anesthesic phenotypes, and because ssu-1 is only expressed in the ASJ neurons, it is likely that the ablative rescue of the unc-1 phenotypes is due to elimination of SSU-1. Cutting the ASJ dendrites or axons in L4 has seemingly the opposite effect as ablating the cell body, decreasing the locomotory velocity of unc-1(e580) worms. This suggests two conclusions. First, since the locomotory velocity does not increase after axon surgery (i.e., no locomotion rescue), and since unc-1 is broadly expressed, this suggests that the ASJ cell bodies secrete an SSU-1-modified signal to affect other cells involved in the unc-1 locomotion pathway. Second, the decrease in velocity might suggest that the ASJ neuron takes input through its neuronal fibers which is inhibitory for the SSU-1-modified signal.
Our dauer entry and exit experiments in the ASJ neuron under daf-11(sa195) and wild-type backgrounds suggest that food and pheromone are sensed by DAF-11 and a putative pheromone receptor in the ASJ cilia, respectively. For dauer entry, the dendritic signal from DAF-11 is mediated by cGMP and inhibits the ASJ cell body. The dendritic signal along the pheromone pathway excites the ASJ cell body. The cell bodies presumably integrate the signals with synaptic input from the axons and send out a signal through their axons promoting dauer entry. For dauer exit, the DAF-11 dendritic signal excites the ASJ cell bodies and the pheromone pathway dendritic signal inhibits the ASJ cell bodies. The cell bodies secrete a signal to promote dauer exit. While the ASJ neuron has also been implicated in endocrine signaling for dauer exit, anesthetic sensitivity & locomotion, and longevity, to our knowledge, our study is the first to present direct evidence supporting that claim. Both the locomotion and the dauer exit data indicate that the ASJ neuron secretes signals to affect other cells.
