Femtosecond Laser Ablation Reveals Antagonistic Sensory and Neuroendocrine Signaling that Underlie C. elegans Behavior and Development

Abstract

SUMMARY The specific roles of neuronal subcellular components in behavior and development remain largely unknown, even though advances in molecular biology and conventional whole-cell laser ablation have greatly accelerated the identification of contributors at the molecular and cellular levels. We systematically applied femtosecond laser ablation, which has submicrometer resolution in vivo, to dissect the cell bodies, dendrites, or axons of a sensory neuron (ASJ) in Caenorhabditis elegans to determine their roles in modulating locomotion and the developmental decisions for dauer, a facultative, stress-resistant life stage. Our results indicate that the cell body sends out axonally mediated and hormonal signals in order to mediate these functions. Furthermore, our results suggest that antagonistic sensory dendritic signals primarily drive and switch polarity between the decisions to enter and exit dauer. Thus, the improved resolution of femtosecond laser ablation reveals a rich complexity of neuronal signaling at the subcellular level, including multiple neurite and hormonally mediated pathways dependent on life stage.

Novel DLK-independent neuronal regeneration in Caenorhabditis elegans shares links with activity-dependent ectopic outgrowth

“…Femtosecond laser surgery has submicrometer precision in the sample bulk (23) and permits cell-specific surgery of the ASJ dendrite within the amphid bundle and axon proximal to the amphid commissure (18). Unless otherwise noted, we performed laser surgery in L2 or L3 life stage with 3-nJ pulses at 10-kHz repetition rate, severing axons within several micrometers of the cell body and dendrites near their midpoint.…”

“…1 A2 and B), whereas dendrite transection results in little or no growth, even after 4 days (18,23). Consistent with previous studies of several neuron types in C. elegans (12,13,18), the dlk-1(ju476) mutation eliminates conventional DLK-1-mediated axon regeneration after axotomy alone, with <5% of postsurgery neurons regenerating ( Fig. 1 A3 and B).…”

“…laser surgery to establish their role in development and behavior (18). ASJ axons rapidly regenerate to the vicinity of their original synaptic targets, but regeneration is prevented by mutation of dlk-1, which plays a crucial role in neuronal regeneration in C. elegans (12), Drosophila (19), and mammals (20,21).…”

Ytterbium-doped fibre femtosecond laser offers robust design with deep and precise microsurgery

“…Specifically Yb2O3-doped fibre lasers emit pulses that are about one hundred to hundreds of femtosecond wide, at the range of 1000-1100 nm, at average power of mW to tens of watts 14 . The basic design is similar to Ti:Sapphire lasers that are very commonly used for multiphoton imaging and axotomy 15,16 . One advantage of Ti:Sapphire laser over Yb-fibre is that the wavelength is tunable, but this advantage is not crucial for ablation applications such as ours.…”

Sensory systems: their impact on C. elegans survival

“…This could then influence organismal homeostasis by modulating hormone release, e.g. , from DCVs in different subcellular compartments of a neuron (Chung et al, 2013). One such peptide hormone affected could be the TGF-β-like DAF-7, which is expressed in ASI sensory neurons and has been shown to inhibit both dauer entry and longevity (Ren et al, 1996; Schackwitz et al, 1996; Shaw et al, 2007).…”