Journal
JOVE-JOURNAL OF VISUALIZED EXPERIMENTS
Volume -, Issue 51, Pages -Publisher
JOURNAL OF VISUALIZED EXPERIMENTS
DOI: 10.3791/2707
Keywords
Neuroscience; Issue 51; C. elegans; axotomy; regeneration; GABA neurons; pulsed laser; in vivo
Categories
Funding
- NIGMS NIH HHS [T32GM007223, T32 GM007223] Funding Source: Medline
- NINDS NIH HHS [R01 NS066082, R01 NS066082-01, R01 NS094219] Funding Source: Medline
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Neurons communicate with other cells via axons and dendrites, slender membrane extensions that contain pre- or post- synaptic specializations. If a neuron is damaged by injury or disease, it may regenerate. Cell- intrinsic and extrinsic factors influence the ability of a neuron to regenerate and restore function. Recently, the nematode C. elegans has emerged as an excellent model organism to identify genes and signaling pathways that influence the regeneration of neurons1- 6. The main way to initiate neuronal regeneration in C. elegans is laser- mediated cutting, or axotomy. During axotomy, a fluorescently- labeled neuronal process is severed using high- energy pulses. Initially, neuronal regeneration in C. elegans was examined using an amplified femtosecond laser5. However, subsequent regeneration studies have shown that a conventional pulsed laser can be used to accurately sever neurons in vivo and elicit a similar regenerative response1,3,7. We present a protocol for performing in vivo laser axotomy in the worm using a MicroPoint pulsed laser, a turnkey system that is readily available and that has been widely used for targeted cell ablation. We describe aligning the laser, mounting the worms, cutting specific neurons, and assessing subsequent regeneration. The system provides the ability to cut large numbers of neurons in multiple worms during one experiment. Thus, laser axotomy as described herein is an efficient system for initiating and analyzing the process of regeneration.
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