Dissecting and Recording from The C. Elegans Neuromuscular Junction

Summary

Application of electrophysiology to accessible synapses provides a quantifiable measure of synaptic activity, useful in analyzing synaptic mutants. This article describes a dissection method used to expose the neuromuscular junctions (NMJ) of Caenorhabditis elegans (C. elegans) and briefly discusses some of the uses to which this preparation can be applied.

Abstract

Neurotransmission is the process by which neurons transfer information via chemical signals to their post-synaptic targets, on a rapid time scale. This complex process requires the coordinated activity of many pre- and post-synaptic proteins to ensure appropriate synaptic connectivity, conduction of electrical signals, targeting and priming of secretory vesicles, calcium sensing, vesicle fusion, localization and function of postsynaptic receptors and finally, recycling mechanisms. As neuroscientists it is our goal to elucidate which proteins function in each of these steps and understand their mechanisms of action. Electrophysiological recordings from synapses provide a quantifiable read out of the underlying electrical events that occur during synaptic transmission. By combining this technique with the powerful array of molecular and genetic tools available to manipulate synaptic proteins in C. elegans, we can analyze the resulting functional changes in synaptic transmission.

The C. elegans NMJs formed between motor neurons and body wall muscles control locomotion, therefore, mutants with uncoordinated locomotory phenotypes (known as unc s) often perturb synaptic transmission at these synapses ¹. Since unc mutants are maintained on a rich supply of a bacterial food source, they remain viable as long as they retain some pharyngeal pumping ability to ingest food. This, together with the fact that C. elegans exist as hermaphrodites, allows them to pass on mutant progeny without the need for elaborate mating behaviors. These attributes, coupled with our recent ability to record from the worms NMJs ^2,3,7 make this an excellent model organism in which to address precisely how unc mutants impact neurotransmission.

The dissection method involves immobilizing adult worms using a cyanoacrylic glue in order to make an incision in the worm cuticle exposing the NMJs. Since C. elegans adults are only 1 mm in length the dissection is performed with the use of a dissecting microscope and requires excellent hand-eye coordination. NMJ recordings are made by whole-cell voltage clamping individual body wall muscle cells and neurotransmitter release can be evoked using a variety of stimulation protocols including electrical stimulation, light-activated channel-rhodopsin-mediated depolarization ⁴ and hyperosmotic saline, all of which will be briefly described.

Protocol

A. Preparation of tools used for the dissection.

1. The dissection/recording chamber: We typically construct our recording chamber out of a 1/16^th inch magnetic sheet, with a circular hole drilled into the center that is large enough to accommodate a 22 mm diameter circular cover glass. The outer dimensions of the chamber will depend on the microscope stage used to do the recordings. On the reverse side of the magnetic sheet , we attach a 48 x 60 mm cover slip, which we adhere to the chamber by placing a pellet of low melt Paraplax tissue embedding wax in each corner of the cover slip sandwiched between the glass and the chamber. The wax is then me....

Discussion

The genetic model organism C, elegans is ideally suited for the study of synaptic transmission, through the functional analysis of mutations in genes encoding synaptic proteins. Here we have described a dissection technique that renders the C. elegans NMJs accessible for electrophysiological analysis. The accompanying video depicts the critical steps in the C. elegans dissection and the typical recording configuration used to measure synaptic activity. The three steps that pose the greates.......