Worm Breeder's Gazette 15(2): 17 (February 1, 1998)
These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author.
UT Southwestern Medical Center at Dallas, 6000 Harry Hines Blvd., Dallas, TX 75235-9148
GluCla2, the product of the avr-15 gene, is a ligand-gated chloride
channel subunit. It forms part of a glutamate-gated chloride channel
expressed in the pharyngeal muscle. This channel mediates
neurotransmission by the M3 inhibitory glutamatergic motor neuron in the
pharynx. By acting as an agonist of this channel in pharyngeal muscle,
the drug ivermectin hyperpolarizes pharyngeal muscle irreversibly, thus
inhibiting pharyngeal pumping. When expressed in Xenopus oocytes,
GluCla2 forms a homomeric channel that is gated weakly by glutamate and
strongly by ivermectin (Dent et al., EMBO J. 16:5867, 1997). Since
GluCla2 forms an ivermectin sensitive homomeric channel, we were curious
whether, by expressing GluCla2 ectopically, we could use ivermectin to
hyperpolarize and thereby turn off the excitable cell of our choice.
This would allow us to observe the effect of turning off an excitable
cell that is necessary for larval development (such as M4 in the
pharynx). It would obviate one of the caveats to laser ablation, namely
that the worm has adapted to the absence of the ablated neuron over the
course of development. This technique would also be useful for looking
at phenomena such as activity-dependent pathfinding of neurons.
One problem is that wild-type worms are already sensitive to ivermectin.
To get around this problem we have created an ivermectin resistant
strain incorporating the avr-15 mutant. These worms are highly resistant
relative to wild-type but, apart from some mildly abnormal foraging
behavior and locomotion, otherwise appear normal. We placed the GluCla2A
("A" represents one of two alternatively spliced transcripts) cDNA under
control of the unc-54 promoter (thanks to Andy Fire and colleagues) and
used this construct to transform our resistant strain. Two transgenic
strains with extrachromosomal arrays were generated using rol-6 as a
cotransformation marker. Both strains were active on normal plates. When
placed on plates containing 100 ng/ml ivermectin, the non-roller worms
were active whereas the roller worms became paralyzed over several
hours. When prodded, the head and tail can contract, indicating that the
voltage clamp generated by opening the GluCla2 chloride channels can be
overcome somewhat by neuronal input and that the muscles are not simply
dead.
Much work needs to be done to make this a generally useful technique. We
are concerned that overexpression can lead to a significant chloride
leak current even in the absence of ivermectin and that opening chloride
channels in a subset of cells can have generalized effects via gap
junctions or by generally messing up the worm's chloride equilibrium. We
are also keen to show that the technique works in neurons. Nevertheless,
ectopic expression of GluCla2 promises to be an effective inducible
means of turning off neurons and muscles in the worm.