Worm Breeder's Gazette 12(2): 21 (January 1, 1992)
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.
We have recorded single channel currents from membrane patches of nerve ring neurons. To expose these neurons, we dissected pharynxes from N2 L4 worms (LA WBG, this issue) glass cover slips precoated with CELL-TAC. The dissection was done in egg salt buffer (113 mM NaCl, 40 mM KCI, 3.4 mM C aCl2 ,3.4 mM M gCl2 ,10 mM HEPES pH 7.2) by placing a no. 15 surgical blade on the worm just behind the terminal bulb of the pharynx and pressing downward. We dissected up to 50 worms per cover slip and got several good ones (most of the dissections are unsuccessful because of the activity of the worms). In a good dissection, the cuticle surrounding the pharynx shrinks up and exposes the pharyngeal terminal bulb and isthmus and the cell bodies of the nerve ring neurons. These neurons are seen with DIC or phase contrast optics and resemble a cluster of grapes. We were able to get gigaohm seals in each of our first 4 tries using 2-10 megaohms resistance pipettes filled with egg salts. While the first 3 patches contained multiple currents, the fourth patch appeared to contain only a single channel. We recorded current amplitudes over a range of command voltages in order to determine the unitary conductance of this channel. Because the current-voltage ratio was constant over the range measured, we conclude that our calculated value for the conductance, 20 picosiemens, is constant as governed by Ohm's law. The reversal potential is not informative because we do not know the resting potential of the cell.
Next, we excised the patch and recorded in the inside-out cell-free patch configuration. Because the ion concentrations on the two sides of the membrane were symmetrical in this mode, all currents reversed at a command potential of zero. At the suggestion of Andy Blatz, we attempted to identify the ions that carry the current by adding 1 M KCl to the bath, producing an electrochemical driving force for both ions to flow into the pipette at a command potential of zero. Since the resulting current flowed into the pipette, we conclude that the current was carried by potassium. In summary, we have developed a dissection that is readily amenable to electrophysiological recording from nerve ring neurons. We have preliminarily characterized a cation selective channel of 20 pS conductance. We are now trying to identify the neuron from which we obtain the record by prelabeling amphid sensory neurons with a fluorescent probe in wild-type and mutant worms.
[See Figure 1]