Slit Worm Dissection

The first preparation for patch-clamp analysis of excitable cells in C. elegans was described by Goodman and Lockery (19). In their approach, worms are glued in place and a small incision is made in the cuticle, allowing a "bouquet" of neurons to emerge. With the aid of a green fluorescent protein marker, individual neurons in this bouquet can be easily identified for recording. Slight modifications have been made to this approach over the past several years in order to extend patch-clamp analysis to C. elegans muscle cells (20), specific interneurons of the locomotory control circuit (24), and touch receptor neurons (27).

Each approach requires that the worm be immobilized prior to dissection and recording. Worms are glued down on glass cover slips coated with sili-cone elastomer (Sylgard 184; Dow Corning) using tissue adhesive. Cyanoacryl glue (www.glustitch.com) rapidly polymerizes on contact with water and works well for this purpose. Glue is applied by mouth pipet using a glass pipet that has been broken back to increase the flow of glue to a suitable rate. Worms can either be incubated on ice to slow their movement prior to gluing or lassoed by applying glue to the head or tail and subsequently glued in place for recording. Slowing down worm movement prior to gluing permits more precise application of glue while lassoing a moving worm alleviates any concerns about temperature effects. Typically, the worm is lying on its right side (preserving right-sided commissural processes) and glue is applied along the dorsal aspect of the worm, taking care not to allow overflow to the area that will be exposed for recordings. A puncture in the cuticle away from the site of recording is then made to relieve hydrostatic pressure. For recording from body wall muscles, the ventral muscle quadrants and ventral cord are exposed by a longitudinal incision along the glue line on the left dorsal side of the worm extending approximately from the vulva to the terminal bulb of the pharynx (Fig. 1). A sharp glass needle works well for this purpose. The intestine and gonad are removed using either a glass needle or by suction and the free flap of cuticle created by the incision is then glued to the sylgard-coated cover slip, exposing the ventral musculature for recording. Following muscle exposure, the preparation is washed briefly (~30 s) with a collagenase solution (1 mg/mL in recording solution) to clean the plasma membrane of adherent extracellular proteins and then rinsed with the extracellular recording solution (150 mM NaCl, 5 mM KCl, 4 mM MgCl2, 1 mM CaCl2, 15 mM HEPES, and 10 mM glucose, pH 7.4. Sucrose is added to adjust the osmolar-ity to 340 mOsm.) Variations on this procedure can be used for recording from head interneurons or touch neurons in the tail. To access these neurons, the worm is glued down in the same fashion as previously described and the area of interest is exposed by dissection. For head interneurons, a transverse incision is made immediately posterior to the terminal bulb and then continued anteriorly along the glue line toward the tip of the nose (Fig. 1; ref. 24). The resulting triangle of cuticle is glued to the sylgard-coated cover slip, exposing the nerve ring and interneurons for recording. For recording from PLM touch neurons, cell bodies are exposed by a ventral incision along the tail (27).

Neuromuscular Junction Nerve ring/lnterneuron

Neuromuscular Junction Nerve ring/lnterneuron

Fig. 1. Caenorhabditis elegans preparations for electrophysiology. Left, Dissected preparation for muscle recordings. The box on the sketch marks the area pictured below, in which C. elegans is dissected open and the body-wall muscles exposed for patch-clamp recording. Green fluorescent protein (GFP) expression in transgenic worms is limited to muscles using the promoter of the myo-3 gene (60). Right, the dissection used for exposing the bilaterally symmetrical command inter-neurons AVA and AVD. These interneurons can be readily identified in transgenic worms that express GFP under the control of the nmr-1 promoter (61). The markings represent the sites of incision and show the the orientation of the final dissected preparation. The positions of the GFP-positive interneuron cell bodies in the dissected preparation are shown and correspond to the labeled cell bodies in the image below.

Fig. 1. Caenorhabditis elegans preparations for electrophysiology. Left, Dissected preparation for muscle recordings. The box on the sketch marks the area pictured below, in which C. elegans is dissected open and the body-wall muscles exposed for patch-clamp recording. Green fluorescent protein (GFP) expression in transgenic worms is limited to muscles using the promoter of the myo-3 gene (60). Right, the dissection used for exposing the bilaterally symmetrical command inter-neurons AVA and AVD. These interneurons can be readily identified in transgenic worms that express GFP under the control of the nmr-1 promoter (61). The markings represent the sites of incision and show the the orientation of the final dissected preparation. The positions of the GFP-positive interneuron cell bodies in the dissected preparation are shown and correspond to the labeled cell bodies in the image below.

The Sugar Solution

The Sugar Solution

Curb Sugar Cravings Once And For All With These Powerful Techniques. Sugar sensitive people might be low in specific neurochemicals that help us feel calm, centered, confident, and optimistic. Sugar is a drug that temporarily makes the sugar sensitive feel better, but with damaging consequences.

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