The bath contained Tyrode's solution of the following composition: 137 mM NaCl, 2.7 mM KCl, 11.9 mM NaHCO3, 0.3 mM NaH2PO4, 0.5 mM MgCl2, 1.8 mM CaCl2, and 5.6 mM glucose. The pH was maintained at 7.3 by bubbling with a gas mixture of 95% O2 and 5% CO2. Muscles were stimulated indirectly via the nerve clamp, bipolar loop–type, or suction-type electrodes using an S88 Grass stimulator. Muscle tension was recorded with Grass FT 03 isometric force-displacement transducers (Grass Instruments, Warwick, RI, USA), displayed on a Gould TA240 EasyGraf chart recorder (Gould Instruments, Valley View, OH, USA), and digitized using pCLAMP software (Molecular Devices, Sunnyvale, CA, USA). A bath concentration of 1 nM BoNT serotype A (BoNT/A; Metabiologics, Madison, WI, USA) results in complete muscle paralysis of mouse, guinea pig, and rabbit muscle in ~1 h. Application of BoNT/A does not decrease force tension in muscles directly stimulated by electrodes, because BoNT/A acts presynaptically.Electrode design
A schematic drawing of the nerve clamp electrode is illustrated in Figure 1. The assembled device, built within a 1-mL syringe, contains an adjustable spring coil at one end and the nerve clamp–stimulating portion at the other. The electrode was constructed from a metal hypodermic tubing and guidewire assembly (Small Parts, Miami Lakes, FL, USA). A groove was filed into the end of the guidewire (0.046-in diameter) to clamp the nerve in place. The guidewire fits inside a sleeve of hypodermic tubing [16 gauge; outer diameter 0.065 in; inner diameter 0.047 in]. The tubing was modified at one end with a notch for a small segment of nerve to pass inside. The sliding action of the guidewire allows nerves to be held in contact with the hypodermic tubing, which is the stimulation portion of the electrode.
The internal guidewire was made adjustable and placed under tension at rest by cementing it to a spring coil at the proximal end of the electrode. Both spring coil and guidewire were placed inside a 1-mL tuberculin syringe, and the spring coil was cemented to the syringe at a point nearest the narrow syringe opening. The internal guidewire was bent and shrink-wrapped with plastic for fine finger/thumb adjustment.
Instructions for the assembly of this electrode are described and illustrated in Figure 2. In Step 1, a notch was cut into the hypodermic tubing at one end using a fine file, and a sheath of polyethylene tubing was placed tightly over the hypodermic tubing as insulation. A platinum/iridium stimulating wire (0.25-mm diameter) was soldered to the exposed hypodermic tubing. This assembly from Step 1 is positioned inside of a second, larger-diameter polyethylene tubing along with a second platinum/iridium wire, which serves as the ground (see Step 2).
The internal guidewire was prepared at both ends in Step 3. A groove was filed at the nerve clamp end, and the wire was bent into a loop at the other. The spring coil was placed inside of the 1.0-mL syringe barrel and cemented inside. The guide was inserted inside the spring and syringe barrel and cemented to the spring coil. The assembled stimulating/ground portion from Step 2 was inserted over the guidewire into the syringe barrel. The hypodermic tubing was cemented with epoxy to the syringe tip. The platinum/ iridium stimulating and ground wires were subsequently connected to a Bayonet-Neill-Concelman (BNC) cable to be used with the S88 Grass stimulator.
The cut end of a motor nerve innervating a muscle can be clamped and fixed in space using the guidewire assembly in the following manner: when the spring is pulled back, the electrode can be positioned such that any segment length of nerve can be fixed in space against the hypodermic tubing. Release of the spring coil will return the guidewire to its normal position inside the hypodermic tubing and clamp the nerve in place. The stimulator generates supramaximal pulses, causing the clamped nerve to generate muscle contractions, provided the nerve was not damaged during dissection. The extent of muscle contraction can be recorded using an isometric force transducer that converts the force of contraction into a digitized wave form to be analyzed later on pCLAMP software. The tensions can be recorded directly onto a chart recorded as analog waveforms. This method of stimulation enables the study of substances that act either presynaptically in the nerve, such as botulinum toxin, or act at the level of the neuromuscular junction, such as nerve agents. The major advantage of the described electrode is its ability to indirectly stimulate any skeletal muscle, regardless of nerve length, for the measurement of muscle force tension using tissue organ baths.