METHOD

2. METHOD

All studies described here were accomplished under protocols approved by the Department of Biology of the University of California, San Diego. The frog sciatic nerve and gastrocnemius muscle were isolated from adult bullfrogs kept inside a specimen cage inside the laboratory. Two frogs were used in this experiment, the first for the verapamil trial, and the second for both the curare and succinylcholine trials.

2.1 DISSECTION AND EXPERIMENTAL PREPARATION

After the frog was killed by pithing, the gastrocnemius muscle attached to the complementary sciatic nerve was extracted. First, the skin on the left leg was removed, and the two thigh muscles were separated to reveal the sciatic nerve. Following the removal of the frog’s spine and dorsal midsection, a suture was tied to the sciatic nerve close to its neck area. Then the nerve was cut above the suture so that the tied suture is included on the nerve portion still attached to the gastrocnemius muscle. Another suture was tied to the frog’s Achilles’ tendon, and the tendon was removed from the frog such that the Achilles tendon, attached to the gastrocnemius muscle and the cut sciatic nerve can be removed completely from the frog.

The nerve and muscle were laid atop the nerve chamber as depicted in Figure 1. The nerve was weaved from electrode “E” backwards towards “A” via a glass hook, alternating over and under each consecutive electrode. The sutures tied to the ends of the sciatic nerve and gastrocnemius muscle are secured with clay on the nerve chamber ends, and help secure the specimen atop the apparatus. The marked letters A through I represent electrodes, whose functions are also depicted in Figure 2. The distances between each successive electrode from A through D is 5mm; the distances between each successive electrode from D through I is 10mm.

An electrical stimulus is introduced by a MacLab stimulator through the stimulating electrodes “A” and “B”, positioned at the sciatic nerve end. The electrical activity of the nerve is recorded monophasically through recording electrode “D”; the electrical activity of the muscle is recorded biphasically through recording electrodes “G” and “H”. The collective extracellular potential difference between an active and inactive site within the nerve and muscle represents a compound action potential (CAP) and muscle action potential (MAP), respectively. The CAP and MAP were amplified using a 2-channel pre-amplifier and transferred to a Macintosh computer. Data was acquired through Scope™ by eDAQ. Gain was taken into account in our reported results.

2.2 THRESHOLD AND MAP MAXIMUM DETERMINATION

First, the nerve and muscle function was verified with a 200mV stimulus (with a duration of 0.1msec), checking for both a muscle twitch and CAP and MAP detection by the computer. Throughout the experiment, Ringer’s solution was introduced to the nerve and muscle.

The stimulus potential was lowered to 50mV, followed by 5mV stimulus potential increments until a CAP was observed. This value is the threshold stimulus – the minimum stimulus potential needed to induce a CAP. While recording, the stimulus potential was further increased in 5mV increments until the MAP no longer increased (a maximum (or simply “max”) MAP.

2.3 DRUG EFFECTS

The effects of verapamil (0.5 mM), curare (7.33 μM), and succinylcholine (14 μM) on MAP recruitment, facilitation, and synaptic delay were examined separately, the drugs tested in that order. All three drug trials were preceded by a unique control, which establishes a control MAP recruitment, facilitation, and synaptic delay. After each drug test, the muscle/nerve was bathed for 10 minutes in pure Ringer’s solution to wash off any drug still present on the specimen.

2.3.1 CONTROL

2.3.1a MAP RECRUITMENT

Ringer’s solution was dripped on the muscle, which was allowed 3 minutes to return to room temperature to avoid temperature transients in data recording. The muscle was then simulated with single stimuli of 10mV increments, starting from the threshold stimulus to the max MAP stimulus. These MAPs were recorded.

2.3.1b FACILITATION

Facilitation was observed by stimulating the nerve with 5 pulses with an interpulse interval of 10msec, and recording the data. The stimulus amplitude was set to 400mV with a duration of 0.1msec.

2.3.1c SYNAPTIC DELAY

A supramaximal MAP stimulus potential was introduced with the nerve recording electrode at position “D”. The nerve recording electrode was then repositioned to position “E” and stimulated using the same stimulus settings. The nerve conduction velocity and therefore, conduction time, can be calculated from these two recordings. The synaptic delay can be calculated from the difference between conduction time and MAP latency, which is the time between stimulus onset and MAP onset.

2.3.2 DRUG

2.3.2a MAP RECRUITMENT

Following the control, the drug was slow dripped on the muscle for 5 minutes, likewise with 3 minutes allowed for the muscle to warm back up. MAP recruitment was similarly measured with single stimuli of 10mV increments, beginning from the threshold stimulus and ending with the max MAP stimulus.

2.3.2b FACILITATION

Facilitation succeeded MAP recruitment, and was observed in the same way as in 2.3.1b.

2.3.2c SYNAPTIC DELAY

Synaptic delay succeeded facilitation, and was measured and calculated in the same manner as in 2.3.1c.

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