Iontophoresis of Acetylcholine

Another prediction of the ACh hypothesis is that it should be possible to mimic the release of ACh from the presynaptic terminal by artificially applying some ACh to the vicinity of the neuromuscular junction. A simple technique called iontophoresis is available to precisely deliver very small amounts of ACh to restricted regions of a cell. The technique is illustrated in Fig. 9. One intracellular microelectrode is used to record the membrane potential. Another extracellular microelec-trode is filled with ACh and is placed close to the neuromuscular junction (but it does not impale either the motor axon or the muscle). ACh is a positively charged molecule. If the positive pole of a battery is connected to the ACh electrode, the positive charge of the battery will force ACh out of the electrode. The small amount of ACh ejected from the tip of the micropipette could then bind with any ACh receptors on the muscle cell. If ACh is the transmitter at the neuromuscular junction, iontophoretic application of

Stimulate Motor Axon

FIGURE 8 Effects of neostigmine on the EPP. In a saline solution containing an agent such as a small dose of curare to keep the EPP subthreshold, a small EPP is produced. When neostigmine (an agent that blocks the actions of AChE) is also added to the bath, the amplitude and duration of the EPP increase.

Stimulate Motor Axon

FIGURE 8 Effects of neostigmine on the EPP. In a saline solution containing an agent such as a small dose of curare to keep the EPP subthreshold, a small EPP is produced. When neostigmine (an agent that blocks the actions of AChE) is also added to the bath, the amplitude and duration of the EPP increase.

Voltmeter

Voltmeter

Muscle Cell

FIGURE 9 Procedure for iontophoretic application of ACh. Standard intracellular recordings are made from a skeletal muscle cell while a second micropipette filled with ACh is positioned near the neuromuscular junction. By applying a positive voltage to the ACh-filled micropipette, small quantities of ACh can be ejected in the vicinity of the motor end plate.

Muscle Cell

FIGURE 9 Procedure for iontophoretic application of ACh. Standard intracellular recordings are made from a skeletal muscle cell while a second micropipette filled with ACh is positioned near the neuromuscular junction. By applying a positive voltage to the ACh-filled micropipette, small quantities of ACh can be ejected in the vicinity of the motor end plate.

ACh should produce a potential change in the muscle cell similar to that produced by stimulation of the motor axon.

Figure 10 illustrates the results. The upper trace is a normal EPP produced by stimulating the motor axon. The small initial downward deflection is the stimulus artifact that indicates the point in time when the motor axon is electrically stimulated. The lower trace shows the result of the iontophoretic application of ACh. The potential changes produced are nearly identical. Experiments similar to Fig. 10 have demonstrated a number of other aspects of synaptic transmission at the neuromus-cular junction. When ACh is applied in the presence of neostigmine, the size of the potential is enhanced. These results are consistent with the cholinergic nature of synaptic transmission. If the preparation is perfused with neostigmine, ACh is not broken down by AChE, more receptors are bound, the permeability changes are greater, and the resultant potential change is greater. The iontophoretic potential is also affected by curare. By adding curare to the experimental preparation, the size of the iontophoretic response is reduced. In addition, the iontophoretically produced response is not affected by tetrodotoxin (TTX). We have already learned that TTX blocks the voltage-dependent change in Na+ permeability underlying the action potential. Because TTX has no effect on the iontophoretic application of ACh, channels in the membrane that are sensitive to ACh must be different from the channels in the membrane that underlie the action potential. It has also been observed that injecting ACh into the muscle cell produces no potential change. Thus, the receptors for ACh must be on the outer surface of the muscle cell. Iontophoretic applications of ACh to points along the muscle distant from the end plate yield no potential changes. A potential change due to local application of ACh is obtained only in the immediate vicinity of the motor end plate. Presumably, there is no potential change produced by ACh at sites more distant

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