Electrophysiological events recorded at presynaptic terminals of the crayfish neuromuscular junction with a voltage indicator.

The water-soluble voltage indicator JPW1114 was used to stain thin axons and terminal varicosities of the crayfish neuromuscular junction. A slow, overnight injection protocol was developed to brightly stain fine structures without cytotoxicity. Fluorescence transients filtered at 2 kHz showed that the duration of terminal action potentials was shorter than that of those recorded in the main trunk of the axons. In addition, the repolarization phases of the terminal and axonal action potentials overlapped in time, suggesting that the entire axonal arborization repolarizes simultaneously. Manipulating resting membrane potential, +/-15-20 mV, did not alter the peak level or duration of action potentials if they fired in isolation. A prolongation of action potential, by 23%, could be induced if a 10-spike burst at 100 Hz was fired from depolarized membrane potential. No such change was observed when the high frequency train was fired from resting or hyperpolarized levels. Microelectrodes in the main trunk of axons typically recorded a depolarizing after-potential (DAP) following an action potential initiated from resting membrane potential. The DAP could be inverted and enlarged by depolarization and hyperpolarization, respectively. Fluorescence transients recorded from terminals exhibited similar DAP characteristics. The ratio of DAP to action potential amplitude recorded from terminals was similar to that recorded from the main axon. Thus, the entire axonal arborization returned to resting level in a spatially uniform manner during the DAP. The functional significance of DAP is discussed in the light of these observations.