The Frog Motor Nerve Terminal Has Very Brief Action Potentials and Three Electrical Regions Predicted to Differentially Control Transmitter Release

Abstract

The action potential (AP) waveform controls the opening of voltage-gated calcium channels and contributes to the driving force for calcium ion flux that triggers neurotransmission at presynaptic nerve terminals. Although the frog neuromuscular junction (NMJ) has long been a model synapse for the study of neurotransmission, its presynaptic AP waveform has never been directly studied, and thus the AP waveform shape and propagation through this long presynaptic nerve terminal are unknown. Using a fast voltage-sensitive dye, we have imaged the AP waveform from the presynaptic terminal of male and female frog NMJs and shown that the AP is very brief in duration and actively propagated along the entire length of the terminal. Furthermore, based on measured AP waveforms at different regions along the length of the nerve terminal, we show that the terminal is divided into three distinct electrical regions: A beginning region immediately after the last node of Ranvier where the AP is broadest, a middle region with a relatively consistent AP duration, and an end region near the tip of nerve terminal branches where the AP is briefer. We hypothesize that these measured changes in the AP waveform along the length of the motor nerve terminal may explain the proximal-distal gradient in transmitter release previously reported at the frog NMJ.

A high-affinity, partial antagonist effect of 3,4-diaminopyridine mediates action potential broadening and enhancement of transmitter release at NMJs

“…Kv3 currents have been shown to mediate the dominant outward current during brief AP depolarizations within many nerve terminals ( 71 , 72 , 73 , 74 ) and have also been shown to have rapid activation and inactivation characteristics that enable nerve terminals to fire APs with short duration and at high frequency ( 75 , 76 , 77 ). These data are consistent with the AP waveforms that were recently optically recorded from frog presynaptic nerve terminals ( 64 ). Here, we report concentration-dependent broadening of the presynaptic AP at the frog and mouse NMJ after exposure to 3,4-DAP.…”

“…Here, we report concentration-dependent broadening of the presynaptic AP at the frog and mouse NMJ after exposure to 3,4-DAP. Even relatively small changes in presynaptic AP duration (15–20%) have been predicted to have significant effects on calcium ion entry and transmitter release ( 64 ). Thus, the roughly 20% broadening reported here at the mouse NMJ would be predicted to underlie the approximate 3-fold increase in transmitter release we observed ( 64 ).…”

“…Even relatively small changes in presynaptic AP duration (15–20%) have been predicted to have significant effects on calcium ion entry and transmitter release ( 64 ). Thus, the roughly 20% broadening reported here at the mouse NMJ would be predicted to underlie the approximate 3-fold increase in transmitter release we observed ( 64 ). Interestingly, we found that 1.5-μM 3,4-DAP broadened the presynaptic AP waveform in the frog NMJ to a greater extent than at the mouse NMJ.…”

“…Voltage imaging was performed as described previously ( 64 ). To load nerve terminals with the dye for the voltage-imaging procedure, a mixture of 5 ml of normal frog Ringer saline (for frog preparations) or normal mammalian Ringer saline (for mouse preparations) with a BeRST 1 voltage-sensitive dye ( 63 ) concentration of 0.5 μM and 10 μg/ml of Alexa Fluor 488–conjugated alpha-bungarotoxin (BTX; to counterstain postsynaptic receptors at the NMJ and block muscle contractions) was freshly made before each experiment.…”

“…Then, an unbiased ROI selection (a subsection of the full imaging ROI) containing the nerve terminal was created by applying an Otsu local imaging threshold ( 85 ) to the average fluorescence z-projection of the BeRST 1 image stack. The average fluorescence inside this ROI was used as the nerve signal (for frog recordings, 20 μm near the end of the nerve terminal and last node of Ranvier were excluded to restrict recordings to the middle electrical region of the terminal; see ( 64 )). The average fluorescence from the region outside of the Otsu-selected ROI was used as the background signal.…”