Spatial gradient in TTX sensitivity of axons at the crayfish opener neuromuscular junction

Abstract

At the crayfish opener neuromuscular junction, axons branch repeatedly before synapsing onto muscle fibers as varicosities. Excitability of these axons was examined with two-electrode current clamp before and after partial block of Na+ channels with 1 nM tetrodotoxin. 4-Aminopyridine (200 μM) was added to homogenize nonuniformity in K+ channel density. The impact of tetrodotoxin was evaluated in terms of action potential (AP) amplitude, rate of rise, and threshold. All three parameters were more severely affected at the secondary than the primary branching point (BP). Both BPs fired continuously during 1-s current steps before tetrodotoxin. After tetrodotoxin, the secondary BP fired only in brief bursts, whereas the primary BP still fired continuously. Despite this diminished excitability at the secondary BP, no failure in orthodromic AP conduction was observed. AP waveform at terminals (APf) was examined with voltage indicators. For orthodromic APs, reduction in AP amplitude and prolongation of AP rise time in tetrodotoxin were more pronounced in terminals than at the secondary BP. For APs initiated at the secondary BP, APf sometimes failed to show a spikelike waveform in tetrodotoxin. This degraded APf was not due to averaging variable AP invasion into terminals, because the variance of APf traces did not increase in tetrodotoxin. Tetrodotoxin applied in the absence of 4-aminopyridine showed an impact on the distal axon similar but less distinct than that recorded with 4-aminopyridine. In conclusion, the distal axon is more sensitive to tetrodotoxin than the proximal axon, such that AP waveform degrades as it propagates toward terminals in tetrodotoxin.