Spatial variation in membrane excitability modulated by 4-AP-sensitive K+ channels in the axons of the crayfish neuromuscular junction

Current-clamp recordings were made from the primary (1°) and secondary (2°) branching points (BPs) of axons at the crayfish neuromuscular junction. Action potential (AP) firing initiated by current injected at the 2° BP showed strong adaptation or high-frequency firing at threshold current, whereas AP firing frequency at the 1° BP exhibited a gradual rise with increasing current amplitude. The voltage threshold for AP ( VTH) was higher at the 2° BP than the 1° BP. 4-Aminopyridine (4-AP) at 200 μM increased AP amplitude and duration at both BPs but reduced threshold current at the 2° BP more than at the 1° BP. This blocker lowered VTH at both BPs, but the difference between the BPs remained. Firing patterns evoked at the 2° BP became similar to those evoked at the 1° BP in 4-AP. Thus 4-AP-sensitive channels may be more concentrated in the distal axon and control AP initiation and firing patterns there. Orthodromic APs between the two BPs were also compared. There was no difference in AP amplitude between the two BPs, but AP half-width recorded at the 2° BP was longer than that at the 1° BP. AP duration at both BPs increased gradually, by ∼17%, during a 100-Hz, 500-ms train (in-train rise). Normalized AP half-widths revealed a smaller fractional in-train rise at the 2° BP. Thus, although distal APs were broader, AP duration there was under more stringent control than that of the proximal axon. 4-AP increased AP amplitude and duration of the entire orthodromic train and reduced the magnitude of the in-train rise in AP half-width at both BPs. However, this blocker did not uncover a clear difference between the two BPs. Thus 4-AP-sensitive channels concentrated in distal axon may be essential in preventing unintended firing and modulating AP waveform without interfering with orthodromic AP propagation.

Role of NSF in Neurotransmitter Release: A Peptide Microinjection Study at the Crayfish Neuromuscular Junction

Peptides that inhibit the SNAP-stimulated ATPase activity of N-ethylmaleimide-sensitive fusion protein (NSF-2, NSF-3) were injected intra-axonally to study the role of this protein in the release of glutamate at the crayfish neuromuscular junction. Macropatch recording was used to establish the quantal content and to construct synaptic delay histograms. NSF-2 or NSF-3 injection reduced the quantal content, evoked by either direct depolarization of a single release bouton or by axonal action potentials, on average by 66 ± 12% (mean ± SD; n = 32), but had no effect on the time course of release. NSF-2 had no effect on the amplitude or shape of the presynaptic action potential nor on the excitatory nerve terminal current. Neither NSF-2 nor NSF-3 affected the shape or amplitude of single quantal currents. Injection of a peptide with the same composition as NSF-2, but with a scrambled amino acid sequence, failed to alter the quantal content. We conclude that, at the crayfish neuromuscular junction, NSF-dependent reactions regulate quantal content without contributing to the presynaptic mechanisms that control the time course of release.