Sodium Channel β2 Subunits Prevent Action Potential Propagation Failures at Axonal Branch Points

Abstract

Neurotransmitter release depends on voltage-gated Na channels (Nas) to propagate an action potential (AP) successfully from the axon hillock to a synaptic terminal. Unmyelinated sections of axon are very diverse structures encompassing branch points and numerous presynaptic terminals with undefined molecular partners of Na channels. Using optical recordings of Ca and membrane voltage, we demonstrate here that Na channel β2 subunits (Naβ2s) are required to prevent AP propagation failures across the axonal arborization of cultured rat hippocampal neurons (mixed male and female). When Naβ2 expression was reduced, we identified two specific phenotypes: (1) membrane excitability and AP-evoked Ca entry were impaired at synapses and (2) AP propagation was severely compromised with >40% of axonal branches no longer responding to AP-stimulation. We went on to show that a great deal of electrical signaling heterogeneity exists in AP waveforms across the axonal arborization independent of axon morphology. Therefore, Naβ2 is a critical regulator of axonal excitability and synaptic function in unmyelinated axons. Voltage-gated Ca channels are fulcrums of neurotransmission that convert electrical inputs into chemical outputs in the form of vesicle fusion at synaptic terminals. However, the role of the electrical signal, the presynaptic action potential (AP), in modulating synaptic transmission is less clear. What is the fidelity of a propagating AP waveform in the axon and what molecules shape it throughout the axonal arborization? Our work identifies several new features of AP propagation in unmyelinated axons: (1) branches of a single axonal arborization have variable AP waveforms independent of morphology, (2) Na channel β2 subunits modulate AP-evoked Ca-influx, and (3) β2 subunits maintain successful AP propagation across the axonal arbor. These findings are relevant to understanding the flow of excitation in the brain.

Signal propagation along the axon

“…In cerebellar stellate cell interneurons, action potential width and the resultant GABA release is determined by the local density of Kv3 channels found at each terminal [11 ]. In a similar way, differential expression of Navb2 subunits induces heterogeneous action potential amplitudes in various hippocampal axonal branches [10 ]. Moreover, expression of Kv7 at nodes of Ranvier in neocortical pyramidal cells strongly hyperpolarizes the membrane potential and thus improves Nav channel availability and optimal spike amplitude [12].…”

“…Yet, the consequences of the Nav-b2 depletion on spike propagation had not been yet evaluated experimentally. The recent study by the group of Michael Hoppa filled this gap by showing with the use of geneticallyencoded voltage-imaging combined with calcium imaging that genetic deletion of Nav-b2 induces collateralselective propagation failures in axons of hippocampal cells [10 ] (Figure 1b).…”

“…(2) After Navb2 knock-down, conduction failures appear at branch points, caused by the decrease in Nav channels membrane expression. Adapted from[10 ].…”

The Potassium Channel Subunit Kvβ1 is Required for Synaptic Facilitation

“…All live imaging experiments were set up as previously described (42, 43). Briefly, images were obtained using an Olympus microscope (IX-83) equipped with a 40x 1.35 NA oil immersion objective (UApoN40XO340-2) and captured with an IXON Ultra 897 EMCCD (Andor).…”

Freeze-frame imaging of synaptic activity using SynTagMA

“…Field stimulation-evoked action potentials were generated by passing 1 ms current pulses, yielding fields of ~12 V cm -2 through the recording chamber bracketed by platinum/iridium electrodes. Electrical stimuli were locked to start according to defined frame number intervals using a custom-built board named "Neurosync" powered by an Arduino Duo chip (Arduino) manufactured by an engineering firm (Sensostar) (Cho et al, 2017). A collimated 405 nm LED light source (Thorlabs) was set on top of the microscope stage with a custom-built plastic case (Bob Robertson, Dartmouth College).…”