Voltage-Gated Potassium Channels Ensure Action Potential Shape Fidelity in Distal Axons.

The initiation and propagation of the action potential (AP) along an axon allows neurons to convey information rapidly and across distant sites. Although AP properties have typically been characterized at the soma and proximal axon, knowledge of the propagation of APs toward distal axonal domains of mammalian CNS neurons remains limited. We used genetically encoded voltage indicators (GEVIs) to image APs with submillisecond temporal resolution simultaneously at different locations along the long axons of dissociated hippocampal neurons from rat embryos of either sex. We found that APs became sharper and showed remarkable fidelity as they traveled toward distal axons, even during a high-frequency train. Blocking voltage-gated potassium channels (Kv) with 4-AP resulted in an increase in AP width in all compartments, which was stronger at distal locations and exacerbated during AP trains. We conclude that the higher levels of Kv channel activity in distal axons serve to sustain AP fidelity, conveying a reliable digital signal to presynaptic boutons.SIGNIFICANCE STATEMENT The AP represents the electrical signal carried along axons toward distant presynaptic boutons where it culminates in the release of neurotransmitters. The nonlinearities involved in this process are such that small changes in AP shape can result in large changes in neurotransmitter release. Since axons are remarkably long structures, any distortions that APs suffer along the way have the potential to translate into a significant modulation of synaptic transmission, particularly in distal domains. To avoid these issues, distal axons have ensured that signals are kept remarkably constant and insensitive to modulation during a train, despite the long distances traveled. Here, we uncover the mechanisms that allow distal axonal domains to provide a reliable and faithful digital signal to presynaptic terminals.

Activity-Dependent Plasticity of Axo-axonic Synapses at the Axon Initial Segment.

The activity-dependent rules that govern the wiring of GABAergic interneurons are not well understood. Chandelier cells (ChCs) are a type of GABAergic interneuron that control pyramidal cell output through axo-axonic synapses that target the axon initial segment. In vivo imaging of ChCs during development uncovered a narrow window (P12-P18) over which axons arborized and formed connections. We found that increases in the activity of either pyramidal cells or individual ChCs during this temporal window result in a reversible decrease in axo-axonic connections. Voltage imaging of GABAergic transmission at the axon initial segment (AIS) showed that axo-axonic synapses were depolarizing during this period. Identical manipulations of network activity in older mice (P40-P46), when ChC synapses are inhibitory, resulted instead in an increase in axo-axonic synapses. We propose that the direction of ChC synaptic plasticity follows homeostatic rules that depend on the polarity of axo-axonic synapses.

In Vitro Modelling of Nerve-Muscle Connectivity in a Compartmentalised Tissue Culture Device.

Motor neurons project axons from the hindbrain and spinal cord to muscle, where they induce myofibre contractions through neurotransmitter release at neuromuscular junctions. Studies of neuromuscular junction formation and homeostasis have been largely confined to in vivo models. In this study we have merged three powerful tools - pluripotent stem cells, optogenetics and microfabrication - and designed an open microdevice in which motor axons grow from a neural compartment containing embryonic stem cell-derived motor neurons and astrocytes through microchannels to form functional neuromuscular junctions with contractile myofibers in a separate compartment. Optogenetic entrainment of motor neurons in this reductionist neuromuscular circuit enhanced neuromuscular junction formation more than two-fold, mirroring the activity-dependence of synapse development in vivo. We incorporated an established motor neuron disease model into our system and found that coculture of motor neurons with SOD1G93A astrocytes resulted in denervation of the central compartment and diminished myofiber contractions, a phenotype which was rescued by the Receptor Interacting Serine/Threonine Kinase 1 (RIPK1) inhibitor Necrostatin. This coculture system replicates key aspects of nerve-muscle connectivity in vivo and represents a rapid and scalable alternative to animal models of neuromuscular function and disease.