Cortical Axons, Isolated in Channels, Display Activity-Dependent Signal Modulation as a Result of Targeted Stimulation.

Marta K Lewandowska,Jan Müller,David Jäckel,Miloš Radivojević,Andreas R Hierlemann

doi:10.3389/fnins.2016.00083

Abstract

Mammalian cortical axons are extremely thin processes that are difficult to study as a result of their small diameter: they are too narrow to patch while intact, and super-resolution microscopy is needed to resolve single axons. We present a method for studying axonal physiology by pairing a high-density microelectrode array with a microfluidic axonal isolation device, and use it to study activity-dependent modulation of axonal signal propagation evoked by stimulation near the soma. Up to three axonal branches from a single neuron, isolated in different channels, were recorded from simultaneously using 10–20 electrodes per channel. The axonal channels amplified spikes such that propagations of individual signals along tens of electrodes could easily be discerned with high signal to noise. Stimulation from 10 up to 160 Hz demonstrated similar qualitative results from all of the cells studied: extracellular action potential characteristics changed drastically in response to stimulation. Spike height decreased, spike width increased, and latency increased, as a result of reduced propagation velocity, as the number of stimulations and the stimulation frequencies increased. Quantitatively, the strength of these changes manifested itself differently in cells at different frequencies of stimulation. Some cells' signal fidelity fell to 80% already at 10 Hz, while others maintained 80% signal fidelity at 80 Hz. Differences in modulation by axonal branches of the same cell were also seen for different stimulation frequencies, starting at 10 Hz. Potassium ion concentration changes altered the behavior of the cells causing propagation failures at lower concentrations and improving signal fidelity at higher concentrations.

Highlights

The axon is an important element in cell-to-cell communication, since it connects a neuronal cell body with its numerous post-synaptic partners
In the present work we demonstrate how our approach, using a high-density microelectrode array (HD-microelectrode arrays (MEAs)) with an axonal isolation microchannel device on top (Lewandowska et al, 2015), can be used to study axonal modulation of orthodromic action potentials in very thin branches of mammalian cortical axons
That were associated with a putative soma, were spike sorted, and the times were correlated with spikes over the rest of the array to create a profile of a single cell, termed a “footprint.” The number of electrodes monitoring an axon depended on the size and extent of the axonal arbor and ranged from 40 to 90

Summary

Introduction

The axon is an important element in cell-to-cell communication, since it connects a neuronal cell body with its numerous post-synaptic partners. Kole used double and triple patch clamp techniques to patch the soma, axon initial segment (AIS), and an axonal bleb of cortical layer V cells, and examined ion channel distributions in the AIS. They studied K+ channels, and found that Kv1 channels shape the action potential, regulate neurotransmitter release, and are able to integrate subthreshold activity (Kole et al, 2007). Sasaki targeted axons of CA3 pyramidal cells, and studied action potential broadening via local application of glutamate and adenosine A1 receptor antagonists They found that “depolarization-induced K+ channel inactivation is likely to underlie the AP broadening” (Sasaki et al, 2011). Sasaki found that axonal geometry modulated somatic influence differently to proximal and distal synapses, i.e., the observed action potential broadening changed with distance from the soma (Sasaki et al, 2012a)

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