Abstract
The axon initial segment (AIS) is a specialized neuronal compartment involved in the maintenance of axo-dendritic polarity and in the generation of action potentials. It is also a site of significant structural plasticity—manipulations of neuronal activity in vitro and in vivo can produce changes in AIS position and/or size that are associated with alterations in intrinsic excitability. However, to date all activity-dependent AIS changes have been observed in experiments carried out on fixed samples, offering only a snapshot, population-wide view of this form of plasticity. To extend these findings by following morphological changes at the AIS of individual neurons requires reliable means of labeling the structure in live preparations. Here, we assessed five different immunofluorescence-based and genetically-encoded tools for live-labeling the AIS of dentate granule cells (DGCs) in dissociated hippocampal cultures. We found that an antibody targeting the extracellular domain of neurofascin provided accurate live label of AIS structure at baseline, but could not follow rapid activity-dependent changes in AIS length. Three different fusion constructs of GFP with full-length AIS proteins also proved unsuitable: while neurofascin-186-GFP and NaVβ4-GFP did not localize to the AIS in our experimental conditions, overexpressing 270kDa-AnkyrinG-GFP produced abnormally elongated AISs in mature neurons. In contrast, a genetically-encoded construct consisting of a voltage-gated sodium channel intracellular domain fused to yellow fluorescent protein (YFP-NaVII–III) fulfilled all of our criteria for successful live AIS label: this construct specifically localized to the AIS, accurately revealed plastic changes at the structure within hours, and, crucially, did not alter normal cell firing properties. We therefore recommend this probe for future studies of live AIS plasticity in vitro and in vivo.
Highlights
In neurons, the axon initial segment (AIS) is a molecularly-defined portion of the proximal axon with unique structural and functional properties
The YFP-NaVII–III construct was clearly the most suitable—it accurately revealed both baseline and activity-altered AIS structure, and did not affect intrinsic neuronal excitability (Figure 9). This probe may prove useful in future studies investigating alterations in AIS structure in individual neurons over time
There are significant advantages associated with an immunocytochemical approach to live AIS label in cell culture, including comprehensive coverage across all neurons in a given preparation, labeling that is rapidly effective after minutes, and protocols that do not require disruption of neuronal membrane integrity
Summary
The axon initial segment (AIS) is a molecularly-defined portion of the proximal axon with unique structural and functional properties. It serves as a barrier that maintains distinct somatodendritic vs axonal neuronal polarity (Rasband, 2010), and is a key regulator of neuronal excitability—in almost all neuronal cell types, and under almost all circumstances, the AIS is the site of action potential initiation (Bender and Trussell, 2012; Kole and Stuart, 2012). These structural forms of AIS plasticity—which can include changes in AIS length, position and/or ion channel distribution in both excitatory and inhibitory neurons (Grubb and Burrone, 2010a; Kuba et al, 2010; Muir and Kittler, 2014; Chand et al, 2015; Evans et al, 2015; Wefelmeyer et al, 2015)—have been shown to be associated with changes in neuronal excitability, and may form part of a repertoire of compensatory mechanisms acting to maintain network activity within set limits
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