Evaluating Tools for Live Imaging of Structural Plasticity at the Axon Initial Segment

The title sounded useful but the body totally lost me, proving once again that NO research is actually done for stroke survivors.  I really would like to see live monitoring of axon plasticity.
http://journal.frontiersin.org/article/10.3389/fncel.2016.00268/full?

Adna S. Dumitrescu, Mark D. Evans^† and Matthew S. Grubb^*

• Centre for Developmental Neurobiology, King’s College London, London, UK

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 Na_Vβ4-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-Na_VII–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.

Introduction

In neurons, 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).

It is also a highly dynamic structure. Over short-term timescales, the AIS can be modified by intrinsic conductances and intracellular signaling pathways, as well as by extrinsic synaptic and neuromodulatory inputs, to alter the initiation, patterning and spike waveform features of action potential firing (Bender et al., 2010, 2011; Grubb et al., 2011; Cotel et al., 2013; Martinello et al., 2015). Over longer timescales of hours to days, the structural and positional features of the AIS can also undergo modifications in response to sustained perturbations in neuronal activity. 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.

However, the evidence for structural AIS plasticity has thus far been limited to static snapshots, where plasticity is revealed post hoc by comparing fixed AIS label in separate groups of neurons subjected to different activity manipulations. Put simply, no individual AIS has ever been observed to change. This is despite the many potential benefits to be gained from live imaging of structural AIS plasticity. Following AISs live over time would allow us to definitively reveal local structural plasticity in individual neurons. It would also reduce the effects of cell-to-cell and experiment-to-experiment heterogeneity, permitting the detection of fine-scale changes that can be obscured in all but the largest of independent group datasets. It would allow studies of AIS plasticity to be combined with simultaneous live interrogation of neuronal function via electrophysiological and/or functional imaging techniques. And, finally, it has the potential to give us new insight into the mechanisms by which AIS plasticity is produced.

Here we characterize five alternative methodological approaches designed to live-label the AIS for timelapse imaging of activity-dependent plasticity. We find that, unlike other immunofluorescence-based and genetically-encoded probes, the fluorescently-tagged sodium channel motif YFP-Na_VII–III meets our three criteria for a successful AIS live-label tool: (1) it accurately labels AIS structure under baseline conditions; (2) it reveals hours-scale AIS structural plasticity; and (3) it leaves neuronal excitability unperturbed.

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