Live imaging of excitable axonal microdomains in ankyrin-G-GFP mice

Reviewer #1 (Public Review):

This paper introduces a new transgenic mouse line that allows the labelling of the AIS and nodes of Ranvier (noR) by tagging Ank-G with GFP in a Cre-dependent manner. The authors characterise the properties of the AIS and noR when labelled with GFP to show that it has no adverse effects on the properties of the AIS and noR, as well as the intrinsic excitability of neurons. They also show that this mouse line can be used to follow AIS plasticity in vitro and visualise the AIS of neurons in vivo. This is a very useful and timely tool that will make an important impact in the field.

In general, it is clear that this mouse line can label the AIS and noR and will certainly be a useful tool for the community. Although the authors provide a thorough description of the intrinsic properties of neurons and some of the structural properties of the AIS and noR, there are some aspects of these experiments that could be refined to help show that tagging Ank-G with GFP is mostly inert. In particular, some of the basal properties of the AIS (length, position, molecular distribution) following tagging with GFP are not fully explored.

An important advantage of this mouse line is the ability to follow the AIS in live neurons. This is particularly important for imaging the dynamics and plasticity of the AIS, which the authors show is possible both in vitro and ex vivo. Finally, they also show that it is possible to image the AIS in vivo, a finding that opens many experimental doors for the future.

Reviewer #2 (Public Review):

The axon initial segment (AIS) is the axonal domain where most neurons integrate inputs and generate action potentials. Though structural and electrophysiological studies have allowed to better understand the mechanisms of assembly and maintenance of this domain, as well as its functions, there is still a need for efficient tools to study its structural organization and plasticity in vivo.

In this article, the authors describe the generation of a knock-in mouse reporter line allowing the conditional expression of a GFP-tagged version of AnkyrinG (Ank-G), which is a major protein of the axon initial segment and the nodes of Ranvier in neurons. This reporter line can in particular be used to study axon initial segment assembly and plasticity, by combining it with mouse lines or viruses expressing the Cre recombinase under the control of a neuronal promoter. Furthermore, the design of the line should allow to preserve the expression of the main Ank-G isoforms observed in neurons and could thus allow to study Ank-G related mechanisms in various neuronal subcompartments.

Some mouse lines allowing the neuronal expression of AIS/node of Ranvier markers coupled to a fluorescent protein exist, however they correspond to transgenic lines leading to potential overexpression of the tagged protein. Depending on the promoter used, their expression can vary and be absent in some neuronal populations (in particular, the Thy-1 promoter can lead to variable expression depending on the transgene insertion locus). Furthermore, these lines do not allow conditional expression of the protein regarding neuronal subtypes nor controlled temporal expression. Finally, a thorough description of the in vivo expression of the tagged protein at the AIS, and its impact on the structural and electrophysiological properties of the AIS are missing for these lines.

The present reporter line is thus definitely of interest, as the authors convincingly show that it can be used to visualize AIS ans Nodes of Ranvier in various contexts (from in vitro to in vivo). It could in particular be useful to study the assembly and plasticity of the domains where Ank-G is expressed. In this work, the authors thoroughly characterize the Ank-G-GFP reporter line generated and show that the structural and electrophysiological properties of the labeled neurons are not altered by the expression of the tagged Ank-G.