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

View Article: PubMed Central - PubMed

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.

No MeSH data available.


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The 270kDa-AnkG-GFP construct artificially increases AIS length in mature neurons. (A) Diagram of construct used and transfection method timeline. (B) Maximum intensity projection of a DGC expressing 270kDa-AnkG-GFP (cyan) which co-localizes with an anti-AnkG antibody (magenta). Asterisks, nucleus; arrowheads, DGC AIS start and end positions. (C) Maximum intensity projection of an untransfected DGC bearing an AIS of normal length. Arrowheads DGC AIS start and end positions. (D) Distributions of AIS lengths measured from 270kDa-AnkG-GFP transfected neurons and control untransfected DGCs. Each dot or triangle represents a single cell; orange symbols represent cells from panel (B); dark lines show mean ± SEM; paired t-test AnkG-GFP and AnkG ab, p = 0.68; ns, non-significant; unpaired t-test AIS length via AnkG ab from transfected vs. control untransfected cells, ***p = 0.002. (E) Correlation analysis for AISs labeled with both the AnkG-GFP construct and AnkG antibody. Each dot represents one cell; orange symbol, example cell from panel (B); line; best fit linear regression; Pearson’s correlation; ***p < 0.001.
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Figure 6: The 270kDa-AnkG-GFP construct artificially increases AIS length in mature neurons. (A) Diagram of construct used and transfection method timeline. (B) Maximum intensity projection of a DGC expressing 270kDa-AnkG-GFP (cyan) which co-localizes with an anti-AnkG antibody (magenta). Asterisks, nucleus; arrowheads, DGC AIS start and end positions. (C) Maximum intensity projection of an untransfected DGC bearing an AIS of normal length. Arrowheads DGC AIS start and end positions. (D) Distributions of AIS lengths measured from 270kDa-AnkG-GFP transfected neurons and control untransfected DGCs. Each dot or triangle represents a single cell; orange symbols represent cells from panel (B); dark lines show mean ± SEM; paired t-test AnkG-GFP and AnkG ab, p = 0.68; ns, non-significant; unpaired t-test AIS length via AnkG ab from transfected vs. control untransfected cells, ***p = 0.002. (E) Correlation analysis for AISs labeled with both the AnkG-GFP construct and AnkG antibody. Each dot represents one cell; orange symbol, example cell from panel (B); line; best fit linear regression; Pearson’s correlation; ***p < 0.001.

Mentions: Our third genetically-encoded probe consisted of GFP fused to the C-terminus of the 270kDa isoform of AnkG (270kDa-AnkG-GFP). We were aware that overexpressing this AnkG isoform in very young neurons (Galiano et al., 2012) or in AnkG- cells (Jenkins et al., 2015) was previously shown to produce abnormally elongated AISs. However, we reasoned that overexpressing 270kDa-AnkG-GFP in more mature wild-type neurons (Figure 6A; Table 2), in the presence of higher levels of endogenous AnkG and after the establishment of the AnkG-ankyrin-B boundary at the distal AIS (Galiano et al., 2012), might allow accurate live label of normal-length AISs. We also drove 270kDa-AnkG-GFP from the synapsin promoter, in order to achieve both neuronal specificity and activity-independent expression (see “Materials and Methods” Section). With this probe, localization to the AIS was good, with a significant correlation between AIS length revealed by the live marker and immunocytochemical label for total AnkG distribution (Figure 6E; 270kDa-AnkG-GFP vs. AnkG antibody Pearson’s r = 0.59 p = 0.0002 n = 35). However, under baseline conditions overexpression of 270kDa-AnkG-GFP from 7 DIV still produced a marked increase in AIS length when compared to neighboring non-transfected DGCs (Figures 6B–D; unpaired t-test on AIS AnkG antibody length from transfected vs. untransfected cells t = 4.1 p = 0.0001). Since this approach therefore broke our key requirement that live label should leave baseline AIS structure unperturbed, it was rapidly abandoned.


Evaluating Tools for Live Imaging of Structural Plasticity at the Axon Initial Segment
The 270kDa-AnkG-GFP construct artificially increases AIS length in mature neurons. (A) Diagram of construct used and transfection method timeline. (B) Maximum intensity projection of a DGC expressing 270kDa-AnkG-GFP (cyan) which co-localizes with an anti-AnkG antibody (magenta). Asterisks, nucleus; arrowheads, DGC AIS start and end positions. (C) Maximum intensity projection of an untransfected DGC bearing an AIS of normal length. Arrowheads DGC AIS start and end positions. (D) Distributions of AIS lengths measured from 270kDa-AnkG-GFP transfected neurons and control untransfected DGCs. Each dot or triangle represents a single cell; orange symbols represent cells from panel (B); dark lines show mean ± SEM; paired t-test AnkG-GFP and AnkG ab, p = 0.68; ns, non-significant; unpaired t-test AIS length via AnkG ab from transfected vs. control untransfected cells, ***p = 0.002. (E) Correlation analysis for AISs labeled with both the AnkG-GFP construct and AnkG antibody. Each dot represents one cell; orange symbol, example cell from panel (B); line; best fit linear regression; Pearson’s correlation; ***p < 0.001.
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Figure 6: The 270kDa-AnkG-GFP construct artificially increases AIS length in mature neurons. (A) Diagram of construct used and transfection method timeline. (B) Maximum intensity projection of a DGC expressing 270kDa-AnkG-GFP (cyan) which co-localizes with an anti-AnkG antibody (magenta). Asterisks, nucleus; arrowheads, DGC AIS start and end positions. (C) Maximum intensity projection of an untransfected DGC bearing an AIS of normal length. Arrowheads DGC AIS start and end positions. (D) Distributions of AIS lengths measured from 270kDa-AnkG-GFP transfected neurons and control untransfected DGCs. Each dot or triangle represents a single cell; orange symbols represent cells from panel (B); dark lines show mean ± SEM; paired t-test AnkG-GFP and AnkG ab, p = 0.68; ns, non-significant; unpaired t-test AIS length via AnkG ab from transfected vs. control untransfected cells, ***p = 0.002. (E) Correlation analysis for AISs labeled with both the AnkG-GFP construct and AnkG antibody. Each dot represents one cell; orange symbol, example cell from panel (B); line; best fit linear regression; Pearson’s correlation; ***p < 0.001.
Mentions: Our third genetically-encoded probe consisted of GFP fused to the C-terminus of the 270kDa isoform of AnkG (270kDa-AnkG-GFP). We were aware that overexpressing this AnkG isoform in very young neurons (Galiano et al., 2012) or in AnkG- cells (Jenkins et al., 2015) was previously shown to produce abnormally elongated AISs. However, we reasoned that overexpressing 270kDa-AnkG-GFP in more mature wild-type neurons (Figure 6A; Table 2), in the presence of higher levels of endogenous AnkG and after the establishment of the AnkG-ankyrin-B boundary at the distal AIS (Galiano et al., 2012), might allow accurate live label of normal-length AISs. We also drove 270kDa-AnkG-GFP from the synapsin promoter, in order to achieve both neuronal specificity and activity-independent expression (see “Materials and Methods” Section). With this probe, localization to the AIS was good, with a significant correlation between AIS length revealed by the live marker and immunocytochemical label for total AnkG distribution (Figure 6E; 270kDa-AnkG-GFP vs. AnkG antibody Pearson’s r = 0.59 p = 0.0002 n = 35). However, under baseline conditions overexpression of 270kDa-AnkG-GFP from 7 DIV still produced a marked increase in AIS length when compared to neighboring non-transfected DGCs (Figures 6B–D; unpaired t-test on AIS AnkG antibody length from transfected vs. untransfected cells t = 4.1 p = 0.0001). Since this approach therefore broke our key requirement that live label should leave baseline AIS structure unperturbed, it was rapidly abandoned.

View Article: PubMed Central - PubMed

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&mdash;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 NaV&beta;4-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&ndash;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.

No MeSH data available.


Related in: MedlinePlus