Limits...
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.


Related in: MedlinePlus

ExtNF antibody conjugated with Alexa-488 does not track AIS plasticity. (A) Diagram of AIS live label method used and experimental timeline. (B) Maximum intensity projections of example neurons labeled live with the extNF antibody conjugated to Alexa-488, after which they were subjected to either a 3 h control or depolarization treatment. Following fixation, cells were stained with antibodies against AnkG (magenta) and Prox1 (dark blue). Asterisks, nucleus; arrowheads, DGC AIS start and end positions. (C) Distributions of AIS lengths measured with extNF-488 conjugated antibody and AnkG from control or depolarized DGCs. Each dot or triangle represents a single cell; orange symbols, values from example cells displayed in (B); dark lines show mean ± SEM; Bonferroni post-test after two-way repeated measures ANOVA; ns, non-significant; *p < 0.05. (D) Correlation analysis for AISs labeled with both the extNF-488 conjugated antibody label and AnkG under control or depolarized conditions. Orange symbols, values from example cells displayed in (B); lines show best linear regression fit for control (black) or depolarized cells (gray); **p < 0.01; ***p < 0.001.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC5120105&req=5

Figure 3: ExtNF antibody conjugated with Alexa-488 does not track AIS plasticity. (A) Diagram of AIS live label method used and experimental timeline. (B) Maximum intensity projections of example neurons labeled live with the extNF antibody conjugated to Alexa-488, after which they were subjected to either a 3 h control or depolarization treatment. Following fixation, cells were stained with antibodies against AnkG (magenta) and Prox1 (dark blue). Asterisks, nucleus; arrowheads, DGC AIS start and end positions. (C) Distributions of AIS lengths measured with extNF-488 conjugated antibody and AnkG from control or depolarized DGCs. Each dot or triangle represents a single cell; orange symbols, values from example cells displayed in (B); dark lines show mean ± SEM; Bonferroni post-test after two-way repeated measures ANOVA; ns, non-significant; *p < 0.05. (D) Correlation analysis for AISs labeled with both the extNF-488 conjugated antibody label and AnkG under control or depolarized conditions. Orange symbols, values from example cells displayed in (B); lines show best linear regression fit for control (black) or depolarized cells (gray); **p < 0.01; ***p < 0.001.

Mentions: Live immunolabeling approaches using standard, multivalent probes suffer the potential risk of crosslinking antigen molecules at the levels of both primary and secondary antibodies, thereby altering mobility or function of the bound antigen. If neurofascin molecules live-labeled with extNF were somehow rendered more stable by such crosslinking, this may explain the inaccuracy of the approach in labeling plastic changes in AIS structure. We therefore ruled out the possibility of cross-linking at the secondary antibody level by pre-conjugating the extNF primary antibody with an Alexa 488 fluorophore before applying it to our neurons in a single labeling step (Figure 3A). However, this pre-conjugated extNF-488 live label was no more accurate in reporting rapid activity-dependent AIS shortening. While AnkG-defined DGC AIS lengths were again significantly shorter in the 3 h-depolarized group, distributions of pre-conjugated, live-applied extNF-488 label did not differ between treatment groups (Figures 3B,C; two-way repeated measured ANOVA; treatment, F(1,73) = 0.77 p = 0.38; label type, F(1,73) = 7.42 p = 0.008; interaction F(1,73) = 13.73 p = 0.0004; Bonferroni post-test, extNF-488 control vs. depolarized treatments t = 1.07 p > 0.05; AnkG control vs. depolarized treatment t = 2.6 p < 0.05). Moreover, correlations between AIS length measured by AnkG and pre-conjugated extNF-488 label were relatively weak in both control and depolarized cells (Figure 3D; control cells Pearson’s r = 0.47 p = 0.003 n = 38; depolarized cells Pearson’s r = 0.56 p = 0.0003 n = 37).


Evaluating Tools for Live Imaging of Structural Plasticity at the Axon Initial Segment
ExtNF antibody conjugated with Alexa-488 does not track AIS plasticity. (A) Diagram of AIS live label method used and experimental timeline. (B) Maximum intensity projections of example neurons labeled live with the extNF antibody conjugated to Alexa-488, after which they were subjected to either a 3 h control or depolarization treatment. Following fixation, cells were stained with antibodies against AnkG (magenta) and Prox1 (dark blue). Asterisks, nucleus; arrowheads, DGC AIS start and end positions. (C) Distributions of AIS lengths measured with extNF-488 conjugated antibody and AnkG from control or depolarized DGCs. Each dot or triangle represents a single cell; orange symbols, values from example cells displayed in (B); dark lines show mean ± SEM; Bonferroni post-test after two-way repeated measures ANOVA; ns, non-significant; *p < 0.05. (D) Correlation analysis for AISs labeled with both the extNF-488 conjugated antibody label and AnkG under control or depolarized conditions. Orange symbols, values from example cells displayed in (B); lines show best linear regression fit for control (black) or depolarized cells (gray); **p < 0.01; ***p < 0.001.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5120105&req=5

Figure 3: ExtNF antibody conjugated with Alexa-488 does not track AIS plasticity. (A) Diagram of AIS live label method used and experimental timeline. (B) Maximum intensity projections of example neurons labeled live with the extNF antibody conjugated to Alexa-488, after which they were subjected to either a 3 h control or depolarization treatment. Following fixation, cells were stained with antibodies against AnkG (magenta) and Prox1 (dark blue). Asterisks, nucleus; arrowheads, DGC AIS start and end positions. (C) Distributions of AIS lengths measured with extNF-488 conjugated antibody and AnkG from control or depolarized DGCs. Each dot or triangle represents a single cell; orange symbols, values from example cells displayed in (B); dark lines show mean ± SEM; Bonferroni post-test after two-way repeated measures ANOVA; ns, non-significant; *p < 0.05. (D) Correlation analysis for AISs labeled with both the extNF-488 conjugated antibody label and AnkG under control or depolarized conditions. Orange symbols, values from example cells displayed in (B); lines show best linear regression fit for control (black) or depolarized cells (gray); **p < 0.01; ***p < 0.001.
Mentions: Live immunolabeling approaches using standard, multivalent probes suffer the potential risk of crosslinking antigen molecules at the levels of both primary and secondary antibodies, thereby altering mobility or function of the bound antigen. If neurofascin molecules live-labeled with extNF were somehow rendered more stable by such crosslinking, this may explain the inaccuracy of the approach in labeling plastic changes in AIS structure. We therefore ruled out the possibility of cross-linking at the secondary antibody level by pre-conjugating the extNF primary antibody with an Alexa 488 fluorophore before applying it to our neurons in a single labeling step (Figure 3A). However, this pre-conjugated extNF-488 live label was no more accurate in reporting rapid activity-dependent AIS shortening. While AnkG-defined DGC AIS lengths were again significantly shorter in the 3 h-depolarized group, distributions of pre-conjugated, live-applied extNF-488 label did not differ between treatment groups (Figures 3B,C; two-way repeated measured ANOVA; treatment, F(1,73) = 0.77 p = 0.38; label type, F(1,73) = 7.42 p = 0.008; interaction F(1,73) = 13.73 p = 0.0004; Bonferroni post-test, extNF-488 control vs. depolarized treatments t = 1.07 p > 0.05; AnkG control vs. depolarized treatment t = 2.6 p < 0.05). Moreover, correlations between AIS length measured by AnkG and pre-conjugated extNF-488 label were relatively weak in both control and depolarized cells (Figure 3D; control cells Pearson’s r = 0.47 p = 0.003 n = 38; depolarized cells Pearson’s r = 0.56 p = 0.0003 n = 37).

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